Статті в журналах: "MSC, GvHD, IMMUNOTHERAPY"

1

Lim, Jisun, Jinbeom Heo, Hyein Ju, Ji-Woong Shin, YongHwan Kim, Seungun Lee, Hwan Yeul Yu, et al. "Glutathione dynamics determine the therapeutic efficacy of mesenchymal stem cells for graft-versus-host disease via CREB1-NRF2 pathway." Science Advances 6, no. 16 (April 2020): eaba1334. http://dx.doi.org/10.1126/sciadv.aba1334.

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Glutathione (GSH), the most abundant nonprotein thiol functioning as an antioxidant, plays critical roles in maintaining the core functions of mesenchymal stem cells (MSCs), which are used as a cellular immunotherapy for graft-versus-host disease (GVHD). However, the role of GSH dynamics in MSCs remains elusive. Genome-wide gene expression profiling and high-throughput live-cell imaging assays revealed that CREB1 enforced the GSH-recovering capacity (GRC) of MSCs through NRF2 by directly up-regulating NRF2 target genes responsible for GSH synthesis and redox cycling. MSCs with enhanced GSH levels and GRC mediated by CREB1-NRF2 have improved self-renewal, migratory, anti-inflammatory, and T cell suppression capacities. Administration of MSCs overexpressing CREB1-NRF2 target genes alleviated GVHD in a humanized mouse model, resulting in improved survival, decreased weight loss, and reduced histopathologic damages in GVHD target organs. Collectively, these findings demonstrate the molecular and functional importance of the CREB1-NRF2 pathway in maintaining MSC GSH dynamics, determining therapeutic outcomes for GVHD treatment.

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Karlsson, Helen, Sujith Samarasinghe, Lynne M. Ball, Berit Sundberg, Arjan C. Lankester, Francesco Dazzi, Mehmet Uzunel, et al. "Mesenchymal stem cells exert differential effects on alloantigen and virus-specific T-cell responses." Blood 112, no. 3 (August 1, 2008): 532–41. http://dx.doi.org/10.1182/blood-2007-10-119370.

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Abstract Mesenchymal stem cells (MSCs) suppress alloantigen-induced T-cell functions in vitro and infusion of third-party MSCs seems to be a promising therapy for graft-versus-host disease (GVHD). Little is known about the specificity of immunosuppression by MSCs, in particular the effect on immunity to pathogens. We have studied how MSCs affect T-cell responses specific to Epstein-Barr virus (EBV) and cytomegalovirus (CMV). We found that EBV- and CMV-induced proliferation and interferon-γ (IFN-γ) production from peripheral blood mononuclear cells (PBMCs) was less affected by third-party MSCs than the response to alloantigen and that MSCs had no effect on expansion of EBV and CMV pentamer-specific T cells. Established EBV-specific cytotoxic T cells (CTL) or CMV-CTL cultured with MSCs retained the ability to proliferate and produce IFN-γ in response to their cognate antigen and to kill virally infected targets. Finally, PBMCs from 2 patients who received MSCs for acute GVHD showed persistence of CMV-specific T cells and retained IFN-γ response to CMV after MSC infusion. In summary, MSCs have little effect on T-cell responses to EBV and CMV, which contrasts to their strong immunosuppressive effects on alloreactive T cells. These data have major implications for immunotherapy of GVHD with MSCs and suggest that the effector functions of virus-specific T cells may be retained after MSC infusion.

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3

Sangiorgi, Bruno, and Rodrigo Alexandre Panepucci. "Modulation of Immunoregulatory Properties of Mesenchymal Stromal Cells by Toll-Like Receptors: Potential Applications on GVHD." Stem Cells International 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/9434250.

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In the last decade, the immunomodulatory properties of mesenchymal stromal cells (MSCs) have attracted a lot of attention, due to their potential applicability in the treatment of graft-versus-host disease (GVHD), a condition frequently associated with opportunistic infections. The present review addresses how Pathogen-Associated Molecular Patterns (PAMPS) modulate the immunosuppressive phenotype of human MSCs by signaling through Toll-like receptors (TLRs). Overall, we observed that regardless of the source tissue, human MSCs express TLR2, TLR3, TLR4, and TLR9. Stimulation of distinct TLRs on MSCs elicits distinct inflammatory signaling pathways, differentially influencing the expression of inflammatory factors and the ability of MSCs to suppress the proliferation of immune system cells. The capacity to enhance the immunosuppressive phenotype of MSCs through TLRs stimulation might be properly elucidated in order to improve the MSC-based immunotherapy against GVHD.

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Boni, Andrea, Pawel Muranski, Claudia Wrzesinski, Andrew Kaiser, Chrystal Paulos, Douglas Palmer, Luca Gattinoni, and Nicholas P. Restifo. "Partly MHC Matched Allogeneic Tumor Specific T Cells Mediate Tumor Regression without Inducing GVHD in Immunosuppressed Host." Blood 108, no. 11 (November 16, 2006): 5210. http://dx.doi.org/10.1182/blood.v108.11.5210.5210.

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Abstract Adoptive cell transfer (ACT) immunotherapy following lymphodepleting conditioning is a promising strategy for the treatment of metastatic solid tumors, however the difficulties in generating autologous tumor specific lymphocytes for every patient has significantly limited its applications. Allogeneic partly matched tumor specific T cells could be used for patients in whom autologous cells are not available, however their rapid rejection by the host restricts this approach. When CD8+ pmel-1 T cells from B6-BALB-C F1 (b/d) were transferred into B16 tumor-bearing B6 mice (b/b) or into a different F1 (b/k), these tumor-specific T cells were rapidly rejected, and had no impact on the tumor regression. Here we show that following myeloablative conditioning, the adoptive transfer of allogeneic, major histocompatibility mismatched tumor-specific T lymphocytes resulted in the regression of large vascularized tumors. The ability of adoptively transferred allogeneic T cells to mediate tumor regression was directly proportional to the dose of irradiation given prior T cell transfer which also correlated with the in vivo survival of the transferred cells. At the highest irradiation dose used (i.e. 11 Gy) allogeneic T cells could survive for as long as 4 weeks and their efficacy was comparable to syngeneic tumor-reactive T cells. In addition we found that the risk of inducing a graft versus host (GVH) reaction was minor when the specificity of transferred TCR is confined. In fact co-transfer of transgenic cells and small amounts of open repertoire T cells (2*10e4) able to react with the host did not result in any measurable toxicity whereas co transfer of greater quantities (2*10e5 or more) could cause fatal GVHD effect. Interestingly GVH reaction was associated with an improved tumor treatment, though this effect was transient as most of the animals succumbed to GVHD. Here we demonstrate that allogeneic T cells might represent an important tool in cancer immunotherapy allowing treatment of patients for whom it is not possible to obtain autologous cells. Furthermore the possible synergy between tumor specific allogeneic T cells and GVH effect could be exploited in bone marrow transplant protocols.

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Fang, Ying, Yichen Zhu, Adam Kramer, Yuning Chen, Yan-Ruide Li, and Lili Yang. "Graft-versus-Host Disease Modulation by Innate T Cells." International Journal of Molecular Sciences 24, no. 4 (February 17, 2023): 4084. http://dx.doi.org/10.3390/ijms24044084.

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Allogeneic cell therapies, defined by genetically mismatched transplantation, have the potential to become a cost-effective solution for cell-based cancer immunotherapy. However, this type of therapy is often accompanied by the development of graft-versus-host disease (GvHD), induced by the mismatched major histocompatibility complex (MHC) between healthy donors and recipients, leading to severe complications and death. To address this issue and increase the potential for allogeneic cell therapies in clinical practice, minimizing GvHD is a crucial challenge. Innate T cells, encompassing subsets of T lymphocytes including mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, and gamma delta T (γδ T) cells, offer a promising solution. These cells express MHC-independent T-cell receptors (TCRs), allowing them to avoid MHC recognition and thus GvHD. This review examines the biology of these three innate T-cell populations, evaluates research on their roles in GvHD modulation and allogeneic stem cell transplantation (allo HSCT), and explores the potential futures for these therapies.

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6

Kwiatkowska-Borowczyk, Eliza P., Anna Kozłowska, Klaudia Maruszak, Luiza Kańczuga-Koda, Mariusz Koda, Monika Dajnowiec, Andrzej Mackiewicz, and Dariusz W. Kowalczyk. "Adoptive transfer of tumor specific T cells from allogeneic donors is feasible, effective and safe alternative to autologous T cell based tumor immunotherapy." Journal of Medical Science 83, no. 1 (March 30, 2014): 21–28. http://dx.doi.org/10.20883/medical.e39.

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Donor lymphocyte infusion is used to increase the graft versus tumor (GVT) effect after allogeneic hematopoietic cell transplant. The limited spectrum of activity and high risk of graft versus host disease (GVHD) remain major limitations of this approach. The finding of new cell populations for adoptive immunotherapy, with the ability to separate GVT from GVHD, would be useful. In the present manuscript, we tested in mouse model the use of allogeneic MHC partially matched effector cells for adoptive T cell immunotherapy of cancer. We sought to maximize graft-versus-tumor effect while minimizing GVHD using tumor-specific allogeneic effector T cells rather than open-repertoire T cells. A F1 hybrid (Balb/c x C57BL/6) -MethA-EGFP–bearing mice received a preparative regimen of nonmyeloablating cyclophosphamide lymphodepletion followed by adoptive transfer of bulk Balb/c derived allogeneic T cells specific for the MethA-EGFP tumor cells. Adoptively transferred allogeneic tumor-specific T lymphocytes prevented tumor formation without graft versus host disease – like symptoms. We found that the risk of GVHD was low even with high number of transferred tumor-specific T cells. These data indicate that the use of tumor-specific allogeneic T cells is feasible, effective and safe alternative to autologous T cell based tumor immunotherapy.

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7

Marcus, Assaf, Tova Waks, and Zelig Eshhar. "Tumor immunotherapy using chimeric receptor redirected allogeneic T cells across MHC barriers (131.39)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 131.39. http://dx.doi.org/10.4049/jimmunol.184.supp.131.39.

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Abstract We present a strategy for adoptive immunotherapy using fully mismatched allogeneic chimeric receptor redirected T cells (allo-'T-bodies') in the absence of Graft vs. Host disease (GvHD). The strategy hinges on creating a therapeutic time-window using mild preconditioning which on one hand allows the allogeneic T-bodies enough time to effectively attack the tumor but on the other hand does not ablate the Host vs. Graft response (HvG) ensuring the eventual rejection of the allogeneic T cells thus preventing GvHD. We found that transfer of fully mismatched allogeneic T cells expressing a HER2/neu specific chimeric receptor following mild preconditioning roughly tripled the median survival of mice bearing pulmonary Renca-HER2 metastases with around 50% of mice surviving long term (>200 days) in the absence of GvHD mortality. We show that allo-T-bodies provide comparable benefit to syngeneic T-bodies, and are far superior to non-specific allogeneic T cells, demonstrating that the response is indeed tumor-specific and not merely allo-specific. Taken together these data provide proof of principle for safe and effective allogeneic adoptive therapy allowing for the use of 'universal effector cells' as a standardized treatment for cancer.

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8

Luznik, Leo, Sanju Jalla, Laura W. Engstrom, Robert Iannone, and Ephraim J. Fuchs. "Durable engraftment of major histocompatibility complex–incompatible cells after nonmyeloablative conditioning with fludarabine, low-dose total body irradiation, and posttransplantation cyclophosphamide." Blood 98, no. 12 (December 1, 2001): 3456–64. http://dx.doi.org/10.1182/blood.v98.12.3456.

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Abstract Treatment of leukemia by myeloablative conditioning and transplantation of major histocompatibility complex (MHC)–mismatched stem cells is generally avoided because of the high risk of graft rejection or lethal graft-versus-host disease (GVHD). This study shows that MHC-incompatible cells can engraft stably after nonmyeloablative conditioning with immunosuppressive chemotherapy and low-dose total body irradiation (TBI). Long-term mixed hematopoietic chimerism, clonal deletion of donor-reactive T cells, and bidirectional cytotoxic T-cell tolerance were achieved by transplanting MHC-mismatched marrow cells into recipients conditioned with pretransplantation fludarabine or cyclophosphamide (Cy), 50 to 200 cGy TBI on day −1, and Cy 200 mg/kg intraperitoneally on day 3. In this model, long-term donor chimerism was proportional to the dose of TBI or donor marrow cells. Pretransplantation fludarabine and posttransplantation Cy were both required for alloengraftment, but the drugs had additional effects. For example, fludarabine sensitized host stem cells to the toxicity of TBI, because animals conditioned with both agents had higher chimerism than animals conditioned with TBI alone (P < .05). Also, posttransplantation Cy attenuated lethal and nonlethal GVH reactions, because F1 recipients of host-reactive, parental spleen cells survived longer (P < .05) and had lower donor cell chimerism (P < .01) if they received posttransplantation Cy than if they did not. Finally, delayed infusions of donor lymphocytes into mixed chimeras prolonged survival after leukemia challenge (P < .0001) without causing lethal GVHD. These results indicate that stable engraftment of MHC-incompatible cells can be induced after fludarabine-based, nonmyeloablative conditioning and that it serves as a platform for adoptive immunotherapy with donor lymphocyte infusions.

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9

Waller, Edmund K., Alan M. Ship, Stephen Mittelstaedt, Timothy W. Murray, Richard Carter, Irina Kakhniashvili, Sagar Lonial, Jeannine T. Holden, and Michael W. Boyer. "Irradiated Donor Leukocytes Promote Engraftment of Allogeneic Bone Marrow in Major Histocompatibility Complex Mismatched Recipients Without Causing Graft-Versus-Host Disease." Blood 94, no. 9 (November 1, 1999): 3222–33. http://dx.doi.org/10.1182/blood.v94.9.3222.

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Abstract Graft rejection in allogeneic bone marrow transplantation (BMT) can occur when donor and recipient are mismatched at one or more major histocompatibility complex (MHC) loci. Donor T cells can prevent graft rejection, but may cause fatal graft-versus-host disease (GVHD). We tested whether irradiation of allogeneic donor lymphocytes would preserve their graft-facilitating activity while inhibiting their potential for GVHD. Infusions of irradiated allogeneic T cells did not cause GVHD in MHC-mismatched SJL → (SJL × C57BL6) F1, C57BL6 → B10.RIII, and C57BL6 → B10.BR mouse donor → recipient BMT pairs. The 60-day survival among MHC-mismatched transplant recipients increased from 2% (BM alone) to up to 75% among recipients of BM plus irradiated allogeneic splenocytes. Optimal results were obtained using 50 × 106 to 75 × 106 irradiated donor splenocytes administered in multiple injections from day −1 to day +1. Recipients of an equal number of nonirradiated MHC-mismatched donor splenocytes uniformly died of acute GVHD. The graft facilitating activity of the irradiated allogeneic splenocytes was mediated by donor T cells. Irradiation to 7.5 Gy increased nuclear NFκB in T cells and their allospecific cytotoxicity. Irradiated T cells survived up to 3 days in the BM of MHC-mismatched recipients without proliferation. Recipients of irradiated allogeneic splenocytes and allogeneic BM had stable donor-derived hematopoiesis without a significant representation of donor splenocytes in the T-cell compartment. Irradiated allogeneic T cells thus represent a form of cellular immunotherapy with time-limited biologic activity in vivo that can facilitate allogeneic BMT without causing GVHD.

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10

Waller, Edmund K., Alan M. Ship, Stephen Mittelstaedt, Timothy W. Murray, Richard Carter, Irina Kakhniashvili, Sagar Lonial, Jeannine T. Holden, and Michael W. Boyer. "Irradiated Donor Leukocytes Promote Engraftment of Allogeneic Bone Marrow in Major Histocompatibility Complex Mismatched Recipients Without Causing Graft-Versus-Host Disease." Blood 94, no. 9 (November 1, 1999): 3222–33. http://dx.doi.org/10.1182/blood.v94.9.3222.421k06_3222_3233.

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Graft rejection in allogeneic bone marrow transplantation (BMT) can occur when donor and recipient are mismatched at one or more major histocompatibility complex (MHC) loci. Donor T cells can prevent graft rejection, but may cause fatal graft-versus-host disease (GVHD). We tested whether irradiation of allogeneic donor lymphocytes would preserve their graft-facilitating activity while inhibiting their potential for GVHD. Infusions of irradiated allogeneic T cells did not cause GVHD in MHC-mismatched SJL → (SJL × C57BL6) F1, C57BL6 → B10.RIII, and C57BL6 → B10.BR mouse donor → recipient BMT pairs. The 60-day survival among MHC-mismatched transplant recipients increased from 2% (BM alone) to up to 75% among recipients of BM plus irradiated allogeneic splenocytes. Optimal results were obtained using 50 × 106 to 75 × 106 irradiated donor splenocytes administered in multiple injections from day −1 to day +1. Recipients of an equal number of nonirradiated MHC-mismatched donor splenocytes uniformly died of acute GVHD. The graft facilitating activity of the irradiated allogeneic splenocytes was mediated by donor T cells. Irradiation to 7.5 Gy increased nuclear NFκB in T cells and their allospecific cytotoxicity. Irradiated T cells survived up to 3 days in the BM of MHC-mismatched recipients without proliferation. Recipients of irradiated allogeneic splenocytes and allogeneic BM had stable donor-derived hematopoiesis without a significant representation of donor splenocytes in the T-cell compartment. Irradiated allogeneic T cells thus represent a form of cellular immunotherapy with time-limited biologic activity in vivo that can facilitate allogeneic BMT without causing GVHD.

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Cho, Seok-Goo, Hyunsil Park, Min Jung Park, Ho-Youn Kim, Jong-Wook Lee, and Woo-Sung Min. "Bone Marrow T Cells Are More Effective Than Peripheral T Cells in Inducing Chimeric Conversion Following MHC-Mismatched Nonmyeloablative Bone Marrow Transplantation." Blood 112, no. 11 (November 16, 2008): 4593. http://dx.doi.org/10.1182/blood.v112.11.4593.4593.

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Abstract Background & Objectives: Recently, T cells in BM have attracted renewed interest because they are now known to have different surface phenotypes, subsets, and activation states from those in the periphery. Memory T cells undergo extensive migration from the blood to the BM and vice versa. The BM plays an important role in preferential homing and extensive proliferation of memory T cells, and contributes considerably to the longlived memory T cell pool. BM T cells are more activated than their splenic counterparts and have a higher rate of local proliferation. Although BM-T (NK1.1– CD4+ or CD8+) cells did not induce lethal GVH disease, even at high cell numbers, BM-T cells mediated vigorous graft-versus-tumor activity and facilitated engraftment of hematopoietic progenitor cells. These studies suggested that BM-T cells could be a useful cellular source for adoptive immunotherapy following ABMT, instead of peripheral T cells. Non-myeloablative bone marrow transplantation (NMT) and allogeneic mixed chimerism can provide an environment adequate for diminishing susceptibility to DLI-mediated GVHD and an immunological platform for DLI in both mouse and human models. In patients treated with DLI, a successful GVL effect is often associated with conversion to complete donor chimerism, supporting the concept of a graft-versus-host (GVH) response as part of the GVL effect. Thus, a quiet chimeric conversion following DLI is desirable to reach an optimal DLI-mediated GVL effect, without the occurrence of GVHD. Although in a mouse model, the administration of non-tolerant donor spleen cells to established mixed chimeras has been shown to convert mixed hematopoietic chimerism to full donor chimerism, without the concomitant development of GVHD, DLI in humans frequently results in serious GVHD and life-threatening complications. However, the use of BM-T cells, as compared with spleen T cells (SP-T), as the DLI source has not been investigated in allogeneic mixed chimerism prepared with NMT. In this study, we evaluated the beneficial alloreactivity of DLI using cryopreserved BM-T cells, a by-product obtained during the T cell depletion (TCD) procedure in BM grafting, to effectively induce chimeric conversion without the occurrence of GVHD in MHC-mismatched NMT. Methods: Cells were prepared using established procedures. During the T cell depletion (TCD) procedure in BM grafting, BM-T cells were obtained as a by-product and then cryopreserved for subsequent DLI using BM-T cells 21 days after the bone marrow transplant. Results: The administration of 5–10 × 105 BM-T (Thy1.2+) cells in mixed chimeras resulted in complete chimeric conversion, with self-limited graft-versus-host disease (GVHD) and no pathological changes. However, the administration of 5–10 × 105 SP-T (Thy1.2+) cells resulted in persistent mixed chimerism, with pathological GVHD signs in the liver and intestine. Conclusion: Our results suggest that DLI using BM-T cells, even in small numbers, could be more potent for inducing chimeric conversion in mixed chimerism than DLI using SP-T cells. Further study is needed to determine whether cryopreserved BM-T cells are an effective cell source for DLI to consolidate donor-dominant chimerism in clinical practice, without concerns about GVHD.

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12

Spitzer, Thomas R., Steven L. McAfee, Dey R. Bimalangshu, Karen Ballen, Eyal Attar, Christine Colby, Fred Preffer, et al. "In Vivo and Ex Vivo T-Cell Depleted (TCD) NonmyeloablativeHaploidentical Stem Cell Transplantation (NSCT) for Hematologic Malignancy (HM)." Blood 106, no. 11 (November 16, 2005): 5431. http://dx.doi.org/10.1182/blood.v106.11.5431.5431.

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Abstract Based on our nonmyeloablative MHC-mismatched murine bone marrow transplant (BMT) models, in which mixed lymphohematopoietic chimerism (MLC) is reliably induced as a platform for adoptive cellular immunotherapy via delayed donor lymphocyte infusions (DLI), we developed a similar clinical strategy for haploidentical NSCT. Conditioning for NSCT has consisted of high dose cyclophosphamide, in vivo T-cell depletion (TCD) with anti-T-cell antibody therapy, pre-transplant thymic irradiation and cyclosporine (CYA) for GVHD prophylaxis (with taper and discontinuation by day 35 for patients with MLC and no evidence of GVHD). Because of a high incidence of ≥ grade II GVHD (10 of 14 patients) using equine anti-thymocyte globulin for in vivo TCD, we substituted MEDI-507 (an anti-CD2 monoclonal antibody) for ATG. Of 8 patients who underwent BMT with MEDI-507 for in vivo TCD, 5 experienced graft loss. The protocol was then modified to include ex vivo T-cell depleted high dose peripheral blood stem cell transplantation (PBSCT). Graft loss occurred in 2 of 4 patients. 2 pts who received DLI for recurrent disease converted to full donor chimerism (FDC) with no (n=1) or grade II (n=1) GVHD. Fludarabine was then added to the conditioning regimen. Ten patients with advanced HM (NHL, n=4; HD, n=3; MDS, n=1; CLL, n=1 CML, n=1) have received NSCT from HLA 5/6 (n=5) or 4/6 (n=5) matched haploidentical donors on a protocol using cyclophosphamide 60 mg/kg x 2, MEDI-507 0.1 mg/kg on day - 8 and 0.6 mg/kg on day 7 and - 6, fludarabine 25 mg/m2 on days - 5 through -1, thymic irradiation on day - 1, and ex vivo TCD (via CD34+ cell selection using the Isolex® device) PBSCT on day 0. Median #/kg (range) of infused CD34+ and CD3+ cells were 6.6X10E6 (3.2X10E6-1.5X10E7) and 3.5X10E4 (6.3X10E2-1.18X10E5), respectively. Of 9 evaluable patients, all initially achieved MLC without GVHD, but 2 patients subsequently lost detectable chimerism. 2 patients with MLC have received DLI, one in conjunction with chemotherapy, for disease progression. 5 pts (n=3 following rapid tapering of immunosuppression to induce a GVT effect, n=2 after DLI) have developed ≥ grade II acute GVHD (3 grade II, 2 grade III-IV). 5 patients have experienced disease progression, of whom one achieved a CR following DLI. Two pts have died due to transplant related complications (GVHD, n=1;late idiopathic pneumonitis, n=1) Split lineage MLC has occurred in each case, with a predominance of early donor granulocyte chimerism (mean 64%,77%,61%,>99% at 2,4,12,26 weeks) and a lower percentage of donor T-cell chimerism (mean 14%,37%,53%,>99% at 2,4,12,26 weeks). Conversion to FDC or near FDC occurred spontaneously in 4 pts and in 2 pts following DLI. Of 22 patients treated with MEDI-507 based haploidentical NSCT regimens, 8 are alive from < 1 to 70 months post-transplant. In conclusion, in vivo and ex vivo TCD haploidentical NSCT reliably leads to split lineage MLC with the potential for spontaneous chimerism conversion or conversion following DLI. Sustained anti-tumor responses in patients with advanced, chemorefractory HM have been achieved.

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Ghosh, Arnab, Martin Hapke, Florian Laenger, Ralf Baumann, Christian Koenecke, Karl Welte, and Martin Sauer. "Ex Vivo Generated Cytotoxic T Cells (CTL) for Adoptive Immunotherapy across MHC Barriers Retain Anti-Leukemic Capacity (GVL) without Eliciting Graft Versus Host Disease (GVHD): Crucial Role of Recipient-Derived Antigen Presenting Cells (APC)." Blood 108, no. 11 (November 16, 2006): 3168. http://dx.doi.org/10.1182/blood.v108.11.3168.3168.

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Abstract BACKGROUND: While recipient-derived APC have proven to be crucial in mediating GVL after allogeneic hematopoietic stem cell transplantation (HCT), this desired effect seemed to be inevitably linked to the risk of developing severe GVHD. It has been elegantly shown in murine models that delayed donor lymphocyte infusions (DLI) into mixed rather than full hematopoietic chimeras produce dramatically improved GVL effects and virtually no GVHD. However, avoidance of GVHD has been more difficult when this intelligent concept was transferred into the clinical setting. Here we demonstrate that donor-derived T cells of MHC-mismatched origin retain strong anti-leukemic activity and lose nonspecific alloreactivity upon specific in vitro priming and consecutive short term expansion. METHODS: We chose a fully MHC-mismatched allogeneic bone marrow transplantation model using B10.A mice (H-2a) as donors and C57BL/6 (H-2b) mice as recipients to produce either full (controls) or stable mixed hematopoietic chimeras. After full hematopoietic recovery mice were challenged with a lethal dose of a C57BL/6-derived leukemic cell line (C1498) and consecutively injected with either naive DLI, ex vivo generated CTL or PBS only. CTL were generated by specifically priming lymphocytes of MHC-mismatched donor origin on bone marrow-derived dendritic cells (DC) (either donor- or recipient-derived) that had been pulsed with recipient-derived leukemia cell lysates. This was followed by expansion using αCD3/αCD28 coated microspheres. RESULTS: We show that ex vivo generated CTL of allogeneic origin can mediate anti-leukemic effects only when primed on recipient-derived DC that had been pulsed with leukemia cell lysates (CTL versus controls, p < 0.05 ). In contrast, loading recipient-derived leukemia lysates on donor-derived DC (T cells and DC syngeneic to each other) abrogates the anti-leukemia effect of adoptively transferred CTL in vitro and in vivo. CTL exhibit a strong proliferative response in vitro when stimulated with irradiated leukemic cells of allogeneic origin (C1498), however, this response is significantly weaker (3-fold) when compared to naive DLI. Remarkably, the nonspecific proliferative response (alloreactivity) of CTL is completely abolished when irradiated C57BL/6 splenocytes are used as stimulators. CTL maintain their responsiveness to leukemia in vivo as demonstrated by cure rates from leukemia in up to 40% of mice. Importantly, upon adoptive transfer into leukemia bearing mice after MHC-mismatched HCT, CTL lose their unspecific alloreactivity in vivo as well as proven on microscopic sections of GVH target organs. Anti-leukemia effects of CTL across MHC-barriers are comparable to naive DLI in leukemia bearing mice (40% versus 50% cure rate). CTL are able to convert mixed hematopoietic chimerism into full hematopoietic chimerism (30% donor →100% donor). In contrast, mixed chimerism remains unchanged when CTL are transferred that had been primed ex vivo on donor-derived DC. This data is encouraging in that it provides further evidence that strong GVL effects can be obtained across MHC barriers without eliciting severe GVHD.

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14

Pai, Chien-Chun, Annie Mirsoian, Steven Grossenbacher, Erik Ames, Kai Sun, Mingyi Chen, Bruce R. Blazar, Mehrdad Abedi, and William J. Murphy. "Organ-Specific Protection By Bortezomib On The Treatment Of Cutaneous Chronic and Acute GvHD." Blood 122, no. 21 (November 15, 2013): 4473. http://dx.doi.org/10.1182/blood.v122.21.4473.4473.

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Allogeneic hematopoietic stem cell transfer (HSCT) can act as a powerful immunotherapy and is a quintessential treatment for many malignant hematologic diseases. However, development of graft versus host disease (GvHD) remains as a major complication after HSCT, and affects numerous organs. Furthermore, chronic GvHD (cGvHD) is emerging as a predominant cause of morbidity. Chronic GvHD has a distinctive pathology and pathogenesis and can develop in the form of scleroderma manifested with cutaneous sclerosis, loss of hair follicles, epidermal atrophy and replacement of peri-eccrine fat. We and others have previously demonstrated that bortezomib, a proteasome inhibitor, can prevent acute GvHD if given immediately after HSCT but that continuous treatment of mice resulted in accelerated GvHD-induced gut pathology due to CD4+ T cells. We therefore wanted to assess the effects of bortezomib on chronic GvHD or in particular acute GvHD models where CD8+ T cells were responsible for the disease. In this study, we use two major-MHC identical, minor-MHC mismatched strain combinations to mimic the clinical manifestations of cutaneous GvHD. In two model HSCT systems, treatment of ongoing GvHD resulted in organ specific protection effect on skin GvHD pathology. Treatment of acute GvHD as prevention with early bortezomib administration resulted in skin but not liver protection using the H-2b strain combination (C3SW donors into B6 recipients; minor MHC mismatch). Furthermore, using a chronic GvHD model, treatment of ongoing GvHD with bortezomib also resulted in skin, but not intestine pathology protection using the H-2d strain combination (B10D2 donors into Balb/c recipients; minor MHC mismatch). The contrasting organ specific effects of bortezomib are further contingent on the timing of administration. In marked contrast to the effects observed with early bortezomib administration in acute GvHD prevention, in cGvHD models, early administration after HSCT worsened the scleroderma pathogenesis, while the later administration of bortezomib during active cGvHD ameliorated the cutaneous lesions. The divergent dose dependent characteristic of bortezomib also suggests a narrow therapeutic window for the treatment of sclerodermatous chronic GvHD. Continuous administration of bortezomib led to down-regulation of serum IL-6 and which was also correlated with skin pathology. Total numbers of donor-derived spleen B cells were significantly reduced and treatment also correlated with lower BAFF gene expression levels in the peripheral skin tissues. We further observed lower donor CD8+ T cell infiltration in the skin of the bortezomib treatment group. Importantly, later bortezomib administration also preserved graft versus tumor (GvT) effects when challenged with a B-cell lymphoma tumor model. Thus bortezomib can produce skin specific protection effects in both acute and chronic GvHD responses. The organ specificity and time sensitivity of bortezomib treatment on sclerodermatous GvHD can provide valuable insights for future clinical trials. Disclosures: Abedi: Millennium: Research Funding.

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Krause, Daniela S., and Richard A. Van Etten. "Adoptive immunotherapy of BCR-ABL–induced chronic myeloid leukemia–like myeloproliferative disease in a murine model." Blood 104, no. 13 (December 15, 2004): 4236–44. http://dx.doi.org/10.1182/blood-2004-06-2229.

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Abstract Donor leukocyte infusion (DLI) can induce graft-versus-leukemia (GvL) reactions in patients with chronic myeloid leukemia (CML) relapsing after allogeneic bone marrow transplantation (BMT), but the mechanisms of the antileukemic effect of DLI are unknown, and the procedure is complicated by graft-versus-host disease (GvHD) and graft failure. Here, we adapted a murine retroviral BMT model of Philadelphia+ leukemia by combining allogeneic bone marrow (BM) from C57Bl/6 (H-2b) mice with BCR-ABL–transduced Balb/c (H-2d) BM, inducing mixed chimerism and myeloproliferative disease in recipients resembling relapse of CML following allogeneic BMT. Infusions of allogeneic splenocytes eliminated BCR-ABL–induced CML-like disease in the majority of mixed chimeras, with significant GvL effects mediated by both CD4+ and CD4- cells. BCR-ABL–induced acute B-lymphoblastic leukemia was also eradicated by DLI in major histocompatibility complex (MHC)–mismatched chimeras. Most DLI-treated mice converted to full allogeneic chimerism but succumbed frequently to GvHD or graft failure. When MHC-matched B10.D2 (H-2d) mice were the allogeneic donors, CML-like disease was more resistant to DLI. These results suggest that depletion of CD8+ cells from DLI could impair GvL against CML, while increased MHC disparity between donor and recipient may improve the responsiveness of Philadelphia+ B-lymphoblastic leukemia to DLI.

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Gao, Liquan, Tian-Hui Yang, Sophie Tourdot, Elena Sadovnikova, Robert Hasserjian, and Hans J. Stauss. "Allo-Major Histocompatibility Complex–Restricted Cytotoxic T Lymphocytes Engraft in Bone Marrow Transplant Recipients Without Causing Graft-Versus-Host Disease." Blood 94, no. 9 (November 1, 1999): 2999–3006. http://dx.doi.org/10.1182/blood.v94.9.2999.

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Abstract Previous experiments in humans and mice have shown that allogeneic donors can serve as a source of cytotoxic T lymphocytes (CTL) specific for proteins, such as cyclin-D1 and mdm-2, expressed at elevated levels in tumor cells. In vitro, allo-major histocompatibility complex (MHC)–restricted CTL against these proteins selectively killed allogeneic tumor cells, including lymphoma, but not normal control cells. This suggested that these CTL may be useful for adoptive tumor immunotherapy, provided that they (1) survive in MHC-disparate hosts, (2) maintain their killing specificity, and (3) do not attack normal host tissues. Here, we used cloned allo-restricted CTL isolated from BALB/c mice (H-2d) that killed H-2b–derived tumor cells expressing elevated levels of the mdm-2 target protein. When these CTL were injected into bone marrow transplanted (BMT) C57BL/6 (H-2b) recipients, they consistently engrafted and were detectable in lymphoid tissues and in the bone marrow (BM). Long-term survival was most efficient in spleen and lymph nodes, where CTL were found up to 14 weeks after injection. The administration of CTL did not cause graft-versus-host disease (GVHD) normally associated with injection of allogeneic T cells. These data show that allo-restricted CTL clones are promising reagents for antigen-specific immunotherapy in BMT hosts, because they engraft and retain their specific killing activity without causing GVHD.

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Gao, Liquan, Tian-Hui Yang, Sophie Tourdot, Elena Sadovnikova, Robert Hasserjian, and Hans J. Stauss. "Allo-Major Histocompatibility Complex–Restricted Cytotoxic T Lymphocytes Engraft in Bone Marrow Transplant Recipients Without Causing Graft-Versus-Host Disease." Blood 94, no. 9 (November 1, 1999): 2999–3006. http://dx.doi.org/10.1182/blood.v94.9.2999.421k12_2999_3006.

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Анотація:

Previous experiments in humans and mice have shown that allogeneic donors can serve as a source of cytotoxic T lymphocytes (CTL) specific for proteins, such as cyclin-D1 and mdm-2, expressed at elevated levels in tumor cells. In vitro, allo-major histocompatibility complex (MHC)–restricted CTL against these proteins selectively killed allogeneic tumor cells, including lymphoma, but not normal control cells. This suggested that these CTL may be useful for adoptive tumor immunotherapy, provided that they (1) survive in MHC-disparate hosts, (2) maintain their killing specificity, and (3) do not attack normal host tissues. Here, we used cloned allo-restricted CTL isolated from BALB/c mice (H-2d) that killed H-2b–derived tumor cells expressing elevated levels of the mdm-2 target protein. When these CTL were injected into bone marrow transplanted (BMT) C57BL/6 (H-2b) recipients, they consistently engrafted and were detectable in lymphoid tissues and in the bone marrow (BM). Long-term survival was most efficient in spleen and lymph nodes, where CTL were found up to 14 weeks after injection. The administration of CTL did not cause graft-versus-host disease (GVHD) normally associated with injection of allogeneic T cells. These data show that allo-restricted CTL clones are promising reagents for antigen-specific immunotherapy in BMT hosts, because they engraft and retain their specific killing activity without causing GVHD.

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Stevanovic, Sanja, Bart Nijmeijer, Marianke LJ Van Schie, Roelof Willemze, Marieke Griffioen, and J. H. Frederik Falkenburg. "Human T Cells with Distinct Specificities Mediate Graft-Versus-Leukemia Reactivity and Xenogeneic Graft-Versus-Host Disease in a NOD/Scid Mouse Model for Human Acute Leukemia." Blood 114, no. 22 (November 20, 2009): 1330. http://dx.doi.org/10.1182/blood.v114.22.1330.1330.

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Abstract Abstract 1330 Poster Board I-352 Immunodeficient mice inoculated with human leukemia can be used as a model to investigate Graft-versus-Leukemia (GvL) effects of donor lymphocyte infusions (DLIs). In addition to GvL reactivity, treatment with DLI induces xenogeneic Graft-versus-Host Disease (GvHD) in mice, characterized by pancytopenia and weight loss. In patients treated with DLI for relapsed or residual leukemia after allogeneic stem cell transplantation, immune responses against non-leukemic cells may also cause GvHD. It has been suggested that GvL reactivity and GvHD, which co-develop in vivo, can be separated and that distinct T cells exist with the specific capacity to mediate GvL reactivity or GvHD. Since adoptive T cell transfer models that allow analysis of separation of GvL and GvHD are rare, we aimed to establish whether GvL reactivity and xenogeneic GvHD could be separated using our model of human leukemia-engrafted NOD/scid mouse after treatment with human donor T cells. In this study, non-conditioned NOD/scid mice engrafted with primary human acute lymphoblastic leukemic cells were treated with CD3+ DLI. Established tumors were effectively eliminated by emerging human T cells, but also induced xenogeneic GvHD. Flowcytometric analysis demonstrated that the majority of emerging CD8+ and CD4+ T cells were activated (HLA-DR+) and expressed an effector memory phenotype (CD45RA-CD45RO+CCR7-). To investigate whether GvL reactivity and xenogeneic GvHD were mediated by the same T cells showing reactivity against both human leukemic and murine cells, or displaying distinct reactivity against human leukemic and murine cells, we clonally isolated and characterized the T cells during the GvL response and xenogeneic GvHD. T cell clones were analyzed for reactivity against primary human leukemic cells and primary NOD/scid hematopoietic (BM and spleen cells) and non-hematopoietic (skin fibroblasts) cells in IFN-g ELISA. Isolated CD8+ and CD4+ T cell clones were shown to recognize either human leukemic or murine cells, indicating that GvL response and xenogeneic GvHD were mediated by different human T cells. Flowcytometric analysis demonstrated that all BM and spleen cells expressed MHC class I, whereas only 1-3 % of the cells were MHC class II +. Primary skin fibroblasts displayed low MHC class I and completely lacked MHC class II expression. Xeno-reactive CD8+ T cell clones were shown to recognize all MHC class I + target cells and xeno-reactive CD4+ T cells clones displayed reactivity only against MHC class II + target cells. To determine the MHC restriction of xeno-reactive T cell clones, NOD/scid bone marrow (BM) derived dendritic cells (DC) expressing high levels of murine MHC class I and class II were tested for T cell recognition in the presence or absence of murine MHC class I and class II monoclonal antibodies in IFN-g ELISA. Xeno-reactive CD8+ T cell clones were shown to be MHC class I (H-2Kd or H-2Db) restricted, whereas xeno-reactive CD4+ T cell clones were MHC class II (I-Ag7) restricted, indicating that xeno-reactivity reflects genuine human T cell response directed against allo-antigens present on murine cells. Despite production of high levels of IFN-gamma, xeno-reactive CD8+ and CD4+ T cell clones failed to exert cytolytic activity against murine DC, as determined in a 51Cr-release cytotoxicity assay. Absence of cytolysis by CD8+ T cell clones, which are generally considered as potent effector cells, may be explained by low avidity interaction between human T cells and murine DC, since flowcytometric analysis revealed sub-optimal activation of T cells as measured by CD137 expression and T cell receptor downregulation upon co-culture with murine DC, and therefore these results indicate that xenogeneic GvHD in this model is likely to be mediated by cytokines. In conclusion, in leukemia-engrafted NOD/scid mice treated with CD3+ DLI, we show that GvL reactivity and xenogeneic GvHD are mediated by separate human T cells with distinct specificities. All xeno-reactive T cell clones showed genuine recognition of MHC class I or class II associated allo-antigens on murine cells similar as GvHD-inducing human T cells. These data suggest that our NOD/scid mouse model of human acute leukemia may be valuable for studying the effectiveness and specificity of selectively enriched or depleted T cells for adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.

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Har-Noy, Michael, Wirote Lausoontornsiri, Reuven Or, and Emmanual Katsanis. "Response of HER2+ breast cancer patients to allogeneic cell immunotherapy." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): e13013-e13013. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e13013.

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e13013 Background: The immune mechanism of non-myeloablative allogeneic stem cell transplantation has been shown to be effective in debulking chemotherapy-resistant metastatic cancer despite the presence of immunoavoidance mechanisms, but this immune effect is intimately related to GVHD toxicity limiting the clinical application. An allogeneic cell therapy (aCT) with intentionally mismatched, CD3/CD28 microbead-conjugated CD4+ memory cells exhibiting both Th1- and NK-like properties designed to elicit autologous anti-tumor effects in the context of graft rejection was evaluated in a FDA-approved phase I/II clinical trial of 42 refractory metastatic solid tumor patients with a variety of indications. No objective tumor response was observed, however there was evidence of enhanced survival and immune-mediated tumor debulking without GVHD toxicity. RECIST overestimated tumor burden, as responding tumors swelled and appeared larger on CT. Multi-parameter analysis identified serum IL-12 as a predictor of enhanced survival. 50% were IL-12+ and survived a median of 211 days vs. 131 days for IL-12- patients (p<.009). Methods: An analysis of IL-12+ responders was conducted to determine the indication with the highest IL-12 response. Results: 9 of 16 refractory metastatic breast cancer (mBC) patients were IL-12+. The Her2+ subset of mBC were 100% IL-12+ (5 of 5) with a median survival of 416 days, while the 11 Her2- patients had a median survival of only 134 days (p<0.05). Conclusions: Normally, Her2+ tumors are more aggressive and have a poorer prognosis than Her2- tumors. The enhanced response to aCT in Her2+ mBC may be due to high titers of anti-Her2 IgG1 antibodies known to be present in these patients. aCT upregulates host FcgR expression on host monocytes through a CD40L-CD40 mechanism. The FcgR bind IgG1 antibodies and mediate ADCC tumor lysis. We hypothesize that patients expressing tumor-specific IgG1 autoantibodies are more likely to respond to aCT. Serum autoantibodies to tumor-associated antigens thus may be a predictive biomarker for aCT responders. The combination of natural or exogenous tumor-specific IgG1 Abs with aCT may be a platform for refractory metastatic tumor debulking leading to increased overall survival.

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Ritchie, David S., and Victoria Watt. "Resting B Cells Suppress CD8+ T Cell Function and Prevent the Induction of Graft Versus Host Disease." Blood 106, no. 11 (November 16, 2005): 3111. http://dx.doi.org/10.1182/blood.v106.11.3111.3111.

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Abstract B cells have been variously shown to induce direct tolerance of antigen specific CD8+ T cells, induce T cell anergy via TGF-b production, down regulate IL-12 production by dendritic cells (DC) and influence Th1/Th2 differentiation via the production of regulatory cytokines. Through these mechanisms, B cells can exert a regulatory function in in vivo models of T cell immunity including, experimental autoimmune encephalitis (EAE) and rheumatoid arthritis (RA). Recently, B cells have been shown to be essential in the prevention of effector T cell differentiation in a model of autoimmunity. We have previously shown that resting B cells inhibited tumor protection induced by dendritic cells vaccination. Inhibition of DC immunity by B cells was independent of presentation of major histocompatibility molecule (MHC) class-I bound tumor antigen but dependent on the expression of class-II MHC. Furthermore the inhibitory effect of B cells was lost if the B cells were activated by CD40L or if CD4+/CD25+ regulatory T cells (Treg) were depleted. These studies have been further extended to examine the role of resting B cells on the induction and severity of graft versus host disease (GVHD) induced in a major MHC mismatch model. We have found that mice transplanted with B cell depleted marrow revealed more rapid CD8+ T cell engraftment, higher IL-2 and IFN-γ production, more severe GVHD and shorter survival. Conversely, those who received additional resting B cells at the time of marrow infusion were substantially protected from GVHD. These findings indicate that resting B cells may regulate T cell activation, in part via the suppressive effects of Treg, but also through their important role in T cell homeostasis. Resting B cells may therefore limit the efficacy of DC based immunotherapy or alternatively be used therapeutically to limit CD8+ T cell autoimmunity including GVHD.

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Herblot, Sabine, Valérie Paquin, Paulo Cordeiro, and Michel Duval. "Adoptive Transfers of Plasmacytoid Dendritic Cells after Hematopoietic Stem Cell Transplantation Decrease the Risk Acute Lymphoblastic Leukemia Relapse without Increasing the Risk of Graft-Versus-Host-Disease." Blood 132, Supplement 1 (November 29, 2018): 2053. http://dx.doi.org/10.1182/blood-2018-99-111130.

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Abstract Despite advances in chemotherapy and hematopoietic stem cell transplantation (HSCT), the outcome of children with relapsed acute lymphoblastic leukemia (ALL) has not significantly improved over the last 2 decades. About 50% of children with relapsed leukemia still die from their disease and ALL is still the first cause of death by cancer in children. A new hope of cure for patients with chemo-resistant cancers has emerged with the development of cancer immunotherapy. However, the major risk of post-transplant immunotherapy is the exacerbation of life-threatening Graft-versus-Host Disease (GvHD) mediated by donor-derived T cells. We therefore explored the avenue of innate immune stimulation. Several reports have demonstrated that activated Natural Killer (NK) cells can control acute myeloid leukemia (AML) in transplanted patients, whereas ALL is deemed to be resistant to NK cell killing. We recently challenged this paradigm and demonstrated that the stimulation of NK cells with third-party activated plasmacytoid dendritic cells (pDC) killed most ALL cell lines and patient-derived ALL blasts. We further demonstrated the efficacy of pDC adoptive transfers to cure ALL in a humanized mouse model of HSCT. Collectively, these results uncovered for the first time the unique therapeutic potential of activated pDC as immunotherapeutic tools to stimulate NK cell anti-leukemic activity early after HSCT. The next step toward the clinical translation of pDC-based post-transplant immunotherapy is to verify that adoptive transfers of pDC do not stimulate T cells nor exacerbate GvHD in the presence of mature T cells. We designed a GMP-compliant method for in vitro expansion and differentiation of cord blood progenitors giving rise to sufficient numbers of pDC for adoptive transfers in patients. We showed that after Toll-like receptor (TLR) stimulation, these in vitro differentiated pDC displayed a phenotype of interferon producing cells (CD80neg PDL-1+) but not of antigen presenting cells (CD80+PDL-1neg). Accordingly, in vitro mixed lymphocyte reactions with purified allogeneic T cells demonstrated that TLR-activated pDC induced very low allogeneic T cell proliferation as compared with bona fide antigen presenting cells such as myeloid dendritic cells (mDC - CD11c+) or monocyte-derived dendritic cells (mo-DC) (Figure 1A). To test whether activated pDC could exacerbate GvHD in the presence of mature T cells, we used a xenoGvHD model in which human peripheral blood mononuclear cells (PBMC) were injected in immune-deficient mice (Nod/Scid/gRc-/-, NSG). We monitored GvHD 3-times a week according to a GvHD-assessment scale as previously described. Overt GvHD was characterized by cutaneous and intestinal lesions, weight loss and high numbers of human CD3+ cells in peripheral blood. Mice were sacrificed when endpoints were reached and GvHD was confirmed by immunohistochemistry and flow cytometry. Five weekly injections of TLR-activated in vitro differentiated pDC did not accelerate the GvHD onset and the severity of the lesions were not increased. We did not either observe any difference in survival between control and pDC-treated groups (Figure 1B). Collectively, our results indicate that TLR-activated pDC do not stimulate allogeneic T cells and do not increase the risk of acute GvHD in a mouse model of xenoGvHD. We therefore expect this novel pDC-based immunotherapy to be safe for transplanted patients. These data open the way for the next step: a Phase I clinical trial of in vitro differentiated pDC after transplantation for leukemia. Disclosures No relevant conflicts of interest to declare.

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Marcus, Assaf, Tova Waks, and Zelig Eshhar. "Redirected tumor-specific allogeneic T cells for universal treatment of cancer." Blood 118, no. 4 (July 28, 2011): 975–83. http://dx.doi.org/10.1182/blood-2011-02-334284.

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Abstract Adoptive cell transfer of allogeneic tumor-specific T cells could potentially be used as a universal treatment for cancer. We present a novel approach for adoptive immunotherapy using fully MHC-mismatched allogeneic T cells redirected with tumor-specific, non-MHC–restricted antibody-based chimeric antigen receptor (T-bodies) in the absence of GVHD. Mice bearing systemic metastatic disease were lymphodepleted by irradiation and treated with Her2/neu re-directed T cells. Lymphodepletion created a ‘therapeutic window’, which allowed the allo–T-bodies to attack the tumor before their rejection. A single split dose administration of allogeneic T-bodies extended the survival of tumor-bearing mice similarly to syngeneic T-bodies, and to a significantly greater extent than nonspecific allogeneic T cells. Blocking egress of lymphocytes from lymphoid organs using the sphingosine-1-phosphate agonist, FTY720, extended the persistence of allogeneic T cells such that allogeneic T-bodies provided superior therapeutic benefit relative to syngeneic ones, and dramatically extended the median survival time of the treated mice for more than a year. Therefore, we suggest that ex-vivo generated MHC-mismatched T-bodies can be used universally for off-the-shelf cancer immunotherapy and that their graft-versus-host reactivity can be safely harnessed to potentiate adoptive cell therapy.

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Dey, Bimalangshu R., Andrew J. Yee, Steven McAfee, Christine Dube, Karen Ballen, Eyal Attar, Christine Colby, et al. "Non-Myeloablative T-Cell Depleted (TCD) Haploidentical Hematopoietic Cell Transplantation (HCT) Followed by Donor Leukocyte Infusion(s) for Hematologic Malignancies: The MGH Experience." Blood 110, no. 11 (November 16, 2007): 5088. http://dx.doi.org/10.1182/blood.v110.11.5088.5088.

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Abstract We developed a haploidentical HCT protocol based on MHC-mismatched murine models, in which mixed chimerism (MC) is induced as a platform for adoptive immunotherapy via delayed donor leukocyte infusions (DLI). While the scientific concept has remained the same, this haplo-HCT protocol has undergone multiple revisions since its inception, such as substitution of MEDI-507 (a CD2 monoclonal antibody) for ATG, addition of ex vivo TCD of donor graft (via CD34+ cell selection), changes in MEDI-507 doses/schedules and finally addition of fludarabine-all based on evolving clinical experience and new discoveries from murine models. A total of 14 patients with advanced, mostly therapy-refractory disease (NHL, n=7; HD, n=3; t-AML, n=2; ALL, n=1; MDS, n=1), received our most recent nonmyeloablative strategy consisting of cyclophosphamide, fludarabine, MEDI-507, thymic irradiation on day -1 and cyclosporine for GVHD prophylaxis (with taper and discontinuation by day 35 for patients with MC and no evidence of GVHD) followed by GCSF-mobilized ex vivo TCD haplo-HCT (HLA 1/6 mismatched, n=7; HLA 2/6 mismatched, n=7). Median number/kg (range) of infused CD34+ and CD3+ cells were 6.84 x106 (3.19 x 106 – 14.7 x 106) and 4.73 x 104 (0.06 x 104 – 11.8 x 104), respectively. One patient died shortly after the transplantation due to intracranial hemorrhage. Of 13 evaluable patients, all initially achieved MC without GVHD, but 3 lost detectable donor graft despite multiple DLIs. Split lineage MC occurred in 10 patients, with a predominance of early donor granulocyte chimerism (mean 91%, 87%, 96%) and a lower percentage of early donor T-cell chimerism (57%, 60%, 97%) at 30, 90 and 150 days post-HCT. Ten out of 10 patients ultimately achieved full donor chimerism (FDC) in myeloid lineage and 9 achieved FDC in T-cell lineage (spontaneously, n=8; following DLI, n=2). Natural killer cells recovered relatively early, despite the presence of circulating MEDI-507. Eight patients developed acute GVHD: grade I, n=3; grade II, n=4; grade IV, n=1. One died from GVHD-related complications, 1 died of late idiopathic pneumonitis and 5 died from progressive disease. Median overall survival (OS) of all 14 patients is 195 days (range, 31–1028) post-HCT; at 1 year the OS is 39.2% for all 14 patients. Six patients are alive at a median of 604 days (range, 180–1028) post-HCT, including 3 in complete remission of whom 2 had therapy-related MDS/AML. HLA mismatches in the GVH or HVG direction did not influence graft outcome. We also assessed inhibitory killer immunoglobulin-like receptor (KIR)-HLA epitope mismatches (missing ligand) in the GVH and HVG (host versus graft) directions based on HLA and KIR genotyping of patients and their donors. There was a trend in the association between spontaneous FDC and fewer missing donor HLA ligands for host KIR receptor (<2 v. ≥ 2 missing donor ligands, p = 0.075). In conclusion, using our current haploidentical HCT protocol, we reliably achieved initial MC that converted to FDC, either spontaneously or following DLI with manageable GVHD and with the achievement of sustained anti-tumor responses in some patients with therapy-refractory hematologic malignancies.

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Delisle, Jean-Sebastien, Marie-Christine Meunier, Chantal Barron, Julie Bergeron, and Claude Perreault. "Differential Immune Susceptibility of Malignant Versus Normal Tissue Leads to Tumor Rejection without GVH after Adoptive Immunotherapy." Blood 104, no. 11 (November 16, 2004): 3047. http://dx.doi.org/10.1182/blood.v104.11.3047.3047.

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Abstract Adoptive immunotherapy based on the injection of allogenic cytotoxic T-lymphocytes (CTL) during or after bone marrow transplant (BMT) has established itself as a potent anti-neoplastic treatment for several malignancies. However this approach is limited by the occurence of graft versus host disease (GVH). Using a previously described murine adoptive immunotherapy model where donor and recipient are mismatched for a single dominant minor histocompatibility antigen (H7a), and where a powerful anti-neoplastic effect is seen without GVH, we sought to determine what rendered cancer cells more vulnerable than normal cells to immune attack. B10.H7a mice were lethally irradiated and reconstituted with B10.H7b (H7a negative) T-depleted bone marrow. On the day of transplant, these mice received a B16.F10 (H7a positive) melanoma challenge. Adoptive transfer of splenocytes obtained from B10.H7b mice previously immunized with B10.H7a splenocytes was performed on day 7 post BMT. Following transfer of those splenocytes containing primed anti-H7a CTL, neither GVH nor vitiligo was noted in recipients despite the fact that H7a is expressed in all tissues and organs. 50% of treated mice, versus 0% of controls rejected the tumor and survived 100 days. Overall survival was increased to 80% when adoptive transfer was carried on day 3 post BMT. The injection of anti-H7b CTL had no effect on melanoma growth. Thus, the anti-tumor activity was T-cell receptor recognition dependent and not a mere bystander effect. In treated mice but not in controls, tumor histology and flow cytometry revealed important CTL infiltration (80% of those CTL being MHC-H7a tetramer positive), increased expression of MHC class I molecules (MHC I) at melanoma cell surface, expression of Rae-1 (an NKG2D ligand), tumor necrosis and decreased angiogenesis. Importantly, normal skin in treated or control animals showed no increased expression of MHC I or Rae-1. B16.F10 melanoma cells express almost no MHC I, and no Rae-1 when cultured in vitro. Co-incubation of B16.F10 cells with INFγ leads to increased MHC I expression but no induction of Rae-1 expression, implying that at least a second factor is present in vivo to account for the expression of this stress ligand. An additional role for INFγ was evidenced when anti-H7a CTL were injected in INFγ receptor knock-out recipients. The angiostatic effect noted after anti-H7a CTL injection was abrogated and no mice were cured. Thus, INFγ-mediated angiostasis on the tumor stroma was crucial for the inhibition of cancer progression. Conclusion: in our model, the differential immune susceptibility of tumor versus normal cells appears to stem from the fact that neoplastic cells are induced to express more MHC I and stress ligands such as Rae-1. Those can respectively increase target antigen density at the cell surface and mediate CTL co-stimulation or cytotoxicity, through NKG2D receptors. Strategies exploiting stress ligand induction as well as the effect of INFγ on MHC expression and angiostasis may contribute to successfully separate anti-tumor from GVH effects.

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25

Uberti, J., F. Martilotti, TH Chou, and J. Kaplan. "Human lymphokine activated killer (LAK) cells suppress generation of allospecific cytotoxic T cells: implications for use of LAK cells to prevent graft-versus-host disease in allogeneic bone marrow transplantation." Blood 79, no. 1 (January 1, 1992): 261–68. http://dx.doi.org/10.1182/blood.v79.1.261.261.

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Abstract We have found that murine lymphokine activated killer (LAK) cells have veto and natural suppressor activities in vitro, and prevent graft- versus-host disease (GVHD) in vivo. To determine whether human LAK cells mediate veto and natural suppression we measured their ability to inhibit generation of allospecific cytotoxic T cells (CTL) in mixed lymphocyte culture (MLC). When added to MLCs at low concentrations LAK cells caused veto-type inhibition: stimulator-type LAK cells inhibited generation of CTL but responder or third-party LAK cells did not. At higher concentrations LAK cells caused nonspecific inhibition: all three LAK cell types inhibited generation of CTL. LAK cell veto and natural suppressor activities were largely eliminated by irradiation with 30 Gy and by depletion of CD56+ cells, but increased after depletion of CD3+ cells. LAK cell veto activity is not likely an artifact of cold-target inhibition by the LAK cells themselves or by proliferation of T cells contaminating LAK cell preparations: (1) veto only occurred when LAK cells were added to MLC on days 0 through 2, but not when added on day 5; (2) addition of saturating numbers of labeled targets to fixed numbers of allo-CTL effectors failed to overcome the inhibitory effects of adding stimulator-type LAK cells at the onset of MLC; and (3) CD3-depleted LAK cells showed greater veto activity than threefold greater numbers of control LAK cells. In light of our previous findings in mice, the current results imply that adoptive immunotherapy with LAK cells may be useful in preventing GVHD in human bone marrow transplant recipients.

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26

Uberti, J., F. Martilotti, TH Chou, and J. Kaplan. "Human lymphokine activated killer (LAK) cells suppress generation of allospecific cytotoxic T cells: implications for use of LAK cells to prevent graft-versus-host disease in allogeneic bone marrow transplantation." Blood 79, no. 1 (January 1, 1992): 261–68. http://dx.doi.org/10.1182/blood.v79.1.261.bloodjournal791261.

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We have found that murine lymphokine activated killer (LAK) cells have veto and natural suppressor activities in vitro, and prevent graft- versus-host disease (GVHD) in vivo. To determine whether human LAK cells mediate veto and natural suppression we measured their ability to inhibit generation of allospecific cytotoxic T cells (CTL) in mixed lymphocyte culture (MLC). When added to MLCs at low concentrations LAK cells caused veto-type inhibition: stimulator-type LAK cells inhibited generation of CTL but responder or third-party LAK cells did not. At higher concentrations LAK cells caused nonspecific inhibition: all three LAK cell types inhibited generation of CTL. LAK cell veto and natural suppressor activities were largely eliminated by irradiation with 30 Gy and by depletion of CD56+ cells, but increased after depletion of CD3+ cells. LAK cell veto activity is not likely an artifact of cold-target inhibition by the LAK cells themselves or by proliferation of T cells contaminating LAK cell preparations: (1) veto only occurred when LAK cells were added to MLC on days 0 through 2, but not when added on day 5; (2) addition of saturating numbers of labeled targets to fixed numbers of allo-CTL effectors failed to overcome the inhibitory effects of adding stimulator-type LAK cells at the onset of MLC; and (3) CD3-depleted LAK cells showed greater veto activity than threefold greater numbers of control LAK cells. In light of our previous findings in mice, the current results imply that adoptive immunotherapy with LAK cells may be useful in preventing GVHD in human bone marrow transplant recipients.

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Laport, Ginna G., Kevin Sheehan, Robert Lowsky, Judith A. Shizuru, Keith Stockerl-Goldstein, Laura J. Johnston, David Miklos, Sally Arai, Jeanette Baker, and Robert S. Negrin. "Cytokine Induced Killer (CIK) Cells as Post-Transplant Immunotherapy Following Allogeneic Hematopoietic Cell Transplantation." Blood 108, no. 11 (November 16, 2006): 412. http://dx.doi.org/10.1182/blood.v108.11.412.412.

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Abstract Donor leukocyte infusions induce remissions in some patients(pts) with hematologic malignancies who relapse after allogeneic hematopoietic cell transplantation(AHCT). Response rates are disease-specific and acute graft vs host disease(GVHD) remains the major complication of this strategy. CIK cells are a unique population of cytotoxic T lymphocytes that express the CD3+CD56+ phenotype and show marked upregulation of the NK cell receptor, NKG2D. CIK cells are non-MHC restricted, NKG2D dependent in target recognition and killing and demonstrate cytotoxicity against a broad array of tumor cell lines including leukemia targets. In preclinical studies, CIK cells have exhibited superior antitumor activity compared to IL-2 activated NK cells with a markedly reduced capacity for acute GVHD. The primary objective of this trial was to determine the feasibility of ex vivo expansion of allogeneic CIK cells suitable for clinical application for pts with relapsed hematologic malignancies after AHCT(myeloablative or non-myeloablative) and to determine the maximum tolerated dose of CIK cells based on CD3+ cell dose. CIK cells were generated from CD3+ precursors by culturing unmobilized peripheral blood mononuclear cells (PBMC) from the patient’s matched sibling donor. The PBMCs were cultured for 21–28 days in the Aastrom Replicell® biochamber in the presence of anti-CD3+ monoclonal antibody, IL-2 and IFN-γ. Ten patients with a median age of 50 yo(range 29–63 yo) received CIK cell infusions based on CD3+cells/kg at a dose of 1x107 (n=3), 5x107 (n=6) and 1x108 (n=1). The diagnoses of these pts included acute myeloid leukemia(n=4), non-Hodgkins lymphoma(n=2), multiple myeloma(n=3) and Hodgkins disease(n=1). Prior to CIK infusion, all pts underwent cytoreduction for tumor debulking. One pt experienced infusional toxicities consisting of ventricular arrhythmias and transient hypotension. After infusion, grade 3–4 toxicities were seen at the 2nd dose level and included symptomatic ventricular arrhythmias in two patients with 1 of these pts also experiencing transient elevation of transaminases. These dose limiting toxicities(DLTs) resulted in expansion of the cohort at this dose level. No further DLTs were seen in subsequent pts allowing escalation to the 3rd and current dose level. Grade 1 skin acute GVHD was seen in 1 pt and 2 pts had limited chronic GVHD. After a median follow-up time of 432 days (range 2–649) from CIK infusion, the 1 year event free survival and overall survival was 20% and 76%, respectively. The median time to progression was 90 days(range 9–577). Analysis of cell cultures showed that most recovered cells were CD3+ (median 98%, range 90–99%) with a median viability of 87%( range 73–95%) with the median expansion of CD3+ cells being 16 fold(range 9–91 fold). CD3+CD56+ cells represented a median of 12% (range 8–32%) of the harvested cultures with a median 96 fold (range 26–515 fold) expansion. CD8+NKG2D+ expression ranged from 17–61%(median 38%) of harvested cells. Upregulation of the activation markers, CD25+ and CD69+ was also seen. Significant tumor cell killing was demonstrated in vitro by cytotoxicity assays against a panel of tumor cell lines. This data demonstrates successful ex vivo expansion of allogeneic CIK cells in the clinical setting. This form of adoptive immunotherapy is well tolerated by pts, induces a low incidence of GVHD and shows evidence for clinical efficacy.

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Kunzmann, Volker, Manfred Smetak, Florian Weissinger, Josef Birkmann, Hermann Einsele, and Martin Wilhelm. "Clinical-Scale Combined CD4/CD8 Depletion for Donor Innate Lymphocyte Infusion (DILI)." Blood 108, no. 11 (November 16, 2006): 3657. http://dx.doi.org/10.1182/blood.v108.11.3657.3657.

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Abstract The ability to selectively deplete or enrich cells of specific phenotype by immunomagnetic selection procedures holds significant promise for application in adoptive immunotherapy protocols. In vitro and in vivo studies demonstrated that MHC-independent effector cells of the innate immune system such as natural killer (NK) cells and γδ T cells have potent anti-tumor activity without inducing Graft-versus-Host disease (GvHD). However, current clinical-scale approaches to reduce the risk of GvHD usually use T-cell depletion (such as CD34+ selection or CD3+ depletion). However, both procedures also deplete γδ T cells which may be advantageous in mediating Graft-versus tumor effects and augmenting innate immune response against infections. Here we present a new method for depletion of T cells with potential GvHD reactivity and simultaneously enrichment of innate lymphocytes (NK cells and γδ T cells) from standard leukapheresis products by using a single-step immunomagnetic protocol which efficiently depletes CD4+ and CD8+ αβ T cells under good manufacturing conditions (GMP). Efficiency of CD4+ and CD8+ T cell depletion from (unstimulated) leukapheresis products (n=6) containing up to 2.0 × 1010 white blood cells, was demonstrated by 4-color flow cytometric analysis (mean log depletion of CD4+ cells: 4.12 (3.17–4.9); mean log depletion of CD8+ cells: 3.77 (2.97–4.54)). In addition to efficient depletion of CD4+ and CD8+ cells, immunomagnetic CD4/CD8 depletion resulted in enrichment of NK cells and γδ T cells (mean NK cell recovery: 38%(19–72), mean γδ T cell cell recovery: 50%(34–79)). In vitro assays of the final product demonstrated that NK cells and γδ T cells preserved their proliferative and cytotoxic capacity. We conclude that simultaneous depletion of CD4+ and CD8+ cells is feasible and can be performed in large scale under GMP conditions with sufficient depletion efficacy of αβ T cells and recovery of functionally intact innate effector lymphocytes (NK cells and γδ T cells) for potential use in adoptive immunotherapy studies.

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Lu, Yi-Fen, L. Cristina Gavrilescu, Monica Betancur, Katherine Lazarides, Hans Klingemann, and Richard A. Van Etten. "Distinct graft-versus-leukemic stem cell effects of early or delayed donor leukocyte infusions in a mouse chronic myeloid leukemia model." Blood 119, no. 1 (January 5, 2012): 273–84. http://dx.doi.org/10.1182/blood-2011-01-331009.

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Abstract Among hematologic neoplasms, chronic myeloid leukemia (CML) is exquisitely sensitive to graft-versus-leukemia (GVL) because patients relapsing after allogeneic hematopoietic stem-cell transplantation (alloHSCT) can be cured by donor leukocyte infusion (DLI); however, the cellular mechanisms and strategies to separate GVL from GVHD are unclear. We used a BCR-ABL1 transduction/transplantation mouse model to study the mechanisms of DLI in MHC-matched, minor histocompatibility antigen–mismatched allogeneic chimeras with CML-like leukemia, in which DLI can be administered at the time of transplantation (early) or after recovery of hematopoiesis (delayed). After early DLI, CML-like leukemia cannot be transferred into immunocompetent secondary recipients as soon as 4 days after primary transplantation, demonstrating that cotransplantation of T lymphocytes blocks the engraftment of BCR-ABL1–transduced stem cells. In contrast, in allogeneic chimeras with established CML-like leukemia, combined treatment with delayed DLI and the kinase inhibitor imatinib eradicates leukemia with minimal GVHD. The GVL effect is directed against minor histocompatibility antigens shared by normal and leukemic stem cells, and is mediated predominantly by CD8+ T cells, with minor contributions from CD5− splenocytes, including natural killer cells. These results define a physiologic model of adoptive immunotherapy of CML that will be useful for investigating the cellular and molecular mechanisms of GVL.

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Ruggeri, Loredana, Mauro Di Ianni, Elena Urbani, Antonella Mancusi, Franca Falzetti, Alessandra Carotti, Adelmo Terenzi, et al. "Tregs Suppress GvHD at the Periphery and Unleash the Gvl Effect in the Bone Marrow." Blood 124, no. 21 (December 6, 2014): 842. http://dx.doi.org/10.1182/blood.v124.21.842.842.

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Abstract Posttransplant relapse is still a major cause of treatment failure in high-risk acute leukemia. Evidence from experimental bone marrow transplantation (BMT) showed coinfusion of conventional T lymphocytes (Tcons) with regulatory T lymphocytes (Tregs) suppressed lethal Graft-versus-Host Disease (GvHD) without impairing the Graft-versus-Leukemia (GvL) effect (Edinger et al., Nat Med. 2003;9:1144-1150). In HLA haploidentical transplantation for acute leukemia we demonstrated that donor-derived natural Tregs, coinfused with Tcons, protected recipients against GvHD (Di Ianni et al., Blood 2011, 117:3921-3928) and largely prevented posttransplant leukemia relapse, as only 5% of evaluable patients relapsed (Martelli et al., Blood 2014, 124:638-44). The mechanism by which the GvL effect is maintained in the absence of GvHD is still unknown. In humans, naïve CD45RA+ Tregs express CXCR4 and preferentially localize to the bone marrow while memory CD45RO+ Tregs display lower CXCR4 expression and home to the periphery (Booth et al., J Immunol. 2010;184:4317-4326). Since peripheral blood Tregs to be used for adoptive immunotherapy are CD45RO+, we hypothesized that GvL without GvHD might be due to unopposed Tcon alloreactivity in the bone marrow combined with regulated T cell alloreactivity at the periphery. Immunodeficient NSG mice (a total of 40 for each experimental group) received human myeloid or lymphoblastic leukemia (3x106) and HLA mismatched Tregs/Tcons (3x106). Mice that received leukemia and Tcons (without Tregs) cleared leukemia but died of GvHD. Human T cells harvested from their BM, spleen and liver were predominantly (90%) CD8+ and displayed potent alloreactivity (by 51-chromium release at an E:T ratio of 5:1) against human leukemia (lysis = 50% ± 15), autologous to leukemia PHA blasts (lysis = 60% ± 10) and mouse Con A blasts (lysis = 40% ± 8). Mice that received human leukemia and Tcons plus Tregs were rescued from leukemia and survived without GvHD. Human T cells harvested from spleen and liver were composed of CD8+ T cells (40%) and CD4+/FOXP3- T cells (60%). Despite their FOXP3 negativity, purified CD4+ T cells had retained their regulatory function as they inhibited mixed lymphocyte reaction (by 50% at a Tcon/Treg ratio of 1:2). Purified CD8+ T cells displayed no alloreactivity against human leukemia, PHA blasts and mouse Con A blasts. In contrast, human T cells harvested from BM were still predominantly CD8+ and still displayed potent alloreactivity against human leukemia and autologous to leukemia PHA blasts and mouse ConA blasts, suggesting Tcons had retained their alloantigen recognition against human and mouse MHC. Finally, in mice treated with Tregs alone, human T cells were recovered only in the spleen and liver, they displayed a CD4+/FOXP3- phenotype and inhibited mixed lymphocyte reaction. No human T cells were found in the bone morrow, thus showing human CD45RO+ Tregs do not home to the bone marrow. In conclusion, Treg/Tcon adoptive immunotherapy mediates GvL effect in the absence of GvHD because Tcons that home to the bone marrow exert unopposed alloantigen recognition, while Tcons that home to the periphery are blocked by Tregs. Disclosures No relevant conflicts of interest to declare.

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Oostvogels, Rimke, Rieuwert Hoppes, Henk Lokhorst, Robbert M. Spaapen, Huib Ovaa, and Tuna Mutis. "Improved Vaccine Design For Adoptive Immunotherapy In Hematological Malignancies Through Chemically Modified Minor Histocompatibility Antigen Epitopes." Blood 122, no. 21 (November 15, 2013): 5435. http://dx.doi.org/10.1182/blood.v122.21.5435.5435.

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Abstract Allogeneic stem cell transplantation (allo-SCT), alone or followed by donor lymphocyte infusion (DLI), is a potentially curative treatment for various hematological malignancies. In an HLA-matched transplantation setting, the therapeutic graft-versus-tumor (GvT) effect is mediated by donor T-cells directed at minor histocompatibility antigens (mHags), which are HLA-bound polymorphic peptides. Unfortunately, most patients don’t achieve complete response or relapse after allogeneic stem cell transplantation and thus still require additional therapies. Immunotherapy aimed at hematopoietically restricted mHags could theoretically provide an ideal method to augment the GvT effect, without causing GvHD. The most relevant mHags for immunotherapy are those antigens that are only expressed on hematopoietic tissue, are presented by frequent HLA molecules and display an equally balanced population frequency. UTA2-1 and HA-1 are two of these most broadly applicable mHags identified up until now and are therefore included in on-going clinical trials of mHag-peptide loaded dendritic cell vaccination in patients with various hematological malignancies. Another method for mHag-based immunotherapy could be adoptive transfer of ex vivo cultured mHag-specific cytotoxic T lymphocytes (CTL). However, initial results of both methods, also from preclinical models and trials in patients with solid tumors, postulate the necessity for improved strategies for efficient ex vivo and in vivo induction of tumour specific CTLs. We here show for the HLA-A*02 restricted epitopes UTA2-1 and HA-1 that their MHC binding and consequent T cell reactivity can be improved through the incorporation of certain newly designed non-proteogenic amino acids at crucial MHC anchoring positions. With this novel approach we designed superior altered peptide ligands (APLs) for both epitopes, of which the best modifications not only increased MHC binding and stability, but also improved recognition by antigen specific T cells. Most importantly, these optimised peptides gave rise to superior antitumor T cell responses in vitro and in vivo in comparison to the native epitope, as they induced significantly enhanced proliferation of peptide-specific T cells with retained cytotoxic potential against malignant targets expressing the natural UTA2-1 antigen. Hence, these APLs designed with non-proteogenic amino acids with enhanced MHC-affinity and immunogenicity may improve the therapeutic outcome of mHag-based vaccination strategies, or can be utilized for ex vivo antigen-specific T cell enrichment and expansion for transfer into patients with haematological malignancies. Disclosures: Lokhorst: Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

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Meinhardt, Kathrin, Ruth Bauer, Irena Kroeger, Julia Schneider, Franziska Ganss, Diana Dudziak, Michael Rehli, Andreas Mackensen, and Evelyn Ullrich. "Tissue-Specific Homing and Different Impact in Gvhd Prevention of NK Cell Subpopulations." Blood 120, no. 21 (November 16, 2012): 3004. http://dx.doi.org/10.1182/blood.v120.21.3004.3004.

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Abstract Abstract 3004 Clinical studies exploiting the impact of natural killer (NK) cells in allogeneic hematopoietic stem cell transplantation (HSCT) have provided promising results. It is known that NK cells are a heterogeneous population and can be divided into functionally distinct NK cell subpopulations. Murine NK cells can be separated along their expression of CD27 and CD11b and CD117 (c-kit). However, the functional relevance of distinct NK cell subsets in graft-versus-host-disease (GVHD) has not been investigated in detail so far. We have established different protocols for ex vivo isolation and expansion of murine NK cell subpopulations. These NK subsets were further analyzed in vitro and in vivo in an allogeneic murine GVHD model. Here we report on different genomic, phenotypic and functional properties of 4 NK cell subsets. Our data clearly demonstrate that CD27+ NK cells revealed the highest IFN-g production upon coculture with tumor cells and/or IL-2. Interestingly, the CD11b+ NK cells express multiple genes of cytotoxic pathways and develop the highest cytotoxic capacity towards tumor cells. We observed up to 60% tumor lysis by CD27- CD11b+ NK cells compared to 40–45% by CD27+ CD11b+, about 25% by CD27+ CD11b- and 10% by c-kit+ CD11b- NK cells at an effector-target ratio of 5:1, respectively. Furthermore, the CD11b+ NK cell subset significantly reduced T cell proliferation induced by allogeneic dendritic cells in mixed lymphocytes reactions. Next, we analyzed the migratory capacity and tissue-specific homing of FACS-sorted NK cell subsets by adoptive transfer of congeneic CD45.1+ and Luc+ NK cell subpopulations in autologous and allogeneic bone marrow transplantation. Of interest, FACS analysis and in vivo imaging showed that CD11b+ NK cells migrated to peripheral GVHD target organs, whereas CD27+ NK cells preferentially homed to the bone marrow. Finally, this study addressed for the first time the role of distinct NK cell subpopulations in the development of GVHD in a fully MHC mismatched HSCT mouse model. Importantly, we identified the CD11b+ NK cell population as the NK cell subset that significantly diminished GVHD. In vivo imaging of Luc+CD11b+ NK cells revealed that this subset migrates to the colonic tissue to prevent development of GVHD colitis as shown by colonoscopy. In summary, our comparative study outlines that only CD11b+ NK cells, migrating to the peripheral GVHD target organs and providing the most efficient cytolytic capacity directed against allogeneic dendritic cells, protect against GVHD. These new insights are highly relevant for the selection of optimal NK cell subsets in the field of cellular immunotherapy. Disclosures: No relevant conflicts of interest to declare.

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Di Ianni, Mauro, Raffaella Giancola, Stefano Baldoni, Francesca Ulbar, Beatrice Del Papa, Stella Santarone, Annalisa Natale, et al. "Adoptive Immunotherapy with Regulatory and Conventional T Cells in Haploidentical Transplantation Primes Dendritic Cells to Promote T Cell Alloreactivity in the Bone Marrow and Tolerance in the Periphery." Blood 134, Supplement_1 (November 13, 2019): 3224. http://dx.doi.org/10.1182/blood-2019-126061.

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In high-risk acute leukemia patients undergoing HLA haploidentical T cell-depleted tranplantation, we demonstrated that adoptive immunotherapy with donor T regulatory cells (Tregs; 2x106/kg) co-infused with conventional T cells (Tcon; 1x106/kg ) provided significant protection from acute graft-versus-host disease (aGvHD) and was associated with an almost complete control of leukemia relapse (graft versus leukemia effect, GvL) (Di Ianni et al., Blood 2011; Martelli et al., Blood 2014; Ruggeri et al., ASH 2018). In the present study we investigated whether Tregs interact with bone marrow (BM) and peripheral blood (PB) dendritic cells (DCs) and whether such interaction is responsible for GvHD protection and GvL effect. Twenty six patients (median age 54 ; 20 AML; 4 ALL; 2 MDS) transplanted between July 2016 and April 2019 were evaluated up to one year after the transplant. BM and PB DCs (using CD123 for plasmocitoid DC-pDC; CD11c for myeloid DC-mDC; CD80/CD86 for costimulatory molecules) and T cells (CD3/CD4/CD8; CD4/CD25/CD127; CD28/PD-1/TIM3) were analysed by flow-cytometry. DCs were also sorted and analysed by RT-PCR for a panel of genes involved in activation (IL-6; TNF-a; IL-12; CCR7; NOTCH ligands) vs tolerigenic (TGF-beta; PD-1/PDL1; IDO; IL-10; ICOS) pathways. To study the effects of DCs on T cell proliferation, pre-activated (with GM-CSF at 50 ng/ml, IL-4 at 800 U/ml and TNF-a at 50 ng/ml for 18 hrs) BM and PB CD1c+ DCs were co-cultured for 96 hrs with autologous CFSE labelled BM and PB CD3+ cells at a DC:CD3 ratio of 1:10. mDC numbers were significantly higher in BM than PB during the first 6 months after transplant. BM-derived mDCs expressed higher levels of the co-stimulatory receptor CD86. No differences emerged in pDCs. RT-PCR showed an activation signature in BM-DCs (significantly higher IL-6 level) and a tolerigenic signature in PB-DCs (significantly higher TGF-beta and PDL-1 levels). BM-derived CD8+ T cells displayed a higher expression of the co-stimulatory receptor CD28 than PB-derived CD8+ T cells (30.3±18.8 vs 9.2±4.9; p<0.05 ). In contrast, the expression of the immune checkpoint inhibitor PD-1 was significantly higher in both PB-derived CD4 (69%±29 vs 24±11) and CD8 (65±25 vs 4±3; p<0.05) T cells than BM-derived T lymphocytes. T cells from both BM and PB did not express the T cell exhaustion marker TIM-3. CD3/CFSE+-DCs co-cultures showed a T cell proliferation rate that was significantly higher in BM than in PB (25±7.2 vs 6.7±8.7; p<0.05). These data show that haploidentical transplantation with Treg/Tcon immunotherapy promotes the reconstitution of DCs with an activating signature in the BM and a tolerigenic signature in the PB. Human peripheral blood Tregs that are used for adoptive immunotherapy are largely CD45RO+ and express low level of CxCR4 bone marrow homing receptor. When infused in immunodeficient mice they migrate to the periphery (spleen, gut, liver) but are unable to home to the bone marrow (Ruggeri et al., ASH 2018). In conclusion, Tregs/DC interaction induce tolerance in the periphery (and may protect from GvHD). In the BM, in the absence of Tregs, DCs activate alloreactive Tcon and may favour killing of the leukemic targets. Therefore, Tregs/DC interactions may contribute to the separation between GvL effect and GvHD in the Treg based haploidentical transplantation. Disclosures No relevant conflicts of interest to declare.

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Gandhi, Shreyans A., Victoria T. Potter, Victoria J. Tindell, Rachel Kesse-Adu, Laura Reiff-Zall, Michelle Kenyon, Judith C. W. Marsh, et al. "Alemtuzumab-Based RIC HSCT with Pre-Emptive DLI as An Effective Strategy to Achieve Long-Term Disease Remission In Patients with High Risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukaemia (AML)." Blood 116, no. 21 (November 19, 2010): 1304. http://dx.doi.org/10.1182/blood.v116.21.1304.1304.

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Abstract Abstract 1304 Disease relapse following allogeneic haematopoietic stem cell transplant (HSCT) for MDS/AML remains one of the primary causes of treatment failure, particularly in the setting of T-cell depleted reduced-intensity conditioning (RIC). Pre-emptive immunotherapy such as the use of donor lymphocyte infusions (DLI) is one strategy utilized to prevent relapse, although this approach may be associated with an increased risk of GvHD. We present our experience, evaluating toxicity and efficacy of pre-emptive DLI for the treatment of mixed donor chimerism (MDC) in a cohort of high risk MDS/AML patients who received a uniform alemtuzumab-based RIC HSCT protocol. 37 patients received a RIC protocol using fludarabine, busulphan and alemtuzumab. The stem cell source was BM (n=8) and PBSC (n=29) with a median cell dose of 2.40 and 5.34×106̂ CD34/kg respectively. The donor source was from HLA-matched sibling (n=20), HLA-matched unrelated (n=14) with 3 1-allele mismatched unrelated donors. Peripheral blood CD3 chimerism was monitored at day 30, 60, 100, 180 and 1 year post-HSCT. Patients with MDC (defined as CD3 <50%, or fall of CD3 of greater than 20% in a month) received pre-emptive DLI at a starting dose of 5×105̂CD3/kg or 1×106̂CD3/kg. In order to receive pre-emptive DLI, patients had to be in cytogenetic and morphological remission, to be off immunosuppression and have no evidence of active GvHD. Escalating doses of DLI were administered at 6–8 week intervals in the absence of improvement in donor chimerism (increase of <20% donor CD3). The median age of the cohort at transplant was 59 yrs (25-71). At the time of initial diagnosis 70% of the patients had an advanced stage disease (RAEBI/II (24%) or AML(46%)). Mean donor CD3 chimerism at the time of DLI was 44% (range: 0–76%) and median time to DLI was 195 days (range: 22 to 732 days). The median number of DLI doses was 3 (range:1-5) with a median total CD3 dose of 4.9×106̂ CD3/kg (range: 5×105̂ to 4.15×108̂). The majority of patients (83%) achieved full donor chimerism (FDC, >95%) at a median time of 193 days following the start of DLI (range: 47–732 days). The incidence of GVHD was 54%, however the incidence of severe (acute grade III,IV or chronic extensive) GvHD was limited at 21%. Skin was the commonest site of involvement with GvHD. The number of DLI doses, the total DLI dose infused, recipient age, donor source and disease stage all had no association with the development of GvHD or attainment of FDC post DLI. At last follow-up, 10 patients had died due to disease relapse(4), PTLD(1), GvHD/CMV(1), multi-organ failure(1), cerebral haemorrhage(1), infection(1) and encephalopathy(1). Evidence of relapsed disease was seen in 7 patients, and 3 patients proceeded to a second RIC HSCT. The 2-yr actuarial OS, TRM and DFS was 81%+/−6%, 14%+/−6%, and 68%+/−8% respectively. Encouragingly, the median OS for survivors post-DLI was 2537 days (range: 805–3287 days). Neither donor source, disease stage nor the presence of GvHD post-DLI had any impact on TRM, relapse or OS. Patients who achieved FDC post DLI had a 2-yr OS of 84%+/−7% when compared with those who failed to achieve FDC: 60%+/−21%, p=0.23. In conclusion, the use of pre-emptive DLI HSCT is associated with a low incidence of severe GvHD and a low TRM and relapse. In combination with an alemtuzumab-based RIC HSCT, this approach facilitates the attainment of durable long-term remission in patients whilst reducing the procedure related toxicity. Disclosures: No relevant conflicts of interest to declare.

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Morandi, Fabio, Mahboubeh Yazdanifar, Claudia Cocco, Alice Bertaina та Irma Airoldi. "Engineering the Bridge between Innate and Adaptive Immunity for Cancer Immunotherapy: Focus on γδ T and NK Cells". Cells 9, № 8 (22 липня 2020): 1757. http://dx.doi.org/10.3390/cells9081757.

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Most studies on genetic engineering technologies for cancer immunotherapy based on allogeneic donors have focused on adaptive immunity. However, the main limitation of such approaches is that they can lead to severe graft-versus-host disease (GvHD). An alternative approach would bolster innate immunity by relying on the natural tropism of some subsets of the innate immune system, such as γδ T and natural killer (NK) cells, for the tumor microenvironment and their ability to kill in a major histocompatibility complex (MHC)-independent manner. γδ T and NK cells have the unique ability to bridge innate and adaptive immunity while responding to a broad range of tumors. Considering these properties, γδ T and NK cells represent ideal sources for developing allogeneic cell therapies. Recently, significant efforts have been made to exploit the intrinsic anti-tumor capacity of these cells for treating hematologic and solid malignancies using genetic engineering approaches such as chimeric antigen receptor (CAR) and T cell receptor (TCR). Here, we review over 30 studies on these two approaches that use γδ T and NK cells in adoptive cell therapy (ACT) for treating cancer. Based on those studies, we propose several promising strategies to optimize the clinical translation of these approaches.

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36

Warren, Edus H., Philip D. Greenberg, and Stanley R. Riddell. "Cytotoxic T-Lymphocyte–Defined Human Minor Histocompatibility Antigens With a Restricted Tissue Distribution." Blood 91, no. 6 (March 15, 1998): 2197–207. http://dx.doi.org/10.1182/blood.v91.6.2197.

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Abstract Cytotoxic T lymphocytes (CTL) specific for human minor histocompatibility (H) antigens can be isolated from the blood of major histocompatibility complex (MHC)-matched allogeneic bone marrow transplant (BMT) recipients and may play a prominent role in the graft-versus-host (GVH) and graft-versus-leukemia (GVL) reactions (Tsoi et al, J Immunol 125:2258, 1980; Tsoi et al, Transplant Proc 15:1484, 1983; Goulmy et al, Nature 302:159, 1983; Irle et al, Transplantation 40:329, 1985; and Niederwieser et al, Blood 81:2200, 1993). The identification of minor H antigens that are expressed in hematopoietic cells, including leukemic cells, but not in fibroblasts and other tissue types has suggested that such tissue-restricted antigens could potentially serve as targets for T-cell immunotherapy to enhance GVL activity without inducing GVH disease (de Bueger et al, J Immunol 149:1788, 1992; van der Harst et al, Blood 83:1060, 1994; and Dolstra et al, J Immunol 158:560, 1997). To explore the feasibility of this strategy, donor CD3+CD8+ CTL clones specific for recipient minor H antigens were isolated and characterized from allogeneic BMT recipients. CTL clones were obtained from the majority of donor/recipient pairs. Seventeen distinct minor H antigens distinguishable by their MHC-restricting allele, population frequency, and/or distribution of tissue expression were defined by 56 CD3+CD8+ CTL clones isolated from these patients. The MHC-restricting alleles for these CTL clones included HLA-A2 and HLA-B7, which had previously been shown to present minor H antigens to CTL, as well as HLA-A3, -A11, -B8, -B53, and -Cw7, which had not previously been described to present minor H antigens to CTL. Estimated phenotype frequencies for these 17 distinct minor H antigens range from 0.17 to 0.92. In vitro cytotoxicity assays using hematopoietic cells and fibroblasts as target cells showed that 5 of the 17 minor H antigens were expressed in both hematopoietic cells and fibroblasts. However, 12 were presented for CTL recognition only by hematopoietic cells and not by dermal fibroblasts derived from the same donors. These results significantly extend the spectrum of CTL-defined human minor H antigens that could potentially serve as target antigens for cellular immunotherapy to promote GVL activity after allogeneic BMT.

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Warren, Edus H., Philip D. Greenberg, and Stanley R. Riddell. "Cytotoxic T-Lymphocyte–Defined Human Minor Histocompatibility Antigens With a Restricted Tissue Distribution." Blood 91, no. 6 (March 15, 1998): 2197–207. http://dx.doi.org/10.1182/blood.v91.6.2197.2197_2197_2207.

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Cytotoxic T lymphocytes (CTL) specific for human minor histocompatibility (H) antigens can be isolated from the blood of major histocompatibility complex (MHC)-matched allogeneic bone marrow transplant (BMT) recipients and may play a prominent role in the graft-versus-host (GVH) and graft-versus-leukemia (GVL) reactions (Tsoi et al, J Immunol 125:2258, 1980; Tsoi et al, Transplant Proc 15:1484, 1983; Goulmy et al, Nature 302:159, 1983; Irle et al, Transplantation 40:329, 1985; and Niederwieser et al, Blood 81:2200, 1993). The identification of minor H antigens that are expressed in hematopoietic cells, including leukemic cells, but not in fibroblasts and other tissue types has suggested that such tissue-restricted antigens could potentially serve as targets for T-cell immunotherapy to enhance GVL activity without inducing GVH disease (de Bueger et al, J Immunol 149:1788, 1992; van der Harst et al, Blood 83:1060, 1994; and Dolstra et al, J Immunol 158:560, 1997). To explore the feasibility of this strategy, donor CD3+CD8+ CTL clones specific for recipient minor H antigens were isolated and characterized from allogeneic BMT recipients. CTL clones were obtained from the majority of donor/recipient pairs. Seventeen distinct minor H antigens distinguishable by their MHC-restricting allele, population frequency, and/or distribution of tissue expression were defined by 56 CD3+CD8+ CTL clones isolated from these patients. The MHC-restricting alleles for these CTL clones included HLA-A2 and HLA-B7, which had previously been shown to present minor H antigens to CTL, as well as HLA-A3, -A11, -B8, -B53, and -Cw7, which had not previously been described to present minor H antigens to CTL. Estimated phenotype frequencies for these 17 distinct minor H antigens range from 0.17 to 0.92. In vitro cytotoxicity assays using hematopoietic cells and fibroblasts as target cells showed that 5 of the 17 minor H antigens were expressed in both hematopoietic cells and fibroblasts. However, 12 were presented for CTL recognition only by hematopoietic cells and not by dermal fibroblasts derived from the same donors. These results significantly extend the spectrum of CTL-defined human minor H antigens that could potentially serve as target antigens for cellular immunotherapy to promote GVL activity after allogeneic BMT.

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Albring, Jörn C., Matthias Stelljes, Wolfgang E. Berdel, and Joachim Kienast. "Graft-Versus-Tumor (GvT) Effects in the Absence of Alloantigens on Tumor Cells Crucially Depend on Graft-Versus-Host Disease (GvHD) Intensity." Blood 106, no. 11 (November 16, 2005): 1304. http://dx.doi.org/10.1182/blood.v106.11.1304.1304.

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Abstract While a variety of tumors is potentially immunogenic and is capable to trigger tumor-associated antigen (TAA)-specific immune responses in vivo the endogenous immune system usually fails to eradicate established tumors. Despite considerable effort and promising experimental and preliminary clinical data most cellular immunotherapeutic strategies have not evolved into a clinically relevant treatment strategy with allogeneic immune replacement as cancer immunotherapy being the sole exception. At this juncture allogeneic HSCT not only allows for otherwise fatal high-dose chemo- and radiotherapy intended to reduce the tumor cell burden but also encompasses potent immunologically mediated anti-tumor effects, referred to as GvT reaction. This GvT reaction, which is based on donor T and natural killer cells, is interrelated, however, with potentially deleterious GvHD, one of the major limitations of allogeneic HSCT. Mutated tumor-associated antigens, as well as selectively or aberrantly expressed nonmutated antigens, represent potential targets for T cell-mediated GvT effects that are in principle, separable from generalized anti-host responses. Using an allogeneic parent-into-F1 murine transplantation model (BALB/c or C57BL/6 → [C57BL/6 x BALB/c]F1) with different chemically induced tumors derived from either parental strain, we have previously demonstrated that a significant CD8+ T cell-mediated GvT effect accounts for immunological tumor control after allogeneic HSCT in vivo. Remarkably, this effect occurs in the absence of histoincompatibilities between donor immune cells and host tumor (Blood2004; 104:1210–1216). Since most chemically induced tumors have immunogeneic neo-antigens we sought to expand our initial findings to spontaneously occurring tumor cell lines, which often are less immunogenic. In addition, we asked whether the intensity of GvHD directly impacts on GvT effects in the absence of alloantigens on tumor cells. Sublethally irradiated [C57BL/6 x BALB/c]F1 (H-2b/d) mice were transplanted with whole bone marrow with or without splenocytes from allogeneic C57BL/6 (H-2b) and BALB/c (H-2d) donors, respectively. Recipient mice were inoculated subcutaneously with hematological and solid tumors derived from either C57BL/6 (B16, LLC - both H-2b) or BALB/c (RENCA, A20, MPC-11, WEHI-3B - all H-2d) strains. By varying the histocompatibility between donor immune cells, host tissue and tumor tissue, it was possible to study GvT effects in the absence of alloantigens on tumor cells. Furthermore, using different numbers of coinjected donor splenocytes the interrelation of GvHD intensity and alloantigen independent GVT effects was assessed. As compared with allograft recipients having weak GvHD (2x107 bone marrow cells) a significant tumor-alloantigen independent GvT effect was present in animals undergoing intense GvHD (2x107 bone marrow cells and 107 splenocytes). These data indicate that a biologically relevant GvT effect that is independent of alloantigen recognition on tumor cells is crucially influenced by the intensity of GvHD.

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Colonna, Lucrezia, Mareike Florek, Dennis B. Leveson-Gower, Emanuela I. Sega, Robert Zeiser, Martin Buess, and Robert S. Negrin. "IL-17 Gene Ablation Does Not Impact Treg-Mediated Suppression of Graft-Versus-Host Disease Following Bone Marrow Transplantation,." Blood 118, no. 21 (November 18, 2011): 4037. http://dx.doi.org/10.1182/blood.v118.21.4037.4037.

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Abstract Abstract 4037 Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for many hematologic malignancies such as leukemia, lymphoma and myelodysplastic syndromes. A potentially fatal complication of HCT is graft-versus-host disease (GVHD), where conventional T cells of alloreactive-specificities (CD4+ and CD8+ T cells; Tcon) infiltrate and destroy target organs such as the liver, gut and skin. While immunomodulation is necessary to control the adverse GVHD reactions, an effective immune response is required for successful tumor eradication, named graft-versus-tumor effect (GVT). Following allogeneic bone marrow transplantation, our and other laboratories have shown that the transfer of highly purified regulatory T cells (Treg), a key immunoregulatory cell population involved in the maintenance of immune tolerance, can prevent lethal GVHD induced by the donor-derived Tcon in multiple models across both major and minor histocompatibility barriers. Notably, when transferred into recipient mice with established leukemia or lymphoma, Treg cells effectively suppress Tcon cell proliferation and prevent lethal GVHD, while preserving GVT activity. We have recently adapted our pre-clinical model to an ongoing clinical trial, and are currently assessing the efficacy of highly purified human Treg cells in a clinical setting. However, recent evidence suggest that, under the influence of intense inflammatory signals, the Tregsuppressive phenotype can become unstable, a phenomenon that can culminate in Tregconversion into IL-17-producing pro-inflammatory cells. IL-17 is a proinflammatory cytokine that has been reported to promote GVHD in different models for human HCT. We hypothesized that under the influence of the intense proinflammatory signals, such as those released during an ongoing alloreaction, a fraction of the transferred Treg might be redirected to the Th17 cell fate, thereby losing immunosuppressive potential and acquiring proinflammatory capacity. Our preliminary in vitro studies demonstrated that Treg cells do indeed upregulate Il17 gene expression following activation by allogeneic antigen presenting cells (APCs). We, therefore, sought to evaluate the impact of Il17 gene ablation on Treg stability and immunosuppressive capacity in a major MHC mismatch model, in order to ultimately assess the possible therapeutic potential of IL-17 targeting for the enhancement of Treg-mediated GVHD suppressive ability following HCT. On the basis of our preliminary studies we anticipated that Treg cells purified from Il17 knockout (KO) donor mice would have enhanced immunomodulatory capacity compared to wild-type (WT) Treg, given their inability to produce IL-17. To monitor ongoing GVHD our laboratory has developed an imaging system that allows for the in vivo visualization of T cell homing and proliferation over time, without the necessity to sacrifice the experimental animals. Specifically, the allogeneic Tcon cells are isolated from donor mice where the gene coding for the enzyme luciferase (luc) is expressed by all hematopoietic cells. Importantly, the proliferation of the transferred luc+ Tcon can be directly assessed by the number of photons detected by the bioluminescent imaging system (BLI), which gives a measure of ongoing GVHD. Treg were purified from both WT and IL-17 KO animals and their GVHD-suppressive ability was directly compared in vivo following the transfer of luc+ Tcon. IL-17 KO Tregcells were as proficient as WT Treg in suppressing GVHD. Similarly, the GVHD-inhibitory capacity of Treg cells isolated from IL-1R KO mice, that were previously shown to be resistant to Th17 conversion, was comparable to that of WT Treg. Furthermore, upon ex vivo re-isolation of Tregthat were previously transferred into transplanted recipients, we could detect little or no IL-17 production by intracellular cytokine staining. Thus, we conclude that freshly isolated and highly purified Treg do not convert into IL-17 producing cells in our model, suggesting that IL-17 production is not a factor affecting Tregstability during ongoing GVHD, and excluding IL-17 targeting as a valuable strategy to improve Treg immunotherapy following bone marrow transplantation. Disclosures: No relevant conflicts of interest to declare.

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40

Bernhardt, Anna Luise, Julia Zeun, Miriam Marecek, Hannah Reimann, Sascha Kretschmann, Judith Bausenwein, Edith D. van der Meijden, et al. "Influence of DM-sensitivity on immunogenicity of MHC class II restricted antigens." Journal for ImmunoTherapy of Cancer 9, no. 7 (July 2021): e002401. http://dx.doi.org/10.1136/jitc-2021-002401.

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BackgroundGraft-versus-host-disease (GvHD) is a major problem in allogeneic stem cell transplantation. We previously described two types of endogenous human leukocyte antigen (HLA)-II restricted antigens depending on their behavior towards HLA-DM. While DM-resistant antigens are presented in the presence of HLA-DM, DM-sensitive antigens rely on the expression of HLA-DO-the natural inhibitor of HLA-DM. Since expression of HLA-DO is not upregulated by inflammatory cytokines, DM-sensitive antigens cannot be presented on non-hematopoietic tissues even under inflammatory conditions. Therefore, usage of CD4+ T cells directed against DM-sensitive antigens might allow induction of graft-versus-leukemia effect without GvHD. As DM-sensitivity is likely linked to low affinity peptides, it remains elusive whether DM-sensitive antigens are inferior in their immunogenicity.MethodsWe created an in vivo system using a DM-sensitive and a DM-resistant variant of the same antigen. First, we generated murine cell lines overexpressing either H2-M or H2-O (murine HLA-DM and HLA-DO) to assign the two model antigens ovalbumin (OVA) and DBY to their category. Further, we introduced mutations within the two T-cell epitopes and tested the effect on DM-sensitivity or DM-resistance. Furthermore, we vaccinated C57BL/6 mice with either variant of the epitope and measured expansion and reactivity of OVA-specific and DBY-specific CD4+ T cells.ResultsBy testing T-cell recognition of OVA and DBY on a murine B-cell line overexpressing H2-M and H2-O, respectively, we showed that OVA leads to a stronger T-cell activation in the presence of H2-O demonstrating its DM-sensitivity. In contrast, the DBY epitope does not rely on H2-O for T-cell activation indicating DM-resistance. By introducing mutations within the T-cell epitopes we could generate one further DM-sensitive variant of OVA and two DM-resistant counterparts. Likewise, we designed DM-resistant and DM-sensitive variants of DBY. On vaccination of C57BL/6 mice with either epitope variant we measured comparable expansion and reactivity of OVA-specific and DBY-specific T-cells both in vivo and ex vivo. By generating T-cell lines and clones of healthy human donors we showed that DM-sensitive antigens are targeted by the natural T-cell repertoire.ConclusionWe successfully generated DM-sensitive and DM-resistant variants for two model antigens. Thereby, we demonstrated that DM-sensitive antigens are not inferior to their DM-resistant counterpart and are therefore interesting tools for immunotherapy after allogeneic stem cell transplantation.

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Yee, Andrew J., Thomas R. Spitzer, Susan L. Saidman, Juanita Shaffer, Karen A. Power, Steven L. McAfee, Karen K. Ballen, et al. "Minimal HLA Disparity and KIR Ligand Compatibility in Host Versus Graft Direction May Facilitate Donor Engraftment Following In Vivo and Ex Vivo T Cell Depleted (TCD) Nonmyeloablative Haploidentical Stem Cell Transplantation for Hematologic Malignancies." Blood 106, no. 11 (November 16, 2005): 3668. http://dx.doi.org/10.1182/blood.v106.11.3668.3668.

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Abstract Mixed chimerism (MC) can be induced in mice across MHC barriers using nonmyeloablative conditioning and T cell depletion. Delayed donor lymphocyte infusions (DLI) convert this MC state to full donor hematopoiesis and impart a powerful graft versus tumor effect without inducing GVHD. Based on this principle, we developed a clinical trial with a goal of inducing a GVH-free mixed chimeric platform for delayed cellular immunotherapy via DLI following nonmyeloablative haploidentical HSCT. This study analyzed graft outcomes in 13 patients who received in vivo and ex vivo TCD, haploidentical (HLA 1–3 Ag mismatched) HSCT for refractory hematologic malignancies (NHL, n=7; CLL, n=1; HD, n=3; CML, n=1; MDS, n=1). Conditioning consisted of MEDI-507 (siplizumab, an anti-CD2 mAb) for in vivo T cell depletion of both the host and donor, cyclophosphamide, fludarabine, thymic irradiation, and a brief course of cyclosporine. An earlier cohort of 4 patients did not receive fludarabine. All patients received ex vivo TCD of G-CSF-mobilized peripheral blood stem cells using a CD34+ cell-selection device. A median of 7.38 x 106 (3.19 x 106 to 1.49 x 107) CD 34+ cells/kg and a median of 5.24 x 104 (6.31 x 102 to 1.61 x 105) CD3+ cells/kg were given. DLI was given as early as 5 weeks in patients without evidence of GVHD. Inhibitory killer immunoglobulin-like receptor (KIR)-HLA epitope mismatches (missing ligand) in the GVH and HVG (host versus graft) directions were assessed based on HLA and KIR genotyping of patients and their donors. Natural killer (NK) cells recovered relatively early, despite the presence of circulating MEDI-507. Split lineage MC was observed in each case, with a predominance of early donor myeloid chimerism and a lower percentage of donor T cell chimerism. Four of the 13 patients eventually lost their grafts despite DLI, including 2 who were not conditioned with fludarabine. Five patients spontaneously achieved full donor chimerism (FDC) and 4 converted to FDC following DLI. The spontaneous chimerism group had significantly fewer HLA mismatches in the HVG direction. Four out of the 5 had only one mismatch, whereas the patients who had graft loss or required DLI had at least two mismatches (p=0.007 by Fisher’s exact test). This finding remained significant when the subgroup of patients not conditioned with fludarabine was excluded from the analysis (p=0.048). Spontaneous FDC was also associated with the presence of graft HLA-C group 1 (HLA-CAsn80) ligand for host NK cell inhibitory receptor KIR2DL2/3 (KIR-L match/compatible, 4 out of 4 evaluable patients) while this graft HLA ligand tended to be absent in the group that lost the graft or required DLI to achieve chimerism (KIR-L mismatch, 5 out of 8 patients) (p=0.08 by Fisher’s exact test). There was no significant relationship between donor KIR and host HLA types in the GVH direction for predicting engraftment. While these observations are preliminary given the heterogeneity of the conditioning regimens and the small number of patients, the data suggest that the lesser degree of HLA mismatching and the presence of KIR ligand compatibility in the HVG direction may influence donor engraftment following nonmyeloablative TCD haploidentical HSCT and warrant further study.

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42

Zhang, Mei, Jun Qi, Shanxi Liu, Baoyan Wang, Xin Liu, Pengcheng He, Jing Li, Huaiyu Wang, Ruibo Cai, and Lugui Qiu. "Induction of Graft Versus Leukemia Effect by Mixed Bone Marrow Transplantation." Blood 106, no. 11 (November 16, 2005): 5333. http://dx.doi.org/10.1182/blood.v106.11.5333.5333.

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Abstract Mixed bone marrow transplantation (MBMT) is a special kind of stem cell transplant by combining autologous bone marrow transplantation (ABMT) with HLA mismatched haploidentical (3/6 loci mismatched) sibling bone marrow stem cells for the those patients who failed finding HLA matched sibling donor or HLA matched unrelated donor(MUD). As the main aim of MBMT is to induce graft versus leukemia (GVL) effect, there is no any prophylaxis regimen to be used for preventing graft versus host disease (GVHD) during the MBMT. In this study, we explore the relationship of graft versus leukemia effect induced by MBMT and long disease-free survival (DSF) of patients between two different transplant groups. (1) MBMT group: 8 patients treated by MBMT were 5 male and 3 female aged 8–40 years old suffering from acute myelocytic leukemia (AML) 6 cases (M3 2 cases, M4 2cases, M5 2case) and actue lymphoblast leukemia 2 cases (L1 and L2). After getting complete remission(CR), all of patients had received 3–5 courses of chemotherapy and then were treated by MBMT. At day +2 to +5, after autologous stem cell infusion at day 0, 20–50 ml allogenic born marrow stem cells were collected from HLA mismatched-haploidentical (3/6 loci mismatch) and ABO, Rh blood type identical sibling donor of patients with the mean number of mononuclear cell (MNC) 2.39x108 ( 0.5–5.0 x108 ) and then were infused intravenously immediately to receptor. The mean number of MNC from allogenic bone marrow accounts for 1/20 to 1/50 (1/30) of that from autologous bone marrow. Prophylaxes regimen for preventing GVHD had not been used in all cases with MBMT. (2) ABMT group: 10 cases of ABMT were compared as control including 6 male and 4 female aged from 21 to 44 year old suffering from acute myelocytic leukemia (AML) 5 cases (M3 1 cases, M2 2 cases, M5 2 case) and NHL 5 cases. We find quiet different results in two groups: (1) All cases in MBMT group had developed typical or atypical manifestations of acute GVHD in clinic within day +10 to day +100, which were diagnosed by skin pathologic biopsy. 3 patients were died because of severe aGVHD (IIIo–IVo) in the early stage after transplant. 5 cases with mild aGVHD were treated with prednisone, CSA and MTX and got control quickly, whose marrow finally kept mixed chimeris for half a year conformed by HLA gene detection and sex cheomosome analysis. All of these 5 patients are non-relape up to now and have acquired long disease-free survival for 129, 118, 113, 97 and 92 months respectively. The total relapse rate is 0%. (2) AMBT group: 6 cases of 10 were relapsed and died within 2 years after AMBT. 3 cases have get DFS for 119, 100 and 35 months respectively. The total relapse rate in 2 year is 60% in ABMT group. The results and conclusions we have get from the study indicate that MBMT may be an effective and safe therapy with much lower relapse and longer DFS because of inducing and existing of GVL effects. Patients with long DSF in MBMT group have showed close relationship with their mixed chimeris and GVL effects after MBMT. However, there are still some problems should be solved in MBMT: A more suitable infusion ratio of MNC from HLA mismatched haploidentical sibling donor and autologous marrow, A better strategy for co-infusion or delayed infusion of allogeneic stem cell, Does MBMT need GVHD prophylaxis or not? Immunotherapy for post-MBMT with DLI or DSI.

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Sutton, Kathryn S., Anindya Dasgupta, David McCarty, Christopher B. Doering, and H. Trent Spencer. "Expansion and Bioengineering of Peripheral Blood-Derived Anti-Cancer Gd T Cells in Serum Free Media." Blood 126, no. 23 (December 3, 2015): 5553. http://dx.doi.org/10.1182/blood.v126.23.5553.5553.

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Abstract Conventional immunotherapy relies on expansion and, in some settings, transduction of αβ T cells. Success is dependent on several highly variable patient-specific parameters including i) cell number obtained from harvest and ex vivo expansion, ii) targeting capacity of cells to tumors, iii) ability of infused T cells to survive and function in a immune evading tumor microenvironment (e.g., down-regulation of major histocompatibility complex (MHC) on the tumor cell surface), and iv) on-target and off-target adverse events such as graft vs host disease (GvHD). Following the the early success of a few anti-leukemia products, some progress has been made on expanding the applicability to a wider variety of hematologic and solid neoplasms but rapid progress has been hindered due to lack of efficacy and significant adverse event profiles. Thus, alternative cellular immunotherapy approaches such as gd T cells are being pursued. Although gd T cells represent a small fraction (2-5%) of circulating peripheral blood mononuclear cells (PBMC), they are ideal candidates for immunotherapy applications because they i) possess intrinsic anti-tumorigenicity, ii) require no priming to mediate tumor cell lysis in an MHC-independent manner, iii) direct tumor killing via recognition of stress-responsive ligands frequently up-regulated by tumor cells, and, as we show, iv) can be expanded to clinical cell doses in cGMP serum free media (SFM). Although tremendous effort has been devoted to αβ T cell processing, little focus has been directed toward the clinical development of genetically engineered gd T cells. Herein, we describe the optimization of drug and cytokine dosing and, culture in SFM, robust expansion and genetic modification necessary for clinical applications. Peripheral blood mononuclear cells (PBMC) isolated from healthy donors were cultured in several SFMs, including: OpTmizer, X-Vivo, SCGM, and AIM V as well as commonly used media containing serum, such as RPMI1640 supplemented with either 10% FBS or human serum. Complete growth media also included 2mM L-glutamine (if not present in the commercial product), Zoledronic Acid (5µM added only at the start of culture), and IL-2 (either 100 or 1000 IU/ml added twice weekly). Of the SFM cultures, PBMC isolated from 7 of 8 donors had robust expansion only in OpTmizer (supplemented with high-dose IL-2) with a concomitant rise in gd T cell percentage to levels sufficient for clinical use. Specifically, the percentage of gd T cells increased from a mean of 3% (2.4-3.6%) to 61% (21-84%), resulting in an 88 fold expansion at 14 days. While gd T cell numbers were lower in SFM compared to serum-containing RPMI, the final product starting with PBMC harvested during a single apheresis procedure when cultured in OpTmizer can yield therapeutic doses. The expanded gd T cells were evaluated for their transduction efficiency using a 3rd generation self-inactivating lentiviral vector (LV) to introduce a reporter gene encoding EGFP. Two different LVs were tested: HIV and SIV harboring the transgene under the control of EF1α and MSCV promoters, respectively. The timing of LV addition was optimized with respect i) gd T cell expansion, ii) LDL Receptor (LDLR) expression (a known receptor utilized by VSV-G pseudotyped LV) and iii) LV dose (3-4 consecutive transductions over 48 hours). Transductions remained consistent using approximately 30% LV concentrated stock by volume. As expected, greater transduction was achieved in cultures with the greatest expansion. Interestingly, higher transduction efficiency was observed in gd T cell populations with the highest LDLR levels. In gd T cells, an MSCV promoter provided greater GFP expression compared to an EF1α promoter. As expected a range of transduction efficiencies was observed for various donors spanning from 13-33%, which is similar to LV transduction levels observed for αβ T cell transductions with similar multiplicity of infection and expansion levels. Therefore, an optimized method of gd T cell expansion and transduction was developed that can be tested in early phase clinical trials. The absence of MHC-restriction affords the opportunity, with appropriate elimination of the αβ T cell component, for use in the allogeneic setting with limited risk of GvHD, and the use of SFM provides clinically safer, widely applicable, and potentially more efficacious cellular immunotherapy. Disclosures Doering: Expression Therapeutics: Equity Ownership; Bayer Healthcare: Consultancy, Honoraria, Research Funding. Spencer:Expression Therapeutics: Equity Ownership.

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44

Faber, L. M., S. A. van Luxemburg-Heijs, R. Willemze, and J. H. Falkenburg. "Generation of leukemia-reactive cytotoxic T lymphocyte clones from the HLA-identical bone marrow donor of a patient with leukemia." Journal of Experimental Medicine 176, no. 5 (November 1, 1992): 1283–89. http://dx.doi.org/10.1084/jem.176.5.1283.

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Allogeneic bone marrow transplantation (BMT) has been associated with a graft-vs.-leukemia (GVL) reactivity. Since T cell depletion of the bone marrow graft has decreased the risk of graft-vs.-host disease (GVHD), but has been associated with higher rates of leukemia relapse, GVL reactivity is probably caused by donor-derived T lymphocytes. Previously, we demonstrated that minor histocompatibility (mH) antigen-specific cytotoxic T lymphocyte (CTL) clones, generated from patients after BMT, are capable of major histocompatibility complex-(MHC) restricted lysis of (clonogenic) myeloid leukemic cells. Here, we investigated whether donor-derived leukemia-specific CTL clones can be generated in vitro, before BMT, using irradiated leukemic cells from a patient with acute myeloid leukemia as stimulator cells, and peripheral blood or bone marrow from the HLA genotypically identical sibling donor as responder cells. Several CTL lines were generated that showed specific lysis (> 50%) of the recipient leukemic cells in a 51Cr-release assay. Two of these CTL lines were cloned by limiting dilution in the presence of the irradiated recipient cells. Multiple leukemia-reactive, HLA class I and II-restricted clones with various specificities could be established. These alloreactive, antileukemic CTL clones may cause GVL reactivity after BMT, and may be used as adjuvant immunotherapy in the treatment of leukemia.

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Li, Jingxia, Reema Panjwani, Jian-Ming Li, Cynthia R. Giver, Maryellen Malone, and Edmund K. Waller. "T Cell Activation By Plasmacytoid Dendritic Cells Is Augmented By Inhibition of Vasoactive Intestinal Polypeptide Signaling: Implications for Cellular Immunotherapy." Blood 126, no. 23 (December 3, 2015): 3438. http://dx.doi.org/10.1182/blood.v126.23.3438.3438.

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Abstract Introduction: Data from clinical allogeneic bone marrow transplant (allo-BMT) and pre-clinical murine models of allo-BMT have shown that donor plasmacytoid dendritic cells (pDC) have important roles in regulating graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activities of donor T cells. Using murine models of allo-BMT we have previously shown that 1) donor pDCs induce Th1 polarization of donor T cells and augment the GvL activity of T cells; and 2) the addition of pDC to grafts composed of purified T cells and HSC limited the subsequent development of GvHD. VIP is an immunosuppressive neuropeptide that regulates adaptive immune responses. We reasoned that VIP signaling may regulate activation of allo-specific T-cells, and the VIP pathway may be target for regulating GvHD and GvL in allo-BMT. Methods: To explore the mechanisms by which pDC and VIP signaling regulate T cell activation we used: 1) transgenic mice expressing GFP under the control of the VIP promoter to measure VIP expression in vivo in allo-BMT recipients; 2) one-way mixed lymphocyte reaction (MLR) to measure the proliferative response of transgenic luciferase positive T cells in response to allo-antigens via bioluminescence imaging (BLI); and 3) a model system of indirect presentation of allo-peptides derived from a H2-Ab MHC class II molecule by pDC to transgenic T cells expressing the TEa TCR. The effect of blocking vasoactive intestinal polypeptide (VIP) signaling during activation of allo-reactive T cells was assessed by using VIP-KO cells and by the addition of VIP peptide or a peptide antagonist of VIP (VIPhyb) to the one-way MLR. T cell proliferation and activation was measured by flow cytometry. Results: Analysis of VIP expression in donor pDC in murine models of allo-BMT showed >100-fold induction of VIP promoter activity in donor pDC and donor T cells during the first two weeks post-transplant, indicating that VIP expression in donor pDC may regulate T cell activation.Addition of endogenous native VIP suppressed T cell proliferation in one-way MLR but was reversed by addition of a 10x concentration of the VIP antagonist peptide (Figure 1B). Furthermore, adding 3 uM of the VIP antagonist to the MLR cultured significantly enhanced T cell proliferation. TEa peptide-primed T cells cultures with peptide-primed pDC from VIP knock-out mice had increased proliferation and expressed more of the activation markers CD69 and CD71 compared with T cells cultured with VIP-WT pDCs. Comparing pDC purified from marrow versus spleen, we found significantly more proliferation in T cells cultured with bone marrow VIP-KO pDCs than splenic VIP-KO pDCs, indicating that the less mature marrow pDC have greater antigen presenting ability than the more mature splenic pDC. Conclusion: These data suggest that 1) VIP is produced by donor pDC early after allo-BMT; 2) VIP inhibits T cell allo-proliferation in one-way MLR's; and 3) blocking VIP signaling using donor cells that cannot produce VIP or through the use of pharmacological inhibitors of VIP can augment activation and proliferation of T cells in response to indirect antigen presentation.The present findings support studies of VIP antagonist in allo-BMT to augment the GvL activity of T cells through indirect antigen presentation. Future studies include using the BLI analysis of MLR to determine the effect of novel drugs on T cell proliferation. **Jingxia Li and Reema Panjwani were equal contributors to this abstract. Disclosures No relevant conflicts of interest to declare.

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46

Lee, Kyoo-Hyung, Seong-Jun Choi, Jung-Hee Lee, Ho-Jin Shin, Young-Shin Lee, Young-Ah Kang, Miee Seol, Seong-Gil Ryu, and Je-Hwan Lee. "HLA-Haploidentical Familial Donor Hematopoietic Cell Transplantation without Ex Vivo- T Cell Depletion after Reduced-Intensity Conditioning of Busulfan, Fludarabine, and Anti-Thymocyte Globulin: A Preliminary Report." Blood 110, no. 11 (November 16, 2007): 3077. http://dx.doi.org/10.1182/blood.v110.11.3077.3077.

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Abstract Animal hematopoietic cell transplantation (HCT) models and several small clinical trials showed that successful engraftment can be achieved across HLA-haplotype difference after reduced-intensity conditioning (RIC). Furthermore, decreased graft-versus-host disease (GVHD) and transplantation-related mortality (TRM) after RIC was shown in a swine leukocyte antigen-haploidentical HCT experiment. Therefore, a protocol investigating the role of RIC in HLA-haploidentical familial donor HCT was initiated in April 2004 and 20 patients [13 male and 7 female; median age 26.5 years (16–65)] without HLA-matched donor enrolled until June 2007. The diagnosis were AML (n=9), ALL (n=4), acute biphenotypic leukemia (n=1), MDS (n=4), and SAA (n=2), and all patients had high-risk features, i.e. first complete remission (CR) but with high-risk chromosomal abnormality (n=1), first CR after salvage (n=1), second CR (n=6), recurrent/refractory state (n=7), immunotherapy failure (n=4), and high-risk MDS (RAEB-1, n=1). The RIC included iv busulfan 3.2 mg/kg × 2, fludarabine 30 mg/m2 × 6, plus anti-thymocyte globulin [Thymoglobuline 3 mg/kg (n=17) or Lymphoglobuline 15 mg/kg (n=3)] × 4. After receiving G-CSF, the donors (13 mothers; 5 offsprings; and 2 HLA-haploidentical siblings) underwent 2 or 3 daily leukapheresis, and the collected cells were given to patients without T cell depletion [medians of; 7.9 (3.7–12.1)×108/kg MNC, 6.9 (3.6–73.5)×106/kg CD34+ cells, and 4.6 (1.8–8.5)×108/kg CD3+ cells]. GVHD prophylaxis was cyclosporine 3 mg/kg/day iv from day -1 and a short course of methotrexate. As a part of separate phase 1 study, the two most-recently enrolled patients received additional donor CD34+ cell-derived NK cells 6 weeks after HCT. Except one patients with SAA who died due to K. pneumoniae sepsis on day 18, all 19 evaluable patients engrafted with ANC> 500/μl median 17 days (12–53) and platelet> 20,000/μl median 23 days (12–100) after HCT. Eight patients experienced acute GVHD (grades I, II, III, and IV; 2, 3, 2, and 1, respectively). Cumulative incidences (CI) of overall and grade II-IV acute GVHD were 40 and 30%, respectively. Eight patients experienced chronic GVHD (limited, 4; extensive, 4; CI, 51%). Fourteen showed positive CMV antigenemia, while 2 suffered CMV colitis, which resolved after treatment. As early as 2 weeks after HCT, 15 of 16 evaluable patients, and, by 4 weeks, all of 17 evaluable patients showed donor chimerism ≥95% on STR-PCR, which was maintained until 24 weeks in all 11 patients tested. Thirteen patients are alive after median follow-up of 13.6 months (1.5–37.9; Kaplan-Meier survival, 55.6%). Of 16 patients with acute leukemia and high-risk MDS, 8 remain alive without recurrence (event-free-survival, 40.9%). Two patients died of K. pneumoniae sepsis and grade IV acute GVHD, respectively (CI of TRM, 11%). Immune recovery in 10 patients without relapse for > 6 months showed robust lymphocyte contents and immunoglobulin levels at 6 months (means of; 1,060/ul CD3+, 222/ul CD4+, 767/ul CD8+ cells, and 1,317 mg/dl IgG) and 12 months. After RIC, consistent engraftment and durable complete donor hematopoietic chimerism can be achieved from HLA-haploidentical familial donor. The frequencies of GVHD and TRM were low.

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Tsai, Schickwann, H. Christopher Leukel, Chikako Morita, Marc J. Heikens, and Thai M. Cao. "Functional Characterization of a Continuous Natural Killer Cell Line, KIL, In Vitro and in Adoptive Immunotherapy." Blood 108, no. 11 (November 16, 2006): 3693. http://dx.doi.org/10.1182/blood.v108.11.3693.3693.

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Abstract We have recently established a continuous natural killer (NK) cell line, KIL (for Killer Lymphocyte), from a coculture of normal murine whole bone marrow (WBM) and the OP-9 stromal cell line expressing the Notch ligand, Jagged2, in the presence of stem cell factor and IL-7 (Dehart et al. Blood2005;105:3521). A clonal prototype, KIL C.2, was derived from a C57BL/6 mouse and has the CD3−NK1.1+CD122+ phenotype, expresses the NKG2D activating receptor, and proliferates extensively upon in vitro stimulation with IL-2 (20ng/ml). We now show that KIL C.2 stimulated with IL-2 for 48 hours produce large quantities of interferon-gamma (10,300 ± 1850 pg/ml) and modest amounts of tumor necrosis factor-alpha (200 ± 60 pg/ml). IL-4 and IL-10 secretion was below background levels. We then evaluated the cytolytic potential of KIL C.2 using a FACS-based flourolysis assay using GFP-labeled target cells. We observed that KIL C.2 potently lyse allogeneic A20 (H2d) lymphoma tumor targets without prior activation. In addition, KIL C.2 is moderately lytic against two syngeneic (H2b) targets: a myeloma cell line, 5T33MM, and BLL C.2, a tumorigenic leukemia/lymphoma cell line that was established from a WBM culture and has the CD19+CD25+CD43+sIg− pre-B-cell phenotype (S.T. and T.M.C., unpublished data). To study the mechanism of cytolysis we performed killing assays in the presence of anti-NKG2D (MI-6) or anti-NK1.1 (PK136) blocking antibodies. Blockade of either NK1.1 or NKG2D reduced cytotoxicity against BLL C.2 targets by 50%. BLL C.2 has the C57BL/6 background, thus does not express H60 but expresses the other NKG2D ligands Rae1 at high levels and Mult1 at moderate levels as determined by quantitative RT-PCR. In contrast, blocking NK1.1 and NKG2D had no effect on cytolysis of A20 suggesting the presence of alternative activation pathways such as those mediated by MHC class I alloantigen receptors. To examine the in vivo tumoricidal potential of KIL C.2, we used a C57BL/6 → BALB/c T-cell depleted (TCD)-WBM transplantation and A20-luciferase tumor model and monitored tumor progression using the Xenogen bioluminescence imaging system. BALB/c mice lethally irradiated and rescued with 2.5 × 107 TCD-WBM along with A20-luciferase inoculation uniformly died from tumor. Mice given TCD-WBM and 1 × 106 donor splenocytes did not develop tumor but died from lethal graft-versus-host disease (GVHD). In contrast, mice given TCD-WBM and two injections of KIL C.2 at doses of 1 × 107 per mouse were protected from tumor progression without developing signs of GVHD. These results show that KIL C.2 is a highly functional NK cell line with the capacity for proliferation, cytokine secretion, and cytotoxicity against syngeneic as well as allogeneic tumors. As such, KIL C.2 provides a useful cell line model for studies of NK cell biology and suggests a potentially effective strategy for NK cell-based immunotherapy.

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48

Wu, Xiuli, Xinyu Wang, Zhenyi Jin, Ling Xu, Shaohua Chen, Lijian Yang, Piotr Grabarczyk, Grzegorz K. Przybylski, Christian A. Schmidt, and Yangqiu Li. "Specific Gamma Delta T Cells for Cellular Immunotherapy of EBV-Associated Diseases after Allo-HSCT By T-Cell Receptor Gene Modification." Blood 124, no. 21 (December 6, 2014): 5811. http://dx.doi.org/10.1182/blood.v124.21.5811.5811.

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Abstract Epstein-Barr virus (EBV) infection may result in a spectrum of diseases in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially post-transplant lymphoproliferative disease (PTLD). Furthermore, the clinical course of EBV-associated diseases often superimposes with graft-versus-host disease (GVHD) and that increases the difficulty of treatment. Adoptive immunotherapy with EBV-specific cytotoxic T lymphocytes (EBV-CTL) is recommended as one of the first-line therapy for PTLD. Engineering of T cell receptor (TCR)-transferred T lymphocytes could be an attractive strategy to obtain sufficient T cells with an antigen specificity of choice. But TCR-modified alpha beta T cells are restricted by major histocompatibility (MHC) class I expression and low affinity. Because gamma delta T cells (GD T cells) recognize target cells independent of human leukocyte antigen (HLA), the alpha beta TCR engineering of GD T cells forms a feasible strategy to generate antigen-specific effector T cells without HLA restriction. And regulatory GD T cells (GD Tregs) (one of the novel subset of GD T cells), which express Foxp3 and primarily belong to CD27+CD25high phenotype, might have application prospect in the treatment of GVHD. In order to investigate a novel strategy with potential therapeutic benefits in EBV-associated diseases after allo-HSCT, we obtained the monoclonal EBV-CTL clone HLA-A*0201/GLCTLVAML from the PROIMMUNE company (U.K.) and detected peptide-specific CTL activity in chromium release assays, then identified the EBV-CTL associated - TCR Valpha 15 and TCR Vbeta 1 chain genes, and subsequently cloned into the eukaryotic expression vector pIRES to construct the recombinant plasmid; We transferred the EBV specific recombinant plasmid to GD T cells and GD Tregs (generated in vitro by stimulating with anti-TCR gamma delta in the presence of TGF-beta and IL-2). The TCR gene-transduced groups had special TCR Vbeta 1 expression in fluorescence detection, quantitative PCR and western Blot analysis. TCR gene-transduced GD T cells had significant cytotoxicity against the EBV+ target cell lines (including toledo cells (HLA-A2 positive) (38.17±0.86%) and daudi cells (HLA-A2 negative) (61.74±1.84%)) compared with empty vector transfected GD T cells (toledo cells (30.15±0.89%); daudi cells (53.91±4.33%)) (P<0.001, P=0.006). TCR gene-transduced GD Tregs also had significant cytotoxicity against toledo cells (56.21±5.34%) and daudi cells (68.41±2.30%) compared with empty vector transfected GD Tregs (toledo cells (48.47±0.35%); daudi cells (56.23±2.04%)) (P=0.012, P<0.001). Both TCR gene-transduced GD T cells and GD Tregs had high levels of IFN-gamma in culture supernatants. Moreover, TCR gene-transduced GD Tregs had high levels of IL-10 in culture supernatants. Interestingly, TCR-modified GD Tregs showed significant suppressive effect on autologous CD4+ responder cells in CCK8 detection and CFSE assays. In conclusion, GD T cells or GD Tregs co-transduced with TCR alpha beta genes (from EBV CTL clone) could acquire antiviral capacity. Immunomodulatory function of GD Tregs could remain after TCR gene transfection. TCR modified GD Tregs might be an alternative and attractive strategy with potential therapeutic benefits in EBV-associated diseases after allo-HSCT, especially with GVHD. Disclosures Wu: National Natural Science Foundation of China (No. 81200388), the Science and Technology Planning Project of Guangdong Province of China (No. 2012B031800403), the project of the Zhujiang Science & Technology Star of Guangzhou(No. 2013027): Research Funding. Li:National Natural Science Foundation of China (No. 81200388), the Science and Technology Planning Project of Guangdong Province of China (No. 2012B031800403), the project of the Zhujiang Science & Technology Star of Guangzhou(No. 2013027): Research Funding.

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49

Veerapathran, Anandharaman, Joseph Pidala, Francisca Beato, William E. Janssen, Xue-Zhong Yu, and Claudio Anasetti. "Tregs Specific for Minor Histocompatibility Antigens." Blood 120, no. 21 (November 16, 2012): 1891. http://dx.doi.org/10.1182/blood.v120.21.1891.1891.

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Abstract Abstract 1891 Background: The risk of acute GVHD after HSCT is increased in male recipients of female grafts. Disparities for the male-associated H-Y and other minor histocompatibility antigens (mHAs) have the capacity to sensitize alloreactive donor T cells and cause GVHD in HLA-matched recipients. These mHAs are polymorphic proteins that differ between donor and recipient and are presented as peptides by HLA molecules on recipient or donor antigen-presenting cells to donor immune cells. Currently, there is no evidence that minor histocompatibility antigen specific Tregs exist. Earlier in our laboratory, we have measured the frequency, growth requirements, and function of human blood Tregs specific for allo-MHC. In the present study, we sought to detect the frequency, expansion kinetics and characteristics of the minor antigens specific Tregs in the blood of HLA-matched sibling pair. Methods: CD4+CD25+CD127− Tregs were isolated by immunoabsorption from sibling donors, and cultured with HLA-matched sibling recipient antigen-presenting cells in the presence of IL-2, IL-15 and rapamycin. We detected 30–50 fold increase in H-TdR uptake at 6 days in Treg cultures stimulated by HLA-identical sibling compared to self DC. The precursor frequency of mHA-specific Tregs are between 7 and 43 (median - 13) cells per one million blood Tregs. The frequency of mHA-specific conventional CD4 T cells among total blood CD4 T cells is similar in HLA-matched sibling donors. Ex vivo expanded mHA-specific Tregs maintained higher levels of Foxp3 expression, retained the lymphoid homing receptor CD62L and a chemokine receptor, CCR7, suggesting that they are functional and are able to migrate to lymphoid tissue in vivo. Split well assay on day 12 demonstrated the mHA specificity, since Treg responded to restimulation with DC from the original HLA-identical sibling, but not self DC. The mHA-specific Tregs expanded to more than 100 fold in vitro, and exhibited antigen specific suppression. When Tregs were cultured at limiting dilution, we obtained 6 mHA-specific Treg clones that retained TGF-beta secretion in response to the sibling's mHA-disparate DC but not self DC. Conclusion: We demonstrated for the first time that it is possible to detect and expand mHA specific Tregs from HLA-matched sibling pairs, immunotherapy with mHA-specific Tregs may prevent GVHD. Disclosures: No relevant conflicts of interest to declare.

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Dehno, Mojgan Naghizadeh, Yutao Li, Hans Weiher, and Ingo G. H. Schmidt-Wolf. "Increase in Efficacy of Checkpoint Inhibition by Cytokine-Induced-Killer Cells as a Combination Immunotherapy for Renal Cancer." International Journal of Molecular Sciences 21, no. 9 (April 27, 2020): 3078. http://dx.doi.org/10.3390/ijms21093078.

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Cytokine-induced killer (CIK) cells are heterogeneous, major histocompatibility complex (MHC)-unrestricted T lymphocytes that have acquired the expression of several natural killer (NK) cell surface markers following the addition of interferon gamma (IFN-γ), OKT3 and interleukin-2 (IL-2). Treatment with CIK cells demonstrates a practical approach in cancer immunotherapy with limited, if any, graft versus host disease (GvHD) toxicity. CIK cells have been proposed and tested in many clinical trials in cancer patients by autologous, allogeneic or haploidentical administration. The possibility of combining them with specific monoclonal antibodies nivolumab and ipilimumab will further expand the possibility of their clinical utilization. Initially, phenotypic analysis was performed to explore CD3, CD4, CD56, PD-1 and CTLA-4 expression on CIK cells and PD-L1/PD-L2 expression on tumor cells. We further treated CIK cells with nivolumab and ipilimumab and measured the cytotoxicity of CIK cells cocultured to renal carcinoma cell lines, A-498 and Caki-2. We observed a significant decrease in viability of renal cell lines after treating with CIK cells (p < 0.0001) in comparison to untreated renal cell lines and anti-PD-1 or anti-CTLA-4 treatment had no remarkable effect on the viability of tumor cells. Using CCK-8, Precision Count Beads™ and Cell Trace™ violet proliferation assays, we proved significant increased proliferation of CIK cells in the presence of a combination of anti-PD-1 and anti-CTLA-4 antibodies compared to untreated CIK cells. The IFN-γ secretion increased significantly in the presence of A-498 and combinatorial blockade of PD-1 and CTLA-4 compared to nivolumab or ipilimumab monotreatment (p < 0.001). In conclusion, a combination of immune checkpoint inhibition with CIK cells augments cytotoxicity of CIK cells against renal cancer cells.

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