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Foley, Bree, Sarah Cooley, Michael R. Verneris, Michelle Pitt, Julie Curtsinger, Xianghua Luo, Sandra Lopez-Vergès, Lewis L. Lanier, Daniel Weisdorf i Jeffrey S. Miller. "Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function". Blood 119, nr 11 (15.03.2012): 2665–74. http://dx.doi.org/10.1182/blood-2011-10-386995.
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AbstractDuring mouse cytomegalovirus (CMV) infection, a population of Ly49H+ natural killer (NK) cells expands and is responsible for disease clearance through the induction of a “memory NK-cell response.” Whether similar events occur in human CMV infection is unknown. In the present study, we characterized the kinetics of the NK-cell response to CMV reactivation in human recipients after hematopoietic cell transplantation. During acute infection, NKG2C+ NK cells expanded and were potent producers of IFNγ. NKG2C+ NK cells predominately expressed killer cell immunoglobulin–like receptor, and self-killer cell immunoglobulin–like receptors were required for robust IFNγ production. During the first year after transplantation, CMV reactivation induced a more mature phenotype characterized by an increase in CD56dim NK cells. Strikingly, increased frequencies of NKG2C+ NK cells persisted and continued to increase in recipients who reactivated CMV, whereas these cells remained at low frequency in recipients without CMV reactivation. Persisting NKG2C+ NK cells lacked NKG2A, expressed CD158b, preferentially acquired CD57, and were potent producers of IFNγ during the first year after transplantation. Recipients who reactivated CMV also expressed higher amounts of IFNγ, T-bet, and IL-15Rα mRNA transcripts. Our findings support the emerging concept that CMV-induced innate memory-cell populations may contribute to malignant disease relapse protection and infectious disease control long after transplantation.
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Balogh, László, Krisztina Szabó, József Márton Pucsok, Ilona Jámbor, Ágnes Gyetvai, Marianna Mile, Lilla Barna i in. "The Effect of Aerobic Exercise and Low-Impact Pilates Workout on the Adaptive Immune System". Journal of Clinical Medicine 11, nr 22 (17.11.2022): 6814. http://dx.doi.org/10.3390/jcm11226814.
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Growing evidence indicates the pronounced effects of physical activity on immune functions, which may largely depend on the type of exercise, intensity, and duration. However, limited information is available regarding the effects of low-impact exercises, especially on the level of adaptive immune system. Our study aimed to investigate and compare the changes in a broad spectrum of lymphocyte subtypes after 14 weeks of aerobic-type total-body-shaping workouts (TBSW) and Pilates workouts (PW) among healthy individuals. We determined the percentages of peripheral natural killer cells and different T and B lymphocyte subtypes with flow cytometry. At the end of the exercise program, significant changes in naïve and memory lymphocyte ratios were observed in TBSW group. Percentages of naïve cytotoxic T (Tc) cells elevated, frequencies of memory Tc and T-helper cell subsets decreased, and distribution of naïve and memory B cells rearranged. Proportions of activated T cells also showed significant changes. Nonetheless, percentages of anti-inflammatory interleukin (IL)-10-producing regulatory type 1 cells and immunosuppressive CD4+CD127lo/−CD25bright T regulative cells decreased not only after TBSW but also after PW. Although weekly performed aerobic workouts may have a more pronounced impact on the adaptive immune system than low-impact exercises, both still affect immune regulation in healthy individuals.
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Lai, Dazhi, Jinfang Zhu, Tianhong Wang, Jane Hu-Li, Masaki Terabe, Jay A. Berzofsky, Carol Clayberger i Alan M. Krensky. "KLF13 sustains thymic memory-like CD8+ T cells in BALB/c mice by regulating IL-4–generating invariant natural killer T cells". Journal of Experimental Medicine 208, nr 5 (11.04.2011): 1093–103. http://dx.doi.org/10.1084/jem.20101527.
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“Memory-like T cells” are a subset of thymic cells that acquire effector function through the maturation process rather than interaction with specific antigen. Disruption of genes encoding T cell signaling proteins or transcription factors have provided insights into the differentiation of such cells. In this study, we show that in BALB/c, but not C57BL/6, mice, a large portion of thymic CD4-CD8+ T cells exhibit a memory-like phenotype. In BALB/c mice, IL-4 secreted by invariant natural killer T (iNKT) cells is both essential and sufficient for the generation of memory-like T cells. In C57BL/6 mice, iNKT cells are less abundant, producing IL-4 that is insufficient to induce thymic memory-like CD8+ T cells. BALB/c mice deficient in the transcription factor Kruppel-like factor (KLF) 13 have comparable numbers of iNKT cells to C57BL/6 mice and extremely low levels of thymic memory-like CD8+ T cells. This work documents the impact of a small number of KLF13-dependent iNKT cells on the generation of memory-like CD8+ T cells.
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Ndhlovu, Zaza M., Eleni Stampouloglou, Kevin Cesa, Orestes Mavrothalassitis, Donna Marie Alvino, Jonathan Z. Li, Shannon Wilton i in. "The Breadth of Expandable Memory CD8+T Cells Inversely Correlates with Residual Viral Loads in HIV Elite Controllers". Journal of Virology 89, nr 21 (12.08.2015): 10735–47. http://dx.doi.org/10.1128/jvi.01527-15.
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ABSTRACTPrevious studies have shown that elite controllers with minimal effector T cell responses harbor a low-frequency, readily expandable, highly functional, and broadly directed memory population. Here, we interrogated thein vivorelevance of this cell population by investigating whether the breadth of expandable memory responses is associated with the magnitude of residual viremia in individuals achieving durable suppression of HIV infection. HIV-specific memory CD8+T cells were expanded by using autologous epitopic and variant peptides. Viral load was measured by an ultrasensitive single-copy PCR assay. Following expansion, controllers showed a greater increase in the overall breadth of Gag responses than did untreated progressors (P= 0.01) as well as treated progressors (P= 0.0003). Nef- and Env-specific memory cells expanded poorly for all groups, and their expanded breadths were indistinguishable among groups (P= 0.9 for Nef as determined by a Kruskal-Wallis test;P= 0.6 for Env as determined by a Kruskal-Wallis test). More importantly, we show that the breadth of expandable, previously undetectable Gag-specific responses was inversely correlated with residual viral load (r= −0.6;P= 0.009). Together, these data reveal a direct link between the abundance of Gag-specific expandable memory responses and prolonged maintenance of low-level viremia. Our studies highlight a CD8+T cell feature that would be desirable in a vaccine-induced T cell response.IMPORTANCEMany studies have shown that the rare ability of some individuals to control HIV infection in the absence of antiretroviral therapy appears to be heavily dependent upon special HIV-specific killer T lymphocytes that are able to inhibit viral replication. The identification of key features of these immune cells has the potential to inform rational HIV vaccine design. This study shows that a special subset of killer lymphocytes, known as central memory CD8+T lymphocytes, is at least partially involved in the durable control of HIV replication. HIV controllers maintain a large proportion of Gag-specific expandable memory CD8+T cells involved in ongoing viral suppression. These data suggest that induction of this cell subset by future HIV vaccines may be important for narrowing possible routes of rapid escape from vaccine-induced CD8+T cell responses.
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Angelini, Daniela F., Giovanna Borsellino, Mary Poupot, Adamo Diamantini, Rémy Poupot, Giorgio Bernardi, Fabrizio Poccia, Jean-Jacques Fournié i Luca Battistini. "FcγRIII discriminates between 2 subsets of Vγ9Vδ2 effector cells with different responses and activation pathways". Blood 104, nr 6 (15.09.2004): 1801–7. http://dx.doi.org/10.1182/blood-2004-01-0331.
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Abstract Upon recognition of nonpeptidic phosphoantigens, human Vδ2 T lymphocytes enter a lineage differentiation pattern that determines the generation of memory cells with a range of effector functions. Here, we show that within the effector memory Vδ2 population, 2 distinct and complementary subsets with regard to phenotype, mode of activation, and type of responses can be identified: Vδ2 TEMh cells, which express high levels of chemokine receptors, but low levels of perforin and of natural killer receptors (NKRs) and which produce large amounts of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) in response to T-cell receptor (TCR)–specific stimulation by phosphoantigens; and Vδ2TEMRA cells, which constitutively express several NKRs, high amounts of perforin, but low levels of chemokine receptors and of IFN-γ. These NK-like cells are refractory to phosphoantigen but respond to activation via FcγRIII (CD16) and are highly active against tumoral target cells. Thus, circulating Vδ2T lymphocytes comprise 2 functionally diverse subsets of effector memory cells that may be discriminated on the basis of CD16 expression.
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Purton, Jared F., Joyce T. Tan, Mark P. Rubinstein, David M. Kim, Jonathan Sprent i Charles D. Surh. "Antiviral CD4+ memory T cells are IL-15 dependent". Journal of Experimental Medicine 204, nr 4 (9.04.2007): 951–61. http://dx.doi.org/10.1084/jem.20061805.
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Survival and intermittent proliferation of memory CD4+ and CD8+ T cells appear to be controlled by different homeostatic mechanisms. In particular, contact with interleukin (IL)-15 has a decisive influence on memory CD8+ cells, but not memory CD4+ cells. Past studies of memory CD4+ cells have relied heavily on the use of naturally occurring memory phenotype (MP) cells as a surrogate for antigen (Ag)-specific memory cells. However, we show here that MP CD4+ cells contain a prominent subset of rapidly proliferating major histocompatibility complex (MHC) II–dependent cells. In contrast, Ag-specific memory CD4 cells have a slow turnover rate and are MHC II independent. In irradiated hosts, these latter cells ignore IL-15 and expand in response to the elevated levels of IL-7 in the lymphopenic hosts. In contrast, in normal nonlymphopenic hosts where IL-7 levels are low, memory CD4 cells are heavily dependent on IL-15. Significantly, memory CD4+ responsiveness to endogenous IL-15 reflects marked competition from other cells, especially CD8+ and natural killer cells, and increases considerably after removal of these cells. Therefore, under normal physiological conditions, homeostasis of CD8+ and CD4+ memory cells is quite similar and involves IL-15 and IL-7.
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Rambaldi, Benedetta, Haesook T. Kim, Carol Reynolds, Sharmila Chamling Rai, Yohei Arihara, Tomohiro Kubo, Leutz Buon i in. "Impaired T- and NK-cell reconstitution after haploidentical HCT with posttransplant cyclophosphamide". Blood Advances 5, nr 2 (15.01.2021): 352–64. http://dx.doi.org/10.1182/bloodadvances.2020003005.
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Abstract Administration of posttransplant cyclophosphamide (PTCy) has significantly expanded the number of patients undergoing HLA-haploidentical hematopoietic cell transplantation (haplo-HCT). To examine immune reconstitution in these patients, we monitored T- and natural killer (NK)-cell recovery in 60 patients receiving bone marrow or peripheral blood stem cell (PBSC) grafts after haplo-HCT with PTCy and 35 patients receiving HLA-matched donor PBSC grafts with standard graft-versus-host disease (GVHD) prophylaxis. Compared with HLA-matched recipients, early T-cell recovery was delayed in haplo-HCT patients and skewed toward effector memory T cells with markedly reduced naive T cells. We found higher regulatory T (Treg)-cell/conventional T (Tcon)-cell ratios early after HCT and increased PD-1 expression on memory T cells. Within the haplo-HCT, patients who did not develop chronic GVHD (cGVHD) had higher PD-1 expression on central and effector memory CD4+ Treg cells at 1 month after transplant. These findings suggest an immunologic milieu that promotes immune tolerance in haplo-HCT patients. NK cells were decreased early after haplo-HCT with preferential expansion of immature CD56brightCD16− NK cells compared with matched donor transplants. One month after transplant, mass cytometry revealed enrichment of immature NK-cell metaclusters with high NKG2A, low CD57, and low killer-cell immunoglobulin-like receptor expression after haplo-HCT, which partially recovered 3 months post-HCT. At 2 months, immature NK cells from both groups were functionally impaired, but interleukin-15 priming corrected these defects in vitro. Increased immature/mature NK-cell ratios were associated with cytomegalovirus reactivation and increased incidence of cGVHD after haplo-HCT. These homeostatic imbalances in T- and NK-cell reconstitution after haplo-HCT reveal opportunities for early immune-based interventions to optimize clinical outcomes.
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Pfirrmann, Verena, Sarah Oelsner, Eva Rettinger, Sabine Huenecke, Jindrich Cinatl, Winfried Wels, Thomas Klingebiel i Peter Bader. "CMV-Specific Cytokine-Induced Killer Cells Comprise Concomitant Cytotoxicity Against Leukemia and Cytomegalovirus". Blood 124, nr 21 (6.12.2014): 5814. http://dx.doi.org/10.1182/blood.v124.21.5814.5814.
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Abstract Introduction Infection is one of the main causes of mortality and morbidity after allogeneic stem cell transplantation, especially in patients who received T cell depleted haploidentical stem cells. Reactivation or de novo infection of cytomegalovirus (CMV) is amongst the most frequent complications and occur due to a lack of virus-specific T cells post-transplant. Pre-emptive immunotherapy may support both reconstitution of viral specific responses on one hand and may prevent impending leukemic relapse on the other hand. Therefore we established a protocol to generate CMV-specific cytokine-induced killer cells (CIKpp65) with dual cytolytic function against CMV and AML. Protocol CIK cells were generated in vitro from peripheral blood mononuclear cells (PBMC) of CMV-seropositive healthy donors using IFN-γ, activating monoclonal anti-CD3 antibody (MAb), interleukin (IL)-2 and IL-15. An additional single stimulation with human CMVpp65 protein was adequate to increase the amount of cytotoxic CMV-specificcells within CIK cells up to 23%. In total the CMVpp65 stimulation resulted in up to 11.0-fold increased frequency of CMV-dextramer+CD8+cells after 15 days of expansion (n=12). Results Cytotoxicity Next we investigated cell-mediated cytotoxicity against leukemic cell lines THP-1 and K562, pp65 loaded cell line T2 and CMV-infected primary fibroblasts. CIK cell cytotoxicity is described as mediated by activating NK-cell receptor NKG2D. This receptor was blocked in order to determine the specific MHC-mediated cytotoxicity in experiments targeting pp65 loaded cells. The lysis of pp65 loaded cells by CIKpp65 cells was significant higher as compared to conventional CIK cells (effector to target cell ratio of 5:1, 39.9±21.6% to 13.6±10.6%, P<0.01). CIKpp65 cells also induced high cytotoxicity in infected fibroblasts (up to 55%, 10:1 E:T ratio). The anti-leukemic effect was retained in CIKpp65 cells. CIKpp65 cells revealed a mean cytotoxicity of 71.5%, 60.7% and 37.8% against THP-1 and 55.0%, 50.0%, 20.5% against K562 in 40:1, 20:1 and 5:1 E:T ratio, respectively. In contrast, the reactivity against allogeneic PBMC remained low (18% lysis, 40:1 E:T ratio) and allogeneic mock-infected fibroblasts were not lysed at all. This clearly indicates towards the low alloreactive potential of CIKpp65 cells. Phenotype Furthermore we characterized subpopulation and memory phenotype of CIKpp65 cells in detailed flow cytometric analyses and examined the cytokine secretion pattern by cytometric bead array. After expansion the population mainly consisted of a CD3+CD56- T cell (77.6±4.5%) and CD3+CD56+ T-NK cell phenotype (20.0±12.6%). The T-NK cells additionally co-expressed high amounts of CD8 cytotoxic antigen (63.8±16.8%). Interestingly, the T-NK cell compartment contained higher amounts CMV-specific CD8+ cells (mean 5.5%) than the T cell compartment (mean 1.3%). Expression of activating NKG2D and CD25 receptor was strongly positive in both cell fractions. Remarkably, almost 30% of T-NK cells expressed γδ+ T cell receptor, whereas T cells only expressed 4.5% of this receptor type. The cytotoxic T cells within the CIKpp65 cells consisted of a mixed naïve (CD45RA+CD62L+), central memory (CD45RO+CD62L+) and effector memory (CD45RO+CD62L-) phenotype, the cytotoxic T-NK cells mainly of effector memory and EMRA (CD45RA+CD62L-) phenotype. Cytokine secretion (granzyme B, IFN-γ, MIP-1α, TNF-α, Fas-L, IP-10, IL-10, IL-6 and IL-4) were measured during the expansion period and cytotoxic assays and resulting data confirmed the cytotoxic nature of the cells and indicated towards a mainly TH1 cell type character. Conclusion In conclusion CIKpp65 cells can easily be generated from donor PBMC and might represent advantage to conventional CIK cells. Our pre-clinical data demonstrate the concomitant cytotoxicity of generated cells against leukemia cells and CMV, as well as low alloreactivity and limited risk to induce GvHD. Therefore CIKpp65 cells may represent an effective tool for pre-emptive immunotherapy in patients which have both an apparent risk of CMV reactivation and leukemic relapse after allogeneic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.
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Anyanwu, Ebere, Andrew W. Campbell, Joseph Jones, John E. Ehiri i Akpan I. Akpan. "The Neurological Significance of Abnormal Natural Killer Cell Activity in Chronic Toxigenic Mold Exposures". Scientific World JOURNAL 3 (2003): 1128–37. http://dx.doi.org/10.1100/tsw.2003.98.
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Toxigenic mold activities produce metabolites that are either broad-spectrum antibiotics or mycotoxins that are cytotoxic. Indoor environmental exposure to these toxigenic molds leads to adverse health conditions with the main outcome measure of frequent neuroimmunologic and behavioral consequences. One of the immune system disorders found in patients presenting with toxigenic mold exposure is an abnormal natural killer cell activity. This paper presents an overview of the neurological significance of abnormal natural killer cell (NKC) activity in chronic toxigenic mold exposure. A comprehensive review of the literature was carried out to evaluate and assess the conditions under which the immune system could be dysfunctionally interfered with leading to abnormal NKC activity and the involvement of mycotoxins in these processes. The functions, mechanism, the factors that influence NKC activities, and the roles of mycotoxins in NKCs were cited wherever necessary. The major presentations are headache, general debilitating pains, nose bleeding, fevers with body temperatures up to 40�C (104�F), cough, memory loss, depression, mood swings, sleep disturbances, anxiety, chronic fatigue, vertigo/dizziness, and in some cases, seizures. Although sleep is commonly considered a restorative process that is important for the proper functioning of the immune system, it could be disturbed by mycotoxins. Most likely, mycotoxins exert some rigorous effects on the circadian rhythmic processes resulting in sleep deprivation to which an acute and transient increase in NKC activity is observed. Depression, psychological stress, tissue injuries, malignancies, carcinogenesis, chronic fatigue syndrome, and experimental allergic encephalomyelitis could be induced at very low physiological concentrations by mycotoxin-induced NKC activity. In the light of this review, it is concluded that chronic exposures to toxigenic mold could lead to abnormal NKC activity with a wide range of neurological consequences, some of which were headache, general debilitating pains, fever, cough, memory loss, depression, mood swings, sleep disturbances, anxiety, chronic fatigue, and seizures.
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Smith, Samantha L., Philippa R. Kennedy, Kevin B. Stacey, Jonathan D. Worboys, Annie Yarwood, Seungmae Seo, Everardo Hegewisch Solloa i in. "Diversity of peripheral blood human NK cells identified by single-cell RNA sequencing". Blood Advances 4, nr 7 (9.04.2020): 1388–406. http://dx.doi.org/10.1182/bloodadvances.2019000699.
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Abstract Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2–activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16−, CD56dimCD16+CD57−, and CD56dimCD16+CD57+. CD56dimCD16+CD57− cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon–responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.
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Chiu, Yen-Ling, Shih-Chen Chang i Jean-San Chia. "IL-7 Receptor Alpha Chain Expression Identifies Human CD8+ Memory T Cell Subsets with Distinct Gene Expression Profiles and TCR Repertoire Compositions". Journal of Immunology 204, nr 1_Supplement (1.05.2020): 81.11. http://dx.doi.org/10.4049/jimmunol.204.supp.81.11.
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Abstract Heterogeneity of CD8 T cell play crucial roles in cancer patients and reveals the co-evolution between tumor and immunity. While CD127 expression identifies memory precursor cells during acute infections, the role of CD127 expression in defining memory T cell heterogeneity is not investigated. Here, we analyzed the Vβ-CDR3 usage of human effector memory TEM and TEMRA cells accompanied by functional and gene expression patterns based on surface CD127 expression. We found that, CD127hi subsets are associated with higher level of TCR diversity regardless of their TEM or TEMRA phenotype. CD127lo populations are presented with relatively low TCR repertoire diversity. Moreover, there is a high correlation between CD127hi and CD127lo populations which imply possible differentiation tract between CD127hi and CD127lo T cells, but not between TEM and TEMRA cells. On the other hand, both TEM and TEMRA CD127lo populations showed higher expression of effector genes including perforin, granzyme B, granzyme H, and pan-inhibitory killer immunoglobulin-like receptors, exhibited lower phenotype plasticity upon TCR activation and cytokine-driven proliferation, when compared to their CD127hi counterparts. In other words, depending on the expression of CD127, T cells from either TEM or TEMRA are similar in their gene expression and functionality patterns. These results provide the first evidence that T cell repertoire and gene expression signature can be uncoupled based on distinct CD127-dependent phenotype expression patterns but are delicately balanced in the memory T cell pool.
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Bengsch, Bertram, Hans Christian Spangenberg, Nadine Kersting, Christoph Neumann-Haefelin, Elisabeth Panther, Fritz von Weizsäcker, Hubert E. Blum, Hanspeter Pircher i Robert Thimme. "Analysis of CD127 and KLRG1 Expression on Hepatitis C Virus-Specific CD8+ T Cells Reveals the Existence of Different Memory T-Cell Subsets in the Peripheral Blood and Liver". Journal of Virology 81, nr 2 (1.11.2006): 945–53. http://dx.doi.org/10.1128/jvi.01354-06.
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ABSTRACT The differentiation and functional status of virus-specific CD8+ T cells is significantly influenced by specific and ongoing antigen recognition. Importantly, the expression profiles of the interleukin-7 receptor alpha chain (CD127) and the killer cell lectin-like receptor G1 (KLRG1) have been shown to be differentially influenced by repetitive T-cell receptor interactions. Indeed, antigen-specific CD8+ T cells targeting persistent viruses (e.g., human immunodeficiency virus and Epstein-Barr virus) have been shown to have low CD127 and high KLRG1 expressions, while CD8+ T cells targeting resolved viral antigens (e.g., FLU) typically display high CD127 and low KLRG1 expressions. Here, we analyzed the surface phenotype and function of hepatitis C virus (HCV)-specific CD8+ T cells. Surprisingly, despite viral persistence, we found that a large fraction of peripheral HCV-specific CD8+ T cells were CD127+ and KLRG1− and had good proliferative capacities, thus resembling memory cells that usually develop following acute resolving infection. Intrahepatic virus-specific CD8+ T cells displayed significantly reduced levels of CD127 expression but similar levels of KLRG1 expression compared to the peripheral blood. These results extend previous studies that demonstrated central memory (CCR7+) and early-differentiated phenotypes of HCV-specific CD8+ T cells and suggest that insufficient stimulation of virus-specific CD8+ T cells by viral antigen may be responsible for this alteration in HCV-specific CD8+ T-cell differentiation during chronic HCV infection.
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Takahashi, Tsuyoshi, Sussan Dejbakhsh-Jones i Samuel Strober. "Analysis of CD161+ T Cells in Human Peripheral Blood." Blood 104, nr 11 (16.11.2004): 3235. http://dx.doi.org/10.1182/blood.v104.11.3235.3235.
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Abstract CD161 (NKR-P1) is a natural killer receptor of C-type lectin superfamily found on natural killer (NK) cells. A significant number of both human CD4+ and CD8+ T cells express CD161. However, little is known about the extended phenotype or function of CD161+ T cells. Here, we analyzed this population in normal human peripheral blood by immunofluorescent staining, and multi-color flow cytometric analysis (N=15). Amongst CD4+ T cells, a mean of 22% showed intermediate staining for CD161 (CD161int) and the remainder were CD161−. Almost all the CD4+CD161int TCRαβ+ T cells had the CD45RO+ memory phenotype, and did not express the CD16 or CD56 NK markers (<5%). CD4+CD161− T cells were a mixture of CD45RO+ and CD45RA+ cells. Invariant natural killer T (NKT) cells with the Vα24+/Vβ11+ TCR are known to express CD161, but CD4+CD161int T cells contained less than 1 % of these invariant NKT cells. After in vitro stimulation with αCD3 and αCD28 mAb, CD4+CD161int T cells produced larger amounts of both Th1 and Th2 type cytokines compared with CD4+CD161− T cells, especially IFN-γ, IL-4 and IL-10. However, there was no difference in the proliferative response between CD161− and CD161int CD4+ T cells, and CD4+CD161int T cells had no suppressor functions against autologous cell proliferation. In CD8+ T cells, there were two populations that were CD161 positive. One was CD161hi (mean; 11%) and another was CD161int (mean; 9%). Almost all CD161int and CD161hi CD8+ T cells had the CD45RO+ memory phenotype, and few expressed the Vα24+/Vβ11+ TCR NKT cell marker (<1%) or CD16 (<5%). CD8+CD161hi cells contained around 15% of CD56+ and CD8+CD161int cells contained around 5% of CD56+. CD8+CD161hi T cells had decreased expression of CD8β and 40% of CD8+CD161hi T cells expressed only CD8α. Conversely, most of the CD8α+CD8β− T cells in human peripheral blood expressed CD161. After in vitro stimulation with αCD3 and αCD28 mAb, CD8+CD161hi T cells secreted no IFN-γ, TNF-α or IL-2. Both CD8+CD161− and CD8+CD161int T cells produced all three cytokines. CD8+CD161hi T cells failed to proliferate after αCD3+αCD28 stimulation, though CD8+CD161− and CD8+CD161int T cells made vigorous proliferative responses. Thus, it appeared that CD8+CD161hi T cells were anergic. This anergic state could not be reversed by addition of IL-2. CD8+CD161+ T cells had no suppressor function against autologous cell proliferation nor cytotoxicity against the NK or NKT sensitive tumor cell lines, K562 and Jurkat. In conclusion, the CD161 marker can be used to identify subsets of CD4+ and CD8+ T cells that differ in their extended phenotypes and functions. In addition, we identified a unique subset of anergic CD8+CD161hi T cells that express a memory phenotype with a low level of CD8β.
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Riese, Peggy, Stephanie Trittel, Rishi D. Pathirana, Frank Klawonn, Rebecca J. Cox i Carlos A. Guzmán. "Responsiveness to Influenza Vaccination Correlates with NKG2C-Expression on NK Cells". Vaccines 8, nr 2 (5.06.2020): 281. http://dx.doi.org/10.3390/vaccines8020281.
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Influenza vaccination often results in a large percentage of low responders, especially in high-risk groups. As a first line of defense, natural killer (NK) cells play a crucial role in the fight against infections. However, their implication with regard to vaccine responsiveness is insufficiently assessed. Therefore, this study aimed at the validation of essential NK cell features potentially associated with differential vaccine responsiveness with a special focus on NKG2C- and/or CD57-expressing NK cells considered to harbor memory-like functions. To this end, 16 healthy volunteers were vaccinated with an adjuvanted pandemic influenza vaccine. Vaccine responders and low responders were classified according to their hemagglutination inhibition antibody titers. A majority of responders displayed enhanced frequencies of NKG2C-expressing NK cells 7- or 14-days post-vaccination as compared to low responders, whereas the expression of CD57 was not differentially modulated. The NK cell cytotoxic potential was found to be confined to CD56dimCD16+ NKG2C-expressing NK cells in the responders but not in the low responders, which was further confirmed by stochastic neighbor embedding analysis. The presented study is the first of its kind that ascribes CD56dimCD16+ NKG2C-expressing NK cells a crucial role in biasing adaptive immune responses upon influenza vaccination and suggests NKG2C as a potential biomarker in predicting pandemic influenza vaccine responsiveness.
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Capuano, Cristina, Chiara Pighi, Simone Battella, Davide De Federicis, Ricciarda Galandrini i Gabriella Palmieri. "Harnessing CD16-Mediated NK Cell Functions to Enhance Therapeutic Efficacy of Tumor-Targeting mAbs". Cancers 13, nr 10 (20.05.2021): 2500. http://dx.doi.org/10.3390/cancers13102500.
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Natural killer (NK) cells hold a pivotal role in tumor-targeting monoclonal antibody (mAb)-based activity due to the expression of CD16, the low-affinity receptor for IgG. Indeed, beyond exerting cytotoxic function, activated NK cells also produce an array of cytokines and chemokines, through which they interface with and potentiate adaptive immune responses. Thus, CD16-activated NK cells can concur to mAb-dependent “vaccinal effect”, i.e., the development of antigen-specific responses, which may be highly relevant in maintaining long-term protection of treated patients. On this basis, the review will focus on strategies aimed at potentiating NK cell-mediated antitumor functions in tumor-targeting mAb-based regimens, represented by (a) mAb manipulation strategies, aimed at augmenting recruitment and efficacy of NK cells, such as Fc-engineering, and the design of bi- or trispecific NK cell engagers and (b) the possible exploitation of memory NK cells, whose distinctive characteristics (enhanced responsiveness to CD16 engagement, longevity, and intrinsic resistance to the immunosuppressive microenvironment) may maximize therapeutic mAb antitumor efficacy.
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Zhong, RK, AD Donnenberg, J. Rubin i ED Ball. "Differential effect of 4-hydroperoxycyclophosphamide and antimyeloid monoclonal antibodies on T and natural killer cells during bone marrow purging". Blood 83, nr 8 (15.04.1994): 2345–51. http://dx.doi.org/10.1182/blood.v83.8.2345.2345.
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Abstract Autologous bone marrow (BM) transplantation after high dose therapy is widely used to treat acute leukemia, lymphoma, and selected solid tumors. In studies of BM purging with chemical agents, monoclonal antibodies (MoAbs), or other agents, the emphasis has been on the efficacy of tumor cell removal and sparing of hematopoietic progenitor cells. Two commonly used methods of BM purging for patients with acute myeloid leukemia have been the drug 4-hydroperoxycyclophosphamide (4- HC) and (MoAbs) directed to myeloid antigens such as CD14, CD15, and CD33. Although both methods of BM purging have potent activity against leukemia cells, 4-HC is also quite toxic to normal hematopoietic progenitor cells in the same concentrations that are used to deplete leukemia cells. To further characterize the cellular composition of BM after purging, we examined the effects of MoAbs plus complement and 4- HC on cells of the lymphoid lineage in the BM. 4-HC exerted a concentration-dependent cytotoxicity on clonogenic T lymphocytes, natural killer (NK) cells, and lymphokine (interleukin-2)-activated killer (LAK) cells, whereas the anti-CD14 and anti-CD15 MoAbs had little effect. At a concentration of 4-HC commonly used for BM purging (60 micrograms/mL), there were 4 to 5 logs of T-cell depletion and almost complete elimination of NK- and LAK-cell activity. In contrast, 4-HC at low concentrations (eg, 3 micrograms/mL) spared the majority of lymphoid cells suggesting that low concentration 4-HC combined with MoAb purging may be a desirable alternative to higher concentration 4- HC. These data indicate that purging with antimyeloid MoAbs, but not with 4-HC, spares the function of mature graft lymphocytes. Infusion of viable lymphocytes may be important for the transfer of immune memory against microbial and neoplastic antigens and may hasten immune reconstitution. In addition, mature graft lymphocytes may also be selectively activated and expanded in conjunction with interleukin-2 administration after BM transplantation.
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Zhong, RK, AD Donnenberg, J. Rubin i ED Ball. "Differential effect of 4-hydroperoxycyclophosphamide and antimyeloid monoclonal antibodies on T and natural killer cells during bone marrow purging". Blood 83, nr 8 (15.04.1994): 2345–51. http://dx.doi.org/10.1182/blood.v83.8.2345.bloodjournal8382345.
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Autologous bone marrow (BM) transplantation after high dose therapy is widely used to treat acute leukemia, lymphoma, and selected solid tumors. In studies of BM purging with chemical agents, monoclonal antibodies (MoAbs), or other agents, the emphasis has been on the efficacy of tumor cell removal and sparing of hematopoietic progenitor cells. Two commonly used methods of BM purging for patients with acute myeloid leukemia have been the drug 4-hydroperoxycyclophosphamide (4- HC) and (MoAbs) directed to myeloid antigens such as CD14, CD15, and CD33. Although both methods of BM purging have potent activity against leukemia cells, 4-HC is also quite toxic to normal hematopoietic progenitor cells in the same concentrations that are used to deplete leukemia cells. To further characterize the cellular composition of BM after purging, we examined the effects of MoAbs plus complement and 4- HC on cells of the lymphoid lineage in the BM. 4-HC exerted a concentration-dependent cytotoxicity on clonogenic T lymphocytes, natural killer (NK) cells, and lymphokine (interleukin-2)-activated killer (LAK) cells, whereas the anti-CD14 and anti-CD15 MoAbs had little effect. At a concentration of 4-HC commonly used for BM purging (60 micrograms/mL), there were 4 to 5 logs of T-cell depletion and almost complete elimination of NK- and LAK-cell activity. In contrast, 4-HC at low concentrations (eg, 3 micrograms/mL) spared the majority of lymphoid cells suggesting that low concentration 4-HC combined with MoAb purging may be a desirable alternative to higher concentration 4- HC. These data indicate that purging with antimyeloid MoAbs, but not with 4-HC, spares the function of mature graft lymphocytes. Infusion of viable lymphocytes may be important for the transfer of immune memory against microbial and neoplastic antigens and may hasten immune reconstitution. In addition, mature graft lymphocytes may also be selectively activated and expanded in conjunction with interleukin-2 administration after BM transplantation.
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Malard, Florent, Myriam Labopin, Patrice Chevallier, Thierry Guillaume, Alix Duquesne, Fanny Rialland, Sophie Derenne i in. "Larger number of invariant natural killer T cells in PBSC allografts correlates with improved GVHD-free and progression-free survival". Blood 127, nr 14 (7.04.2016): 1828–35. http://dx.doi.org/10.1182/blood-2015-12-688739.
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Abstract We studied the impact of a set of immune cells contained within granulocyte colony-stimulating factor–mobilized peripheral blood stem cell grafts (naïve and memory T-cell subsets, B cells, regulatory T cells, invariant natural killer T cells [iNKTs], NK cells, and dendritic cell subsets) in patients (n = 80) undergoing allogeneic stem cell transplantation (SCT), using the composite end point of graft-versus-host disease (GVHD)-free and progression-free survival (GPFS) as the primary end point. We observed that GPFS incidences in patients receiving iNKT doses above and below the median were 49% vs 22%, respectively (P = .007). In multivariate analysis, the iNKT dose was the only parameter with a significant impact on GPFS (hazard ratio = 0.48; 95% confidence interval, 0.27-0.85; P = .01). The incidences of severe grade III to IV acute GVHD and National Institutes of Health grade 2 to 3 chronic GVHD (12% and 16%, respectively) were low and associated with the use of antithymocyte globulin in 91% of patients. No difference in GVHD incidence was reported according to the iNKT dose. In conclusion, a higher dose of iNKTs within the graft is associated with an improved GPFS. These data may pave the way for prospective and active interventions aiming to manipulate the graft content to improve allo-SCT outcome.
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Kotwal, Anupam, Svetlana Bornschlegl, Michael Gustafson, Allan Dietz, Danae Anastasia Delivanis i Mabel M. Ryder. "Aggressive Thyroid Cancer is Associated With Suppressor Circulating Immunophenotype". Journal of the Endocrine Society 5, Supplement_1 (1.05.2021): A855—A856. http://dx.doi.org/10.1210/jendso/bvab048.1746.
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Abstract Introduction: The influence of the immune system on follicular cell-derived thyroid cancer behavior has been suggested by various studies but this information has not been comprehensively translated into a prediction for thyroid cancer prognosis. We aimed to identify circulating immune cell profiles associated with thyroid cancer prognosis. This information would be a significant advance in the field of thyroid cancer management. Methods: We performed a single-center prospective cohort study of 32 follicular-cell derived thyroid cancer patients enrolled at the time of initial or subsequent surgery. We performed peripheral blood multi-parameter flow cytometry and collected relevant clinicopathologic, biochemical and radiologic data. We classified patients based on the AJCC TNM cancer stage, American Thyroid Association (ATA) initial risk of recurrence, and status of disease during follow-up. Results: On initial immunophenotyping, patients with aggressive/advanced thyroid cancer (ATA high risk vs. intermediate/low risk; AJCC stage 3/4 vs. 1/2; recurrence or distant metastases vs. no evidence of disease during follow-up) demonstrated increased circulating monocytes and granulocytes but decreased all lymphocytes, T cells (specifically CD4+ and gamma delta) and natural killer (NK) T-like cells. Further immunophenotyping revealed that aggressive/advanced thyroid cancer had increased circulating CD4+CD45+RO effector memory but decreased CD4+CD45+RA central memory T cells and increased regulatory T cells. Stage 3/4 thyroid cancer patients additionally had increased circulating CD33+HLADR- myeloid-derived suppressor cells (MDSCs) as compared to stage1/2. Conclusions: Aggressive thyroid cancer at presentation (AJCC stage, ATA risk category) or during follow-up (distant metastases) is characterized by a circulating immunophenotype comprising of increased immune suppressor cells (regulatory T cells, MDSCs, effector memory T cells) but decreased immune activator cells (CD4+ T cells, gamma delta T cells, NK T-like cells, central memory T cells) as compared to less aggressive thyroid cancer. This circulating immunophenotype may serve as a biomarker for cancer prognosis and guide the selection and development of novel immunotherapies for advanced thyroid cancer.
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Greenberg, Steven A., Jack L. Pinkus, Sek Won Kong, Clare Baecher-Allan, Anthony A. Amato i David M. Dorfman. "Highly differentiated cytotoxic T cells in inclusion body myositis". Brain 142, nr 9 (20.07.2019): 2590–604. http://dx.doi.org/10.1093/brain/awz207.
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Abstract Inclusion body myositis is a late onset treatment-refractory autoimmune disease of skeletal muscle associated with a blood autoantibody (anti-cN1A), an HLA autoimmune haplotype, and muscle pathology characterized by cytotoxic CD8+ T cell destruction of myofibres. Here, we report on translational studies of inclusion body myositis patient muscle compared with a diverse set of other muscle disease samples. Using available microarray data on 411 muscle samples from patients with inclusion body myositis (n = 40), other muscle diseases (n = 265), and without neuromuscular disease (normal, n = 106), we identified a signature of T-cell cytotoxicity in inclusion body myositis muscle coupled with a signature of highly differentiated CD8 T-cell effector memory and terminally differentiated effector cells. Further, we examined killer cell lectin-like receptor G1 (KLRG1) as a marker of this population of cells, demonstrated the correlation of KLRG1 gene expression with lymphocyte cytotoxicity across 28 870 human tissue samples, and identified the presence of KLRG1 on pathogenic inclusion body myositis muscle invading T cells and an increase in KLRG1 expressing T cells in inclusion body myositis blood. We examined inclusion body myositis muscle T-cell proliferation by Ki67 immunohistochemistry demonstrating that diseased muscle-invading T cells are minimally or non-proliferative, in accordance with known properties of highly differentiated or terminally differentiated T cells. We found low expression of KLRG1 on infection-protective human lymphoid tissue central memory T cells and autoimmune-protective human blood regulatory T cells. Targeting highly differentiated cytotoxic T cells could be a favourable approach to treatment of inclusion body myositis.
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Fu, Yang-Xin, Guangming Huang, Yang Wang i David D. Chaplin. "B Lymphocytes Induce the Formation of Follicular Dendritic Cell Clusters in a Lymphotoxin α–dependent Fashion". Journal of Experimental Medicine 187, nr 7 (6.04.1998): 1009–18. http://dx.doi.org/10.1084/jem.187.7.1009.
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Lymphotoxin (LT)α is expressed by activated T cells, especially CD4+ T helper type 1 cells, and by activated B and natural killer cells, but the functions of this molecule in vivo are incompletely defined. We have previously shown that follicular dendritic cell (FDC) clusters and germinal centers (GCs) are absent from the peripheral lymphoid tissues of LTα-deficient (LTα−/−) mice. LTα−/− mice produce high levels of antigen-specific immunoglobulin (Ig)M, but very low levels of IgG after immunization with sheep red blood cells. We show here that LTα-expressing B cells are essential for the recovery of primary, secondary, and memory humoral immune responses in LTα−/− mice. It is not necessary for T cells to express LTα to support these immune functions. Importantly, LTα-expressing B cells alone are essential and sufficient for the formation of FDC clusters. Once these clusters are formed by LTα-expressing B cells, then LTα-deficient T cells can interact with B cells to generate GCs and productive class-switched antibody responses. Thus, B cells themselves provide an essential signal that induces and maintains the lymphoid microenvironment essential for GC formation and class-switched Ig responses.
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Jiang, Peiyao, Fangfang Yu, Xiaowei Xu, Yu Cai, Jun Yang, Yin Tong, Chongmei Huang i in. "Impact of Lymphocyte Subsets of Grafts on the Outcome of Haploidentical Peripheral Blood Stem Cell Transplantation". Cell Transplantation 32 (styczeń 2023): 096368972311570. http://dx.doi.org/10.1177/09636897231157054.
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The contribution of lymphocyte subset composition of the graft on the outcomes following haploidentical peripheral blood stem cell transplantation (haploPBSCT) is not fully elucidated. We retrospectively analyzed 314 patients with hematological malignancies who underwent haploPBSCT from 2016 to 2020 in our center. We obtained a cutoff value of CD3+ T cell dose (2.96 × 108/kg) that separated the risk of II–IV acute graft-versus-host disease (aGvHD) and divided patients into the low CD3+ T cell dose group (CD3+ low) and the high CD3+ T cell dose (CD3+ high) group. Significantly higher incidences of I-IV aGvHD, II–IV aGvHD, and III–IV aGvHD were identified in the CD3+ high group (50.8%, 19.8%, and 8.1% in the high group, 23.1%, 6.0%, and 0.9% in the low group, P < 0.0001, P = 0.002, and P = 0.02, respectively). We found that CD4+ T cell and its naïve and memory subpopulations of grafts had a significant impact on aGvHD ( P = 0.005, P = 0.018, and P = 0.044). Besides, we found an inferior reconstitution of natural killer (NK) cells in the CD3+ high group than in the low group within the first-year posttransplant (239 cells/μL vs 338 cells/μL, P = 0.0003). No differences in engraftment, chronic GvHD (cGvHD), relapse rate, transplant-related mortality (TRM), and overall survival (OS) were identified between the two groups. In conclusion, our study found that a high CD3+ T cell dose led to a high risk of aGvHD and inferior reconstitution of NK cells in the haploPBSCT setting. In the future, carefully manipulating the composition of lymphocyte subsets of grafts might reduce the risk of aGvHD and improve the transplant outcome.
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Hansen, Morten, Eva Wieckowski i Pawel Kalinski. "Ex vivo-induction of tumor-specific effector and memory T cells for adoptive T cell therapies using antigen-loaded DCs (TUM4P.910)". Journal of Immunology 192, nr 1_Supplement (1.05.2014): 138.11. http://dx.doi.org/10.4049/jimmunol.192.supp.138.11.
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Abstract Adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes (TILs), or blood-derived T cells genetically-modified to express specific T cell receptors have been used in adoptive cell therapies (ACT) of melanoma and hematologic malignancies. However, wider application of ACT has been limited by poor T cell infiltration of many cancers, terminal differentiation and limited proliferative and functional capacity of TILs from many lesions, or lack of defined tumor-rejection antigens. Here we show that these limitations can be circumvented by in vitro sensitization (IVS) of blood lymphocytes, using antigen-loaded dendritic cells (DCs) to generate CTLs specific against multiple tumor-specific epitopes. Type-1-polarized DCs (aDC1s) consistently induce high frequencies of functional tumor-specific CTLs from PBMCs of healthy donors and cancer patients. Such IVS-induced CTLs can be further expanded up to 1000-fold, in a single cycle of activation with anti-CD3, irradiated feeder cells and low-dose IL-2. Unexpectedly, presence of aDC1 supernatants during the rapid expansion phase improved not only the functional activity of the expanded CTLs, but also the frequencies of tumor-specific cells, their CD62L and IL-7R expression, and ability to survive and perform killer function in the absence of exogenous IL-2. The current data show a new strategy of generating high numbers of functional CTLs for ACT, with several important advantages compared to high-dose-IL-2 expanded TILs.
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McIver, Zachariah A., Andrew Grim, Nicolas Naguib i A. John Barrett. "Low Absolute CD4 Central Memory Count On Day 30 Is Associated with An Increased Incidence of Chronic Graft-Versus-Host Disease." Blood 114, nr 22 (20.11.2009): 516. http://dx.doi.org/10.1182/blood.v114.22.516.516.
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Abstract Abstract 516 Early lymphocyte recovery is an important determinant of hematopoietic stem cell transplant (SCT) outcome. We found that robust day 30 lymphocyte counts are associated with a decreased incidence of relapse, decreased rates of both acute and chronic graft-versus-host disease (a-GvHD, c-GvHD), and increased survival. Natural killer (NK) cells contribute to lymphocyte recovery and may be associated with a strong graft-versus-leukemia and anti-GvHD effect. However, the contribution of the recovery of specific T cell subsets to transplant outcome has not been fully explored. We studied 43 patients undergoing a myeloablative matched-sibling T-cell depleted or selectively depleted SCT for a variety of hematological malignancies including AML, ALL, CML, and MDS. Median age was 43 years (range, 13–68), and median CD34 cell dose was 6.1×106/kg (range, 3.1-10.1). Thirty-one patients developed a-GvHD (12 grade I, 18 grade II, 1 grade II). Twelve patients had c-GvHD (7 limited, 5 extensive). On day 30 after SCT a peripheral blood sample was collected on all patients and cryopreserved. In preparation for analysis, mononuclear cells from these samples were thawed and rested overnight. Flow cytometry was then performed on a BD FACS CantoII flow cytometer with multicolor fluorochrome antibodies to CD3, CD4, CD8, CD27, and CD45RO. Subsets were defined as follows: Naive (N) CD27+CD45RO-, Central Memory (CM) CD27+CD45RO+, Effector Memory (EM) CD27-CD45RO+, and Effectors (E) CD27-CD45RO-. Data was analyzed by BD FACSDiva software, and Kaplan-Meier survival statistical analysis was performed on the readouts. Median subset frequencies were CD3+: 254 /μL (range, 75-2085), CD4+ : 132 /μL (range, 3-501), CD8+ :101 /μL (range, 9-1361), CD4+ EM 49/μL (range, 2-252), CD4+CM 60/μL (range, 1-253), CD8+ EM: 50 cells/μL (range 2-977), CD8+ CM 28 /μL (range, 2-401), Naïve and Effector cells were minimally or non-detected in both CD4+ and CD8+ compartments. When T cell subset recovery was correlated with transplant outcomes (a-GVHD, c-GVHD, relapse and survival) one significant association was identified: low CD4+ CM counts correlated with a higher incidence of c-GvHD. Patients that had less than the median value of 60 CD4+ CM cells/μL had a significantly higher likelihood of developing c-GvHD (HR 4.28, p=0.039, see figure). Additionally, when considering degree of disease, low CD4+ CM counts were associated with the severest manifestations of c-GvHD (p=0.021). As a result, we conclude that low absolute concentrations of CD4+ CM cells on day 30 after SCT reflects a deficiency in regulatory mechanisms important in the control of alloreactivity, and may be used as a surrogate marker for individuals at risk of developing c-GvHD. Disclosures: No relevant conflicts of interest to declare.
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Minamitani, Takeharu, Teruhito Yasui, Yijie Ma, Hufeng Zhou, Daisuke Okuzaki, Chiau-Yuang Tsai, Shuhei Sakakibara, Benjamin E. Gewurz, Elliott Kieff i Hitoshi Kikutani. "Evasion of affinity-based selection in germinal centers by Epstein–Barr virus LMP2A". Proceedings of the National Academy of Sciences 112, nr 37 (24.08.2015): 11612–17. http://dx.doi.org/10.1073/pnas.1514484112.
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Epstein–Barr virus (EBV) infects germinal center (GC) B cells and establishes persistent infection in memory B cells. EBV-infected B cells can cause B-cell malignancies in humans with T- or natural killer-cell deficiency. We now find that EBV-encoded latent membrane protein 2A (LMP2A) mimics B-cell antigen receptor (BCR) signaling in murine GC B cells, causing altered humoral immune responses and autoimmune diseases. Investigation of the impact of LMP2A on B-cell differentiation in mice that conditionally express LMP2A in GC B cells or all B-lineage cells found LMP2A expression enhanced not only BCR signals but also plasma cell differentiation in vitro and in vivo. Conditional LMP2A expression in GC B cells resulted in preferential selection of low-affinity antibody-producing B cells despite apparently normal GC formation. GC B-cell–specific LMP2A expression led to systemic lupus erythematosus-like autoimmune phenotypes in an age-dependent manner. Epigenetic profiling of LMP2A B cells found increased H3K27ac and H3K4me1 signals at the zinc finger and bric-a-brac, tramtrack domain-containing protein 20 locus. We conclude that LMP2A reduces the stringency of GC B-cell selection and may contribute to persistent EBV infection and pathogenesis by providing GC B cells with excessive prosurvival effects.
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Jakubikova, Jana, Efstathios Kastritis, Danka Cholujova, Teru Hideshima, Ludmila M. Flores, Merav Leiba, Jacob P. Laubach i in. "Evaluation of Immune Profile in Patients with Multiple Myeloma Using Cytof Technology". Blood 124, nr 21 (6.12.2014): 3404. http://dx.doi.org/10.1182/blood.v124.21.3404.3404.
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Abstract Introduction: CyTOF (time-of-flight mass cytometry) is a novel high-dimensional technology which permits immunophenotyping and analysis of signaling in single cells. This approach enables simultaneous evaluation of up to 40 parameters using antibodies tagged with distinct elemental isotopes, by combining flow cytometry with atomic mass spectrometry. Since multiple myeloma (MM) is characterized by immune dysfunction, we used CyTOF technology to define the complex immune profile in MM patient bone marrow (BM) samples. Methods: We used 40 different markers to define various B, T, natural killer (NK) subsets, as well as cells of monocytic, granulocytic, erythroid and platelet lineages. Our preliminary data are results from 10 patients with MGUS/ smoldering MM (SMM); 10 newly diagnosed MM; 20 relapsed/refractory MM; and 15 WM patients (5 newly diagnosed and 10 receiving treatment) in comparison with age-matched healthy donors’ BM (HD). A significantly larger cohort of MM (N=150) and WM (N=50) patients is being similarly analyzed and will be presented. To evaluate phenotypic abnormalities in various B cell subsets, we used B lineage markers CD10, CD19, CD20, CD22, CD27, CD34, CD38, CD45, IgA, IgD, IgG and IgM to define B cells maturation stages from hematopoietic stem cells (HSC) to naïve to mature B lymphocytes (pro-B; pre-B-I; pre-B-II; immature B; and mature (naïve) B cells), as well as memory non-switched and memory switched B cells, plasmablasts, normal (CD138+CD38+CD19+CD45+) and clonal plasma cells (CD138+CD38+CD19-CD45-/low), which reside in the specific BM niche. Furthermore, natural killer (NK) subsets (such as NK and NKT cells) and T cells (such as memory CD4T, naive CD4T, memory CD8T, naive CD8T, T regulatory cells and Tg/d cells) were examined. High-dimensional data was obtained using CyTOF technology and analyzed by SPADE and viSNE software. Results: Our data showed significantly decreased HSC in patients with newly diagnosed and relapsed/refractory MM compared to HD (P<0.025). A significant increase in pre-B-I cells was detected in relapsed/refractory MM vs. MGUS/SMM (P<0.028), but the opposite trend was observed in the pre-B-II subpopulation (P<0.005). No differences in immature B cell populations were observed in different stages of MM. However, a significantly higher percentage of immature B cells was present in relapsed/refractory MM compared to HD (P=0.008), and transitional B cells were significantly decreased in newly diagnosed MM compared to HD (P<0.001). Moreover, memory B cells were significant decreased in all MM stages compared to HD (P<0.003). Non-switched memory B cells were significantly increased in MGUS and SMM compared to newly diagnosed MM, while a significant increase of switched memory B cells was present in newly diagnosed MM compared to relapsed/refractory MM. A significant increase in plasmablasts was seen in relapsed/refractory MM in comparison with other MM stages (P<0.011) by CyTOF analyses. Malignant plasma cells (PC) were defined as CD19-, CD38++, CD45-/dim, CD138+ and either cyk or cyl positive. Importantly, a significant increase in clonal PC was found in all MM stages vs. HD, as well as in newly diagnosed MM compared to relapsed/refractory MM (P<0.01). The percentage of PC from CyTOF analyses correlated with % of PC obtained using flow cytometry by Bland-Altman method comparison. We also observed significant differences in T cell subsets including naïve, central memory, effector, and effector memory CD4+ and CD8+T populations between MGUS and newly diagnosed MM, but no significant changes in T regulatory and Tg/d cells. Furthermore, plasmacytoid dendritic (pDC) cells were significantly increased in newly diagnosed MM, and PD-1 expressed on pDC was significantly decreased in newly diagnosed MM compared to MGUS (P=0.007). Interestingly, PD-1 and its ligand PD-L1 were variably expressed on B cells (2-9% and 3-27%) and PC (0.5-46% and 3-41%) from MM BM samples. Other surface molecules including CD269 (4-32%), CD289 (1-8%), CD362 (0.5-1%) and CD329 (1-4%), were variably expressed in PC. Conclusion: A better understanding of the neoplastic BM milieu will provide the framework for identifying and validating novel targeted therapies directed against MM. CyTOF technology represents a novel diagnostic tool to assess the status not only of MM, but also of host immunity, and may allow for the development of rational personalized therapies. Disclosures No relevant conflicts of interest to declare.
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Xiao, Zheng, Kechao Nie, Tong Han, Lin Cheng, Zheyu Zhang, Weijun Peng i Dazun Shi. "Development and Validation of a TNF Family-Based Signature for Predicting Prognosis, Tumor Immune Characteristics, and Immunotherapy Response in Colorectal Cancer Patients". Journal of Immunology Research 2021 (9.09.2021): 1–16. http://dx.doi.org/10.1155/2021/6439975.
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In this study, a comprehensive analysis of TNF family members in colorectal cancer (CRC) was conducted and a TNF family-based signature (TFS) was generated to predict prognosis and immunotherapy response. Using the expression data of 516 CRC patients from The Cancer Genome Atlas (TCGA) database, TNF family members were screened to construct a TFS by using the univariate Cox proportional hazards regression and the least absolute shrinkage and selection operator- (LASSO-) Cox proportional hazards regression method. The TFS was then validated in a meta-Gene Expression Omnibus (GEO) cohort ( n = 1162 ) from the GEO database. Additionally, the tumor immune characteristics and predicted responses to immune checkpoint blockade in TFS-based risk subgroups were analyzed. Eight genes (TNFRSF11A, TNFRSF10C, TNFRSF10B, TNFSF11, TNFRSF25, TNFRSF19, LTBR, and NGFR) were used to construct the TFS. Compared to the high-risk patients, the low-risk patients had better overall survival, which was verified by the GEO data. In addition, a high TFS risk score was associated with high infiltration of regulatory T cells (Tregs), nonactivated macrophages (M0), natural killer cells, immune escape phenotypes, poor immunotherapy response, and tumorigenic and metastasis-related pathways. Conversely, a low TFS risk score was related to high infiltration of resting CD4 memory T cells and resting dendritic cells, few immune escape phenotypes, and high sensitivity to immunotherapy. Thus, the eight gene-based TFS is a promising index to predict the prognosis, immune characteristics, and immunotherapy response in CRC, and our results also provide new understanding of the role of the TNF family members in the prognosis and treatment of CRC.
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Yang, H. Y., P. L. Dundon, S. R. Nahill i R. M. Welsh. "Virus-induced polyclonal cytotoxic T lymphocyte stimulation." Journal of Immunology 142, nr 5 (1.03.1989): 1710–18. http://dx.doi.org/10.4049/jimmunol.142.5.1710.
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Abstract Infections with a variety of viruses (lymphocytic choriomeningitis (LCMV), murine cytomegalovirus, Pichinde virus, vaccinia virus) stimulated C57BL/6 mice to generate allospecific CTL coincidental with the generation of virus-specific CTL. In C57BL/6 (H-2b) mice, LCMV-induced CTL with reactivity against cells from mice bearing gene products of the d, f, k, p, q, and s but not the b MHC loci. Studies with congenic mouse strains indicated that the MHC loci coded for the target of the allospecific killer cells. The targets of the allospecific CTL were further identified as class I MHC Ag by three criteria: 1) target cells from congenic strains of mice differing from effector cells only in the expression of class I Ag were sensitive to lysis; 2) fibroblasts expressing low levels of class I Ag were resistant to lysis but were rendered sensitive after treatment with IFN-beta, which induced higher expression of class I Ag; and 3) antibody specific for class I Ag expressed on the target cell blocked killing. Studies with congenic mouse strains also suggested that the ability to generate high levels of the virus-induced allospecific killer cells was also under MHC regulation, as H-2b mice generated high levels and H-2k mice low levels of the allospecific CTL. Both C3H/St and C57BL/6 mice immunized against LCMV developed detectable LCMV-specific CTL when later challenged with either murine cytomegalovirus, Pichinde virus, or vaccinia virus, indicating that a virus infection can stimulate the reappearance of memory CTL. Cold target competition studies indicated no cross-reactivities between these viruses or allogeneic cells at the CTL level. Both the allospecific CTL and the reactivated LCMV-specific CTL were found in blast-size lymphocyte preparations. Spleen cells taken from LCMV-infected C57BL/6 mice 5 days post-infection spontaneously generated into allospecific and virus-specific CTL after 2 days of culture. The generation of both was dependent on the presence of supernatant factors produced only in the presence of L3T4+ cells. These factors activated allospecific CTL in spleen cells from virus-primed mice but not from control mice. We suggest that lymphokines produced as a consequence of virus infection may act to stimulate the proliferation and activation of CTL not specific to the challenge virus, resulting in a virus-induced polyclonal CTL stimulation.
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Chen, Jun-Yu, Ya-Ru Sun, Tao Xiong, Guan-Nan Wang i Qing Chang. "Identification of HIBCH as a Fatty Acid Metabolism-Related Biomarker in Aortic Valve Calcification Using Bioinformatics". Oxidative Medicine and Cellular Longevity 2022 (7.10.2022): 1–24. http://dx.doi.org/10.1155/2022/9558713.
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Objective. To identify fatty acid metabolism-related biomarkers of aortic valve calcification (AVC) using bioinformatics and to research the role of immune cell infiltration for AVC. Methods. The AVC dataset was retrieved from the Gene Expression Omnibus database. R package is used for differential expression genes analysis and weighted gene coexpression analysis. The differentially coexpressed genes were identified by the Venn diagram, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of differentially coexpressed genes. Functions closely related to AVC were identified by GO and KEGG enrichment analyses of differentially coexpressed genes. Genes related to fatty acid metabolism were retrieved from the Molecular Signatures Database (MSigDB) database. After removing duplicate genes, least absolute shrinkage and selection operator (LASSO) regression analysis, support vector machine recursive feature elimination (SVM-RFE), and random forest were applied to recognize biomarkers related to fatty acid metabolism in AVC. The CIBERSORT tool was used to analyze infiltration of immune cells in normal and AVC samples. Correlations between biomarkers and immune cells were calculated. Finally, HIBCH-related pathway was predicted by single-gene gene set enrichment analysis (GSEA). Results. 2416 differentially expressed genes and one coexpression module were identified. A total of 1473 differentially coexpressed genes were acquired. GO and KEGG enrichment analyses demonstrated that differentially coexpressed genes were closely related to fatty acid metabolism. LASSO regression analysis, SVM-REF, and random forest revealed that 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) was a biomarker of fatty acid metabolism-related genes in AVC. Significant high levels of memory B cells were found in AVC than normal samples, while activated natural killer (NK) cells were significantly low in AVC than normal samples. A significantly positive relevance was observed between HIBCH and activated NK cells, regulatory T cells, monocytes, naïve B cells, activated dendritic cells, resting memory CD4 T cells, resting NK cells, and CD8 T cells. A significantly negative relevance was observed between HIBCH and activated memory CD4 T cells, memory B cells, neutrophils, gamma delta T cells, M0 macrophages, and plasma cells. The single-gene GSEA results suggest that HIBCH may work through the inhibition of multiple immune-related pathways. Conclusion. HIBCH is closely relevant to immune cell infiltration in AVC and could be applied as a diagnostic marker for AVC.
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Jacobs, Miriam T., Pamela Wong, Alice Zhou, Michelle Becker-Hapak, Nancy Marin, Lynne Marsala, Mark Foster i in. "Abstract 2906: Memory-like differentiation, tumor targeting monoclonal antibodies, and chimeric antigen receptors enhance natural killer cell responses to head and neck cancer". Cancer Research 83, nr 7_Supplement (4.04.2023): 2906. http://dx.doi.org/10.1158/1538-7445.am2023-2906.
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Abstract Background: Head and neck cancer (HNC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers, but are frequently dysfunctional in patients with HNC. New strategies are needed to enhance NK cell responses against HNC. Activation of NK cells with IL-12, 15 and 18 results in memory-like (ML) differentiation improving NK cell responses in hematological malignancies. We hypothesized that ML NK cell differentiation, tumor targeting with cetuximab, and engineering with α-ephA2 chimeric antigen receptor (CAR) would enhance NK cell responses against HNC. Methods: ML NK and conventional (c) NK cells were generated from healthy donors (HD) and evaluated for their ability to produce IFNγ, TNF, degranulate (CD107a) and kill HNC cell lines and primary HNC cells alone or in combination with cetuximab in vitro and in xenograft models. To identify activating receptors involved in NK cell recognition of HNC, blocking experiments were used. ML and cNK cells were engineered to express α-EphA2 CAR-CD8A-41BB-CD3z and functional responses were assessed in vitro against HNC cell lines and primary HNC targets. Results: ML NK cells generated from HD displayed enhanced IFNγ response compared to cNK cells against HNC cell lines SCC1(11±2% vs. 24±1%, mean ± SEM p<0.001), SCC47 (13±2% vs. 36±2%, p<0.01) and primary HNC targets (17±2% vs. 32±3%, p<0.01) which were further enhanced by cetuximab, SCC1 (cNK + cetuximab vs. ML + cetuximab 16±2% vs. 28±3%, p<0.001), SCC47 (39±3% vs. 56±2%, p<0.001), primary HNC targets (23±2% vs. 40±2%, p<0.0001). The combination of ML NK cells and cetuximab was superior to cNK and cetuximab in 51 Cr release assay and IncuCyte® killing assays for HNC cell lines (p<0.01) and primary cells (p<0.01). Findings were consistent in SCC1 HNC xenograft model, where tumor bearing NSG mice treated with cetuximab and ML NK cells had significantly lower tumor burden at 21 days compared to untreated mice, mice that received cNK cells + cetuximab or cetuximab only (p<0.001). Mechanistically, NK cell IFNγ, CD107a and cytotoxicity against HNC targets is partially dependent on activating receptors NKG2D, CD2 and DNAM-1. ML NK cells expressing α-ephA2 CAR were specific to ephA2+ cell lines and demonstrated increased IFNγ (37±4% vs. 16±3%, p<0.001) and CD107a (36±4% vs. 20±1%, p<0.001) against NK resistant ephA2+ UD-SCC2, compared to CAR negative cells. EphA2 CAR ML NK cells had increased CD107a (26±4% vs. 39±4%, p<0.01) and IFNγ (45±5% vs. 66±3%, p<0.001) against primary ephA2+ HNC targets. Conclusion: These pre-clinical findings show that ML differentiation alone or in concert with cetuximab directed targeting or ephA2 CAR engineering, was effective against HNC and provide the rationale for investigation in early phase clinical trials for HNC patients. Citation Format: Miriam T. Jacobs, Pamela Wong, Alice Zhou, Michelle Becker-Hapak, Nancy Marin, Lynne Marsala, Mark Foster, Jennifer Foltz, Celia Cubitt, Jennifer Tran, Carly Neal, David Russler-Germain, Lily Chang, Timothy Schappe, Ethan McClain, Samantha Kersting-Schadek, Carl DeSelm, Melissa Berrien-Elliott, Sidarth V. Puram, Todd A. Fehniger. Memory-like differentiation, tumor targeting monoclonal antibodies, and chimeric antigen receptors enhance natural killer cell responses to head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2906.
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Romee, Rizwan, Rosario Maximillian, Melissa M. Berrien-Elliott, Julia A. Wagner, Brea A. Jewell, Timothy Schappe, Jeffrey W. Leong, Stephanie E. Schneider, Sarah Willey i Todd A. Fehniger. "Human Cytokine-Induced Memory-like NK Cells Exhibit in Vivo Anti-Leukemia Activity in Xenografted NSG Mice and in Patients with Acute Myeloid Leukemia (AML)". Blood 126, nr 23 (3.12.2015): 101. http://dx.doi.org/10.1182/blood.v126.23.101.101.
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Abstract Natural killer (NK) cells mediate anti-AML responses and previously published clinical trials of adoptive allogeneic NK cell therapy provide proof-of-principle that NK cells may eliminate leukemia cells in patients. However, complete remissions occur in 30-50% of patients with active AML and are typically of limited duration. Thus, improvements are needed for this promising cellular immunotherapy strategy. Following paradigm-shifting studies in mice, it was established that human NK cells exhibit an innate 'memory-like' responses following a brief, combined pre-activation with IL-12, -15, and -18 (Romee R et. al., Blood, 2012). These long-lived memory-like NK cells have an enhanced ability to produce IFN-g in response to restimulation with cytokines or activating receptor ligation, even following extensive proliferation. We hypothesized that memory-like NK cells exhibit enhanced responses to myeloid leukemia. Compared to control NK cells from the same donor, IL-12/15/18-induced memory-like NK cells produced significantly increased IFN-g upon co-culture with primary AML blasts in vitro (P<0.001), following 7 days of rest in low dose IL-15 vitro. In addition, memory-like NK cells had increased granzyme B expression (P<0.01), and enhanced killing of K562 leukemia targets in vitro (P<0.05). Utilizing an in vivo xenograft model of human NK cells in NSG mice (Leong J et. al., BBMT, 2014), IL-12/15/18-induced memory-like NK cells that differentiated in NSG mice for 7 days exhibited increased IFN-g responses after ex vivo re-stimulation with K562 leukemia, confirming their memory-like functionality (P<0.05). To test in vivo responses to human leukemia in this model, luciferase-expressing K562 cells were engrafted into NSG mice (1x106/mouse, IV), and on day 3, groups of mice were injected with IL-12/15/18-pre-activated or control NK cells from the same donor (4x106/mouse). Mice treated with a single dose of memory-like NK cells exhibited significantly improved in vivo leukemia control measured by whole mouse bioluminescent imaging (P=0.03), as well as overall survival (P<0.05), compared to mice treated with control or no NK cells. Based on these pre-clinical findings, we initiated a first-in-human clinical trial of HLA-haploidentical IL-12/15/18-induced memory-like NK cells in patients with AML (NCT01898793). Relapsed/refractory (rel/ref) AML patients receive lymphodepleting non-myeloablative flu/cy conditioning, infusion of a single dose of CD56+CD3- memory-like donor NK cells, followed by two weeks of low dose rhIL-2. Three patients were treated at dose level 1 (0.5x106 cells/kg) and two patients treated at dose level 2 (1.0x106/kg) with no DLTs observed, and accrual continues. Correlative analyses utilizing donor-specific HLA mAbs allow tracking of donor memory-like NK cell frequency and function following adoptive transfer. Donor memory-like NK cells were detectable in the PB and BM of all tested patients with informative HLA (4/5), peak in frequency at 7-8 days post-infusion, and contract after 14-21 days as expected following recipient T cell recovery (Figure). Memory-like NK cells exhibit significantly increased Ki67%+ as a marker of proliferation at day 7 [97.8+1.0% (donor) vs. 21.6+5.5% (recipient), mean+SEM, P<0.001]. Moreover, functional analyses of NK cells at days 7-8 post-infusion reveal increased numbers of donor IFN-g+ NK cells following restimulation with K562 leukemia cells in the same blood [1009+590 (donor) vs. 8+3 (recipient) IFN-g+ NK cells] or BM [686+423 (donor) vs. 4+2 (recipient) IFN-g+ NK cells] samples. Two of four evaluable patients treated with memory-like NK cells had leukemia free BM and PB at days 14 post-therapy, which correlated with BM NK cell frequency and IFN-g production (Figure). CIML007 had rel/ref AML with 48% BM blasts pre-therapy, and had no evidence of leukemia on day 14, 28, and 100 BM biopsies, and has an ongoing complete remission more than 100 days after this therapy. CIML009 had 80% BM blasts pre-therapy, and had no evidence of leukemia on day 14 BM biopsy post-infusion. Thus, human IL-12/15/18-induced memory-like NK cells expand and have enhanced anti-AML function following adoptive transfer in patients, thereby constituting a promising translational innovation for immunotherapy of AML. Figure 1. Figure 1. Disclosures Fehniger: Celgene: Research Funding.
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Chenchik, Alex, Mikhail Makhanov, Andrey Komarov, Sunitha Sastry i Costa Frangou. "CancerNet: Molecular profiling of tumor microenvironment for biomarker discovery". Journal of Immunology 196, nr 1_Supplement (1.05.2016): 211.18. http://dx.doi.org/10.4049/jimmunol.196.supp.211.18.
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Abstract Human carcinomas consist of a complex mixture of neoplastic epithelial cells, endothelial cells, fibroblasts, myofibroblasts, and immune cells, which collectively form the tumor stroma. These cells are implicated in disease progression, metastasis and drug resistance. The clinical importance of tumor immune infiltrates has been an emerging area in triple negative breast cancer (TNBC) research where increased immune infiltrate predicts both response to chemotherapy and improved survival. Quantitative molecular profiles of tumor-associated normal cells may provide important insights into tumor biology and facilitate the discovery of new biomarkers and therapeutic targets. To this end, we have developed CancerNet, a comprehensive targeted RNA-Seq cancer immuno-panel that profiles ~2,000 genes and distinguishes 37 human hematopoietic cell phenotypes, (including naïve and memory B cells, seven T-cell types, dendritic cells, plasma cells, natural killer (NK) cells and myeloid subsets), genes involved in checkpoint blockade and immunotherapy biomarkers, immune cell activation and canonical immune pathway genes for both innate adaptive and humoral immune responses. In this study, we present an assay that characterizes the cellular composition of the immune/stromal/cancer cell tumor microenvironment and determines the activation status of infiltrating immune cells in primary tumor tissues from TNBC patients. Preliminary studies demonstrate the assay’s unparalleled specificity and sensitivity resulting in better detection of low abundance mRNA transcripts as well as an improved cost-effectiveness for high-throughput clinical Next-Gen applications.
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Ueda, Ryosuke, Kenta Narumi, Reina Miyakawa, Hisayoshi Hashimoto i Kazunori Aoki. "Natural Killer Cell-Mediated Antitumor Effect of Syngeneic Hematopoietic Stem Cell Transplantation". Blood 124, nr 21 (6.12.2014): 1104. http://dx.doi.org/10.1182/blood.v124.21.1104.1104.
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Abstract Introduction: Autologous hematopoietic stem cell transplantation (HSCT) has been thought to be important in tumor eradication induced by high-dose chemotherapy with hematopoietic stem cell rescue. It was reported recently that autologous HSCT can also induce a strong antitumor immunity by homeostatic proliferation (HP) of T cells following preconditioning-induced lymphopenia. It is known that T cell HP is driven by the recognition of self antigens, and that the availability of tumor antigens during HP leads to effective antitumor autoimmunity with specificity and memory. The effect is mediated by a reduction in the activation threshold of low-affinity tumor-specific T cells, leading to their preferential engagement and expansion. However, the mechanisms of antitumor immunity induced by the autologous HSCT are not fully understood. Especially, in addition to adaptive immunity, the role of innate immunity is unclear. In this study, we examined the role of natural killer (NK) cells on the antitumor effect in HSCT recipients using mouse xenograft models. Furthermore, since neutrophils have been reported to be required for homeostasis, maturation, and function of NK cells in human and mice (Jaeger et al. J. Eep. Med. 209:565, 2012), we focused on the interaction between neutrophils and NK cells in tumors after syngeneic HSCT. Results: BALB/c mice were injected subcutaneously with CT26 murine colon cancer cells shortly after lethal irradiation (9Gy), and then bone marrow cells (5 x 106 cells) and splenocytes (4 x 106 cells) from syngeneic donor mice were infused into the recipient mice. Tumor growth was significantly suppressed in the HSCT recipients as previously reported (Narumi et al. Gene Ther 19:34, 2012, Udagawa et al. J. Immunol 191:3440, 2013). To clarify the contribution of NK cells to antitumor effects after syngeneic HSCT, we depleted NK cells by intraperitoneal injection of anti-asialo GM1 antibody in the HSCT recipients. The tumor growth inhibition was largely cancelled by the depletion of NK cells especially during an early period after HSCT (Figure 1a: 2303 mm3 vs. 1383 mm3 at day26; P = 0.0042). Flow cytometry showed that the frequency of surface CD107a+ NK cells, which is an activation marker, was significantly higher in tumors of HSCT recipients (24.4% vs. 6.3%; P = 0.0022) than that in non-HSCT mice, indicating that syngeneic HSCT induces NK cell activation which contributes to the antitumor effect. Next, we examined what factor influences the activation of NK cells in tumors after syngeneic HSCT. We found that a large number of neutrophils accumulated in tumors after HSCT by immunostaining. Thus, we focused on the relationship between neutrophils and NK cells in tumors after HSCT. The cytokine antibody array showed that lungkine (CXCL15), which is one of mouse ELR+ CXC chemokine and recruits neutrophils to lung airspaces during pneumonia, was markedly up-regulated in tumor at day14 after HSCT as compared with non-HSCT tumor. The real-time quantitative polymerase chain reaction confirmed that the lungkine mRNA level was higher in HSCT-tumor at day 6 than that in non-HSCT tumor (Figure 1b: relative expression: 4.0 vs. 0.3; P = 0.0075). Then, to investigate the effects of neutrophils on NK cells in tumors, we depleted neutrophils by an intraperitoneal injection of anti-Ly6G antibody from days 6 to 12 after syngeneic HSCT. Neutrophils were almost depleted in the spleens and tumors. The tumor growth was significantly suppressed in HSCT recipients as previously shown, whereas the depletion of neutrophil significantly increased tumor volumes in HSCT recipients but not in non-HSCT mice at day 10 (Figure 1c: 143 mm3 vs. 84 mm3; P = 0.024). Flow cytometry revealed that the neutrophil-depletion did not change the frequency of NK cells within live cells in tumors at days 14 and 21, however, neutrophil-depletion increased the fraction of dead NK cells at day 14 (Figure 1d: 32.2% vs. 19.2%; P = 0.042). Thus, neutrophils in tumors may prevent NK cells from cell death induction during HP, leading to the significant antitumor effect. Conclusions: NK cells play a major role in the antitumor effect in the early period after syngeneic HSCT. The neutrophils in tumors may support a sustained antitumor effect of NK cells. This novel relationship reveals an important aspect of antitumor immunity induction in HSCT recipients and may contribute to the development of effective therapeutic strategy for cancer using HSCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Phillips, Kate L. E., Joanne E. Dean, Brian F. Flanagan, Steve E. Christmas i Russell D. Keenan. "Potential for Dendritic Cell Stimulated De Novo Anti-Leukaemia CD4 T Cell Response in the Bone Marrow Tumour Microenvironment during Induction Chemotherapy". Blood 128, nr 22 (2.12.2016): 2524. http://dx.doi.org/10.1182/blood.v128.22.2524.2524.
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Abstract In acute lymphoblastic leukaemia (ALL), T cell anergy induced by tumour antigen presentation without co-stimulation contributes to immunological escape and disease progression. Cross priming of T cells by dendritic cells (DCs) may overcome this escape mechanism, restoring T cell activation and promoting a successful anti-tumour immune response. Remission induction chemotherapy induces rapid cytoreduction of leukaemia and has the modifying potential to affect effector-target cell ratios and tumour cell recognition. DCs may take up necrotic and apoptotic tumour cells, process tumour antigens, and present these to T cells alongside the costimulatory molecules required for effective priming. Further, lymphocyte recovery during remission induction chemotherapy has been linked with improved prognosis, leading to postulation that selective lymphocyte expansion may occur as an immune response against tumour cells. In this study, we extensively characterised paediatric B cell ALL patient immune cells and plasma cytokines, at diagnosis and throughout remission induction chemotherapy in peripheral blood (PB) and the bone marrow (BM) tumour microenvironment. 17 paediatric patients diagnosed with precursor B cell ALL were enrolled in this study. Matched PB and BM aspirate tissue samples were collected at time points co-ordinating with treatment protocol (diagnosis, and induction days 8 and/or 15 and 29). Mononuclear cells and plasma samples were obtained by density gradient centrifugation. DCs, monocytes, B cells, natural killer (NK) cells, T cells, invariant natural killer T cells, cytokine induced killer cells and leukaemic blasts were extensively characterised by flow cytometry in matched PB and BM aspirate samples at diagnosis, day 8, day 15 and day 29 of induction therapy. Plasma cytokines from diagnosis and day 29 samples were analysed by Luminex multi-plex immunoassay. PB absolute lymphocyte counts (ALCs) at induction days 8 and 15 were lower than those recorded at diagnosis and lymphocyte recovery was observed at day 29. An equivalent pattern of lower mononuclear cell yields at days 8 and 15 with increased yields at day 29 was recorded in PB and BM samples processed for flow cytometry. All major immune cell populations were identified in PB and BM at all time points. BM DCs were significantly increased at day 29 compared to earlier time points and compared to matched day 29 PB DCs (Figure 1A). Naïve (CCR7+, CD45RA+) T cells dominated the peripheral and BM T cell pools at all time points with only low numbers of effector (CCR7-, CD45RA+), effector memory (CCR7-, CD45RA-) and central memory (CCR7+, CD45RA-) T cells recorded. However, activated (HLA-DR+) naïve CD4 T cells were significantly increased in the BM at day 29 compared to matched PB (Figure 1B) concordant to increased BM DCs. BM soluble cytokine analysis confirmed the presence of IL-1β, TNFα, IL-2, IL-4, IL-12p70 and IFNα in day 29 samples which may provide stimulus in the BM microenvironment for maturation of DCs and subsequent priming and activation of T cells. Immunological alterations in the BM tumour microenvironment may be reflective of an altered capacity to mount an effective anti-tumour immune response. Particularly, T cell reconstitution following induction chemotherapy may have important implications in childhood leukaemia, especially if recovering T cells are able to mediate anti-leukaemia effects. Rapid lymphocyte recovery has previously been associated with improved survival, and has been shown to be accounted for by increasing T cell numbers following induction chemotherapy in paediatric ALL. Here, while ALC recovery was recorded at day 29, extensive immune phenotyping of PB samples confirmed that recovery of T cell numbers is related to an increase in naïve T cells. T cell expansion associated with a specific immune response would be expected to occur in the effector and memory T cell compartments and the increased naïve T cell numbers observed here likely relate to homeostatic proliferation or increased thymic output. However, BM DCs were increased at day 29 alongside increased activation in naïve CD4 BM T cells, which may represent initiation of an anti-leukaemia T cell response. In vitro studies to investigate patient T cell response to autologous tumour cells are required as confirmation and are currently ongoing. Disclosures No relevant conflicts of interest to declare.
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Kim, Chang H., Brent Johnston i Eugene C. Butcher. "Trafficking machinery of NKT cells: shared and differential chemokine receptor expression among Vα24+Vβ11+ NKT cell subsets with distinct cytokine-producing capacity". Blood 100, nr 1 (1.07.2002): 11–16. http://dx.doi.org/10.1182/blood-2001-12-0196.
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Abstract Natural killer T (NKT) cells are important regulators of the immune system, but their trafficking machinery, including expression of chemokine receptors, has been poorly defined. Unlike other conventional T-cell populations, we show that most NKT cells express receptors for extralymphoid tissue or inflammation-related chemokines (CCR2, CCR5, and CXCR3), while few NKT cells express lymphoid tissue–homing chemokine receptors (CCR7 and CXCR5). A population with homing potential for lymph nodes (L selectin+ CCR7+) exists only within a small subset of CD4 NKT cells. We show differential expression of chemokine receptors among NKT cell subsets: CCR4 is mainly expressed by a high cytokine (interleukin-4/interleukin-2)–producing (CD4) NKT subset, while CCR1, CCR6, and CXCR6 are preferentially expressed by the low cytokine-producing CD8 and CD4−CD8− subsets. In line with this, TARC/CCL17 (a CCR4 ligand) induces preferential chemotaxis of the CD4 NKT subset, while chemotactic activities of LARC/CCL20 (a CCR6 ligand) and MIP-1α/CCL3 (a CCR1 ligand) are focused on the CD8 and CD4−CD8− NKT cells. We conclude that, unlike conventional naive, memory, or effector T cells, the entire NKT cell population expresses nonlymphoid tissue homing chemokine receptors, yet NKT cell subsets differ considerably from each other by displaying distinct and reciprocal expression patterns of some chemokine receptors. Our results identify chemokine receptors that are potentially important for trafficking of human blood NKT cell subsets and reveal their function (cytokine production capacity)–dependent differential trafficking potentials.
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Farnault, Laure, Gerard Michel, Herve Chambost, Didier Blaise, Sabine Furst, Felix Montero, Emmanuel Gautherot i in. "Identification of Escape Mechanisms Involved in the Absence of Viral Control Despite Activation of Both Innate (natural killer and gammadelta T cells) and Virus-Specific T Cells in Children with CMV or EBV Disease Following Cord Blood Transplantation." Blood 114, nr 22 (20.11.2009): 2659. http://dx.doi.org/10.1182/blood.v114.22.2659.2659.
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Abstract Abstract 2659 Poster Board II-635 Introduction: Cord blood transplantation (CBT) are being more and more used both for children and adults. However, one of the major limitation remains the low virus- CTL (cytotoxic T lymphocyte) response associated with the delayed immune recovery. Because the large majority of cord blood lymphocytes are naïve and so are not able to rapid clonal expansion, the monitoring of patients undergoing CBT provides a unique opportunity to 1) better understand the development of innate and adaptive human immune system and 2) identify the immune parameters associated to the viral response especially innate immunity (NK and gammadelta T cells) as well as T cells. Specifically, we have analysed their status of activation, function, and the expression of receptors (PD-1, 2B4, BTLA) recently demonstrated to be associated to immune escape. Materials and Methods: We investigated children with severe CMV or EBV disease following CBT and compared them both with children who controlled viral-reactivation following CBT or allogenic bone marrow transplantation (BMT) and with healthy donors. All patients received myeloablative conditioning including ATG and achieved full donor chimerism. Moreover, none of the selected patients received corticosteroid at the time of disease. CMV and EBV-specific CD8 T cells were investigated using HLA class I pentamers or tetramers together with extensive phenotype analyses by multi parametric flow cytometry and functional assay were performed using IFN gamma ELISPOT assay. Results: Kinetic of immune recovery showed a dramatic increase of CD8 but no CD4 T cells in patients with CMV or EBV disease. All CD8 T cells were highly activated (CD69high, CD57 high, PD-1 high, BTLAlow, 2B4 high, CD28 high). Virus-specific CTL were present in variable proportion. The major population of EBV-CTL were differentiated in CD45RA-CD27-CD28- late effector memory (EM) with almost no effector memory CD45RA+ cells (TEMRA) nor EBV specific activated naïve T cell. This is in sharp contrast with EBV-CTL of either: 1) healthy volunteers with controlled viral infection, who were mainly in a CD45RA-CD27+CD28+ Central Memory (CM) and CD45RA-CD27+CD28- early EM stage of differentiation; 2) patients with EBV infection during post transplantation immunosuppression who have activated naïve and TEMRA EBV-CTL. CMV-CTL were also mainly in late EM but no TEMRA differentiation stages in contrast to healthy volunteers and patients who controlled their CMV infection after CBT. Virus- CTL were highly activated (High expression of 2B4, CD28, PD-1, DR) with an overexpression of inhibitory receptors as PD-1 and 2B4 but no BTLA. The T cell response against viruses was highly variable in regard to different virus and patients as analysed by IFNg ELISPOT assay. CD4 T cells although low (<50 /mm3) were differentiated. The innate effectors gamma-delta T cells and NK cells were highly activated. Gamma-delta T cells belonged mainly to VD2- subset with a late EM and EMRA differentiation stage. VD2- subset were activated (CD69high, CD57high, CD8high) with low expression of BTLA. Their status was identical to CBT recipients with controlled viral infection. Similarly, NK cells were activated as determined by NKG2A, NKG2C, NKp30 and NKp46 expression. In addition, they were functional as demonstrated by the expression of CD57, which is associated to perforin and granzyme content. Conclusion: Thus, these reports illustrate a full activation of innate effectors as well as virus-CTL during viral disease after CBT, despite lack of viral clearance. We have identified at least three mechanisms that could explain this apparent immune failure: Perspectives: These data support for the first time the hypothesis that after CBT, the immune system although quantitatively insufficient, is highly activated and so that in-vitro expansion of patient self-lymphocytes or means to expand CD4 and CD8 T cells pools are a potential way to cure patients suffering from uncontrolled viral replication after CBT together with blockade of inhibitory pathways. Disclosures: No relevant conflicts of interest to declare.
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Michael, Husheem, Francine C. Paim, Ayako Miyazaki, Stephanie N. Langel, David D. Fischer, Juliet Chepngeno, Steven D. Goodman, Gireesh Rajashekara, Linda J. Saif i Anastasia Nickolaevna Vlasova. "Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota". PLOS ONE 16, nr 2 (16.02.2021): e0246193. http://dx.doi.org/10.1371/journal.pone.0246193.
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Human rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in low- and middle-income countries (LMICs), where HRV vaccine efficacy is low. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of enteric diseases in humans. However, repeated doses of EcN are required to achieve maximum beneficial effects. Administration of EcN on a microsphere biofilm could increase probiotic stability and persistence, thus maximizing health benefits without repeated administrations. Our aim was to investigate immune enhancement by the probiotic EcN adhered to a dextranomar microsphere biofilm (EcN biofilm) in a neonatal, malnourished piglet model transplanted with human infant fecal microbiota (HIFM) and infected with rotavirus. To create malnourishment, pigs were fed a reduced amount of bovine milk. Decreased HRV fecal shedding and protection from diarrhea were evident in the EcN biofilm treated piglets compared with EcN suspension and control groups. Moreover, EcN biofilm treatment enhanced natural killer cell activity in blood mononuclear cells (MNCs). Increased frequencies of activated plasmacytoid dendritic cells (pDC) in systemic and intestinal tissues and activated conventional dendritic cells (cDC) in blood and duodenum were also observed in EcN biofilm as compared with EcN suspension treated pigs. Furthermore, EcN biofilm treated pigs had increased frequencies of systemic activated and resting/memory antibody forming B cells and IgA+ B cells in the systemic tissues. Similarly, the mean numbers of systemic and intestinal HRV-specific IgA antibody secreting cells (ASCs), as well as HRV-specific IgA antibody titers in serum and small intestinal contents, were increased in the EcN biofilm treated group. In summary EcN biofilm enhanced innate and B cell immune responses after HRV infection and ameliorated diarrhea following HRV challenge in a malnourished, HIFM pig model.
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Benedetti, F. "Circulating immune cell composition and activation status associate with brain white matter microstructure in bipolar depression". European Psychiatry 66, S1 (marzec 2023): S40. http://dx.doi.org/10.1192/j.eurpsy.2023.151.
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AbstractBipolar disorder (BD) has been consistently associated with alterations in the immune system. Evidence suggests a condition of systemic low-grade inflammation due to decreased adaptive, increased innate immunity, with higher levels of circulating cytokines, higher macrophage/monocyte inflammatory activation patterns, and higher neutrophils to lymphocyte counts; and with a dynamic pattern of premature immunosenescence and partial T cell defect starting early in adolescence, involving a reduction of naïve T cells and an expansion of memory and senescent T cells. Quantitative analysis of circulating inflammatory markers suggested persistent low-grade inflammation.A growing literature suggests that the immune system plays a core role in maintaining brain homeostasis, with both adaptive and innate immune support, ensured by cell trafficking across the blood brain barrier, being essential for brain maintenance and repair in healthy conditions, and disrupted in brain disorders including BD. Measured in peripheral blood, these markers of altered immuno-inflammatory setpoints parallel activation of microglia and disruption of white matter (WM) integrity in the brain.Studies in the field are in its infancy, but findings by our group showed that: circulating Th17 cells correlated with higher FA, while regulatory FOXP3+ cells correlated with higher RD and MD, and with lower fMRI neural responses in the right dorsolateral prefrontal cortex; higher circulating cytokine-producing NK cells were fostered by ongoing lithium treatment and directly correlated with better FA, and inversely with RD and MD, also partially mediating the known benefits from lithium on WM; and activation status and expression of killer proteins by cytotoxic CD8+ T cells negatively associated with WM microstructure, thus suggesting that CD8+ T cells can leave the blood stream to migrate into the brain and induce an immune-related WM damage in BD.Implications of these findings for neuroprogression, clinical outcomes, and new treatment strategies of the disorder will be discussed.Disclosure of InterestNone Declared
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Foley, Bree, Michael R. Verneris, Julie Curtsinger, Sandra Lopez-Verges, Lewis L. Lanier, Daniel J. Weisdorf, Sarah Cooley i Jeffrey S. Miller. "Natural Killer (NK) Cells Respond to CMV Reactivation After Allogeneic Transplantation with An Increase in NKG2C+CD57+ Self-KIR+ NK Cells with Potent IFNγ Production". Blood 118, nr 21 (18.11.2011): 356. http://dx.doi.org/10.1182/blood.v118.21.356.356.
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Abstract Abstract 356 In healthy individuals human cytomegalovirus (hCMV) infection is asymptomatic or a mild illness. In immunosuppressed patients after hematopoietic cell transplantation, however, CMV is potentially life-threatening complication. We have previously shown that upon mouse CMV infection, a population of Ly49H+ NK cells expand and are responsible for disease clearance through the induction of a “memory NK cell response”. Whether similar events occur in hCMV infection is unknown. Some studies suggest that the C-type lectin-like receptor NKG2C and the killer cell immunoglobulin-like receptor (KIR) family may be involved. We studied NK cell function after hCMV reactivation. Cohort 1 consisted of patients who received umbilical cord blood grafts (n=23). Recipients were CMV-seronegative (n=10) and -seropositive (n=13), with 8 patients developing detectable hCMV in the blood 22 to 82 days after transplant. For this cohort, peripheral blood mononuclear cells (PBMC) were collected at day 28, day 60 and day 100 after transplant. Cryopreserved PBMC were thawed and rested overnight in cytokine-free media. After incubation with K562 cells a functional analysis was performed using multi-color flow cytometry for CD107a (a surrogate for cytotoxicity), IFNγ, CD56, CD3, CD158a, CD158b, CD158e, CD57, NKG2A, and NKG2C. In cohort 1 the frequency of NKG2C+ NK cells significantly increased between day 28 and day 60 (10±1.3% v 26±5%, p=0.01), and this increase persisted at day 100 in seropositive patients who reactivated CMV (32±4.8%, p=0.001). No change in NKG2C expression was detected in seropositive patients who did not reactivate CMV or in seronegative recipients, showing that this response was specific. There was no difference in the expression of CD107a by NKG2C+ NK cells in the three groups. In marked contrast, IFNγ production by NKG2C+ NK cells was low prior to CMV reactivation (0.8±1.4%) and increased at day 60 and day 100 (7.9±2.1%, p=0.012). The IFNγ response was specific to NKG2C+ NK cells as the NKG2C− population produced minimal IFNγ at day 60 and day 100 (NKG2C+: 7.9±2.1% vs. NKG2C−: 0.4±0.25%, p=0.003). The frequency of NKG2C+ NK cells with increased cytokine production correlated with CMV reactivation in this cohort. The second cohort included PBMCs collected at the time of viral reactivation (monitored weekly) and 2, 4, and 8 weeks later (n=10). The frequency of NKG2C+ NK cells increased following the detection hCMV viremia. The peak response (i.e., rise in NKG2C+ NK cells compared to baseline) was at 4 weeks post infection (23±3.7% vs. 13±1.8%, p=0.046), followed by contraction at 8 weeks. NKG2C+NK cells produced significantly more IFNγ than did NKG2C− cells at 2, 4 (NKG2C+: 9.4±1.2% vs. NKG2C−: 2.4±0.3%, p=0.0002) and 8 weeks post infection. The maximum IFNγ production occurred at 4 weeks, with the peak NKG2C+ frequency. We have previously demonstrated that KIR expression is essential to educate NK cells to produce IFNγ. Therefore, we measured coexpression of NKG2C and KIR in these cohorts. Four weeks after infection, NK cells that coexpressed NKG2C and KIR produced significantly more IFNγ than did the KIR+ NKG2C−NK cells (11.4±1.66% vs. 5.6±0.83%, p=0.0078). Importantly, only the NKG2C+NK cells expressing KIR that recognized recipient MHC ligands made IFNγ. In contrast, NKG2C+ NK cells coexpressing KIR that did not recognize recipient ligands were hyporesponsive. Lastly, we tested whether coexpression of CD57, a phenotypic marker of NK cells maturity, might be present on NKG2C+ cells after CMV reactivation. Over the 8 weeks following CMV reactivation the NKG2C+ NK cells but not the NKG2C−NK cells progressively acquired CD57, suggesting that CD57 may mark the human memory NK cell in response to CMV reactivation. In summary, our data demonstrate that CMV reactivation induces an NK cell IFNγ response specific to NKG2C+cells educated by recipient KIR ligands that then acquire CD57+. These findings support the emerging concept that hCMV specific, innate memory cell populations could be exploited for therapeutic purposes. Disclosures: No relevant conflicts of interest to declare.
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Aigner, Kornelia, Stephan Drothler, Karl R. Aigner i Karl R. Aigner. "Immune response during regional chemotherapy." Journal of Clinical Oncology 40, nr 16_suppl (1.06.2022): e14537-e14537. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e14537.
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e14537 Background: Regional chemotherapy is known to yield high drug concentrations in the treated tumor area while keeping systemic drug concentrations low. Regional Chemotherapy can be performed as an intra arterial infusion or isolated perfusion. Known perfusion techniques are isolated limb perfusion, hypoxic pelvic perfusion, hypoxic abdominal perfusion, upper abdominal perfusion, and isolated thoracic perfusion, Only little is known on the effect of chemotherapy on immune cells, especially not if chemotherapy is applied regionally. Since more and more immuno oncologic treatment possibilities arise, that often are combined with chemotherapy, knowledge on the effects on immune cells is crucial. Since systemic chemotherapy often decreases the number of immune cells in general, the more targeted approach of regional chemotherapy is investigated in this study. Methods: In this work, we investigate subpopulations of T-lymphocytes, Natural Killer cells and B-lymphocytes during regional chemotherapy and report on their changes after cycles of intra-arterial chemotherapy infusion followed by chemofiltration. Surface phenotyping of cells from EDTA blood was performed with combinations of monoclonal antibodies (anti- CD3,CD16, CD56, CD45, CD4, CD19, CD8, CD28, HLA-DR, CD39, CD25, CD127, CD45RA, CD45RO) for FACS analysis. 58 Patients with advanced breast cancer, ovarian cancer, head and neck cancer, lung cancer, or bladder cancer received regional chemotherapy as an intra arterial infusion therapy or an isolated perfusion therapy. Results: Contrary to the mild immune cell depletion during the first two cycles a more activated immune status is seen after treatment cycle three. Non-significant increase or stable numbers are observed for T-Lymphocytes and its subsets of CD4+ T-helper cells, CD8+ cytotoxic T-cells, and terminal memory T-cells (CD8+/CD28-/CD57+). Significant increase of cell numbers is observed for B-lymphocytes, regulative CD8+ T-cells (CD8+/CD28-/CD57-), activated HLA-DR+ T cells, and Natural Killer cells while regulative CD4+ T cells are significantly decreased. Conclusions: The data show that regional chemotherapy has controversial effects on the immune cell subpopulations. As every chemotherapy, it does affect immune cells, however to a limited extend, but importantly has an activating potential. Immune activation prevalently takes place after the third cycle of therapy which might be a hint for auto-vaccination caused by phases of repeated tumor necrosis after locally high drug concentrations. Finding the optimal dosage of chemotherapeutics may be crucial to balance direct tumor response, immune toxicity and immune activation.
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Aigner, Kornelia, Stephan Drothler, Karl R. Aigner i Karl R. Aigner. "Immune response during regional chemotherapy." Journal of Clinical Oncology 40, nr 16_suppl (1.06.2022): e14537-e14537. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e14537.
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e14537 Background: Regional chemotherapy is known to yield high drug concentrations in the treated tumor area while keeping systemic drug concentrations low. Regional Chemotherapy can be performed as an intra arterial infusion or isolated perfusion. Known perfusion techniques are isolated limb perfusion, hypoxic pelvic perfusion, hypoxic abdominal perfusion, upper abdominal perfusion, and isolated thoracic perfusion, Only little is known on the effect of chemotherapy on immune cells, especially not if chemotherapy is applied regionally. Since more and more immuno oncologic treatment possibilities arise, that often are combined with chemotherapy, knowledge on the effects on immune cells is crucial. Since systemic chemotherapy often decreases the number of immune cells in general, the more targeted approach of regional chemotherapy is investigated in this study. Methods: In this work, we investigate subpopulations of T-lymphocytes, Natural Killer cells and B-lymphocytes during regional chemotherapy and report on their changes after cycles of intra-arterial chemotherapy infusion followed by chemofiltration. Surface phenotyping of cells from EDTA blood was performed with combinations of monoclonal antibodies (anti- CD3,CD16, CD56, CD45, CD4, CD19, CD8, CD28, HLA-DR, CD39, CD25, CD127, CD45RA, CD45RO) for FACS analysis. 58 Patients with advanced breast cancer, ovarian cancer, head and neck cancer, lung cancer, or bladder cancer received regional chemotherapy as an intra arterial infusion therapy or an isolated perfusion therapy. Results: Contrary to the mild immune cell depletion during the first two cycles a more activated immune status is seen after treatment cycle three. Non-significant increase or stable numbers are observed for T-Lymphocytes and its subsets of CD4+ T-helper cells, CD8+ cytotoxic T-cells, and terminal memory T-cells (CD8+/CD28-/CD57+). Significant increase of cell numbers is observed for B-lymphocytes, regulative CD8+ T-cells (CD8+/CD28-/CD57-), activated HLA-DR+ T cells, and Natural Killer cells while regulative CD4+ T cells are significantly decreased. Conclusions: The data show that regional chemotherapy has controversial effects on the immune cell subpopulations. As every chemotherapy, it does affect immune cells, however to a limited extend, but importantly has an activating potential. Immune activation prevalently takes place after the third cycle of therapy which might be a hint for auto-vaccination caused by phases of repeated tumor necrosis after locally high drug concentrations. Finding the optimal dosage of chemotherapeutics may be crucial to balance direct tumor response, immune toxicity and immune activation.
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Caushi, Justina X., Jiajia Zhang, Zhicheng Ji, Ajay Vaghasia, Boyang Zhang, Emily Han-Chung Hsiue, Brian J. Mog i in. "Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers". Nature 596, nr 7870 (21.07.2021): 126–32. http://dx.doi.org/10.1038/s41586-021-03752-4.
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AbstractPD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.
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Ansell, Stephen M., Mary Stenson, Thomas M. Habermann, Diane F. Jelinek i Thomas E. Witzig. "CD4+ T-Cell Immune Response to Large B-Cell Non-Hodgkin’s Lymphoma Predicts Patient Outcome". Journal of Clinical Oncology 19, nr 3 (1.02.2001): 720–26. http://dx.doi.org/10.1200/jco.2001.19.3.720.
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PURPOSE: Previous studies in patients with non-Hodgkin’s lymphoma (NHL) and other malignancies have suggested that the presence of host infiltrates in the tumors of these patients may predict a better outcome. This study was undertaken to determine the prognostic importance of the presence of T cells in the biopsy specimens of patients with B-cell NHL. PATIENTS AND METHODS: Seventy-two patients with diffuse large B-cell NHL were prospectively evaluated at a single institution between 1987 and 1994. The percentage of CD3+, CD3+/HLA-DR+, CD4+, CD8+, and natural killer cells was determined by flow cytometry in the pretreatment diagnostic biopsy specimen and correlated with patient outcome. RESULTS: An increase in the percentage CD4+ T cells in the pretreatment tumor biopsies significantly correlated with patient outcome. The percent of CD4+ T cells was also highly correlated with CD3+/HLA-DR+, CD45RO+, and low l-selectin (CD62L) expression, indicating that the CD4+ T cells are activated memory T-helper cells. Those patients with increased numbers of CD4+ T cells, compared with other patients, had a significantly longer 5-year failure-free survival (72% v 43%, respectively; P = .04), as well as a significantly longer 5-year overall survival (65% v 38%, respectively; P = .05). When evaluated in a multivariate model, the International Prognostic Index and more than 20% infiltrating CD4+ T cells in the pretreatment biopsy were significant independent predictors of relapse-free and overall survival. CONCLUSION: The presence of increased numbers of activated CD4+ cells in the area of B-cell diffuse large-cell NHL predicts a better prognosis. This finding provides a strong rationale for the investigation of cellular immunotherapy in B-cell NHL.
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Decker, William Karl, Laurel Douglass, Matthew M. Halpert, Vanaja Konduri, Yunyu Chen, Dan Liang, Jonathan M. Levitt i in. "Active specific immunotherapy for lethal canine hemangiosarcoma: A model system for the treatment of angiosarcoma with curative intent." Journal of Clinical Oncology 35, nr 15_suppl (20.05.2017): e14539-e14539. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e14539.
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e14539 Background: Angiosarcoma is a deadly malignant neoplasm of the vascular endothelium. Metastases are often present at diagnosis, and 5-year survival is only 10-30%. Though it comprises only 1% all soft tissue sarcoma diagnoses, it is a major killer among companion canines, responsible for an estimated 120,000 deaths per year in the US. The canine disease (termed HSA) closely mimics the human disease in both pathology and genetics. It frequently presents in the dog as acute hemoabdomen secondary to splenic rupture. Even if life-saving splenectomy is performed, median OS is only 48 days, and 1 year OS hovers at 0%. Given the accelerated timeline of the canine disease, the outbred dog is an ideal system in which to translate novel therapeutic approaches. Here we report an analysis of a phase I multi-site, open-label veterinary trial of chemo-immunotherapy performed on consecutively-presenting splenectomized companion canines with histopathologically-verified HSA. Methods: Subjects were administered two cycles of 20 mg/m2 (low-dose) doxorubicinand an autologous cell-therapy reported to generate durable CD8+ memory similar to that of physiologic viral infection. Vaccine was generated from mobilized peripheral blood and cryopreserved tumor antigen and was administered with type I interferon. Disease burden was monitored monthly by abdominal ultrasound, chest x-ray, and echocardiogram. Results: At the time of submission, median OS had not been reached in the per protocol population, with 75% of animals alive and healthy (NED or ongoing PR) at up to 1 year post-splenectomy (p < 0.0002). Ultrasound documented resolution of local mesenteric disease in one animal, sternal lymphadenopathy in another, and hepatic metastases in a third. In the intent-to-treat population, median OS was 109 days with 43% of animals alive and healthy at up to 1 year post-splenectomy. Conclusions: Administration of autologous cell therapy with low-dose doxorubicin is feasible, safe, and highly efficacious in the companion canines. If subjects survived long enough to begin treatment, prognosis became favorable. The results suggest that additional clinical studies in both canines and humans are warranted.
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Berrien-Elliott, Melissa M., Wong Pamela, Carly Neal, Julia A. Wagner, Michelle Becker-Hapak, Tim Schappe, Matthew L. Cooper, Emily M. Mace i Todd A. Fehniger. "Primary Human NK Cell Gene-Editing Reveals a Critical Role for NKG2A in Cytokine-Induced Memory-like NK Cell Responses". Blood 134, Supplement_1 (13.11.2019): 3237. http://dx.doi.org/10.1182/blood-2019-129162.
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Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell anti-leukemia potential is unclear. Paradigm-shifting reports have shown that NK cells exhibit "memory-like" properties following hapten exposure, virus infection, or combined cytokine pre-activation. Human cytokine-induced memory-like (ML) NK cells display enhanced re-stimulation responses to numerous activating stimuli, including tumor target cells. This has been translated in clinical trials as cellular therapy for rel/ref AML patients (NCT#), and the dose escalation of a phase 1/2 study has been completed (PMID). Donor memory-like NK cells expanded in patients' blood and bone marrow and retained enhanced functionality ex vivo, with 7 of 11 patients achieving CR/CRi. Since NK cell recognition depend on signals from multiple activating and inhibitory receptors, we developed mass cytometry panels to immunophenotype and track the diversity and effector functions of these human in vivo-differentiated memory-like NK cells. Previous work showed that that in vivo-differentiated memory-like (ML) NK cells were distinct from baseline (BL) NK cells from the same donor, as well as NK cells from normal donor PBMC. Multidimensional analyses revealed a memory-like phenotype: CD56hi CD11blo CD62L+ NKG2Ahi NKp30hi Ki-67+. Furthermore, Citrus analyses revealed that higher NKG2A expression was significantly correlated with treatment failure. NKG2A is a C-type lectin receptor with two immunoreceptor tyrosine-based inhibitory motifs. Signaling through NKG2A is achieved when it engages its ligand, HLA-E. HLA-E is a non-classical major histocompatibility complex class I molecule that is expressed abundantly on many normal tissue types as well as tumors, including AML. Based on these findings that NKG2A is upregulated on memory-like NK cells and the intensity of NKG2A on memory-like NK cells correlated with patient responses, we hypothesized that NKG2A/HLA-E interactions represent a major barrier to memory-like NK cell responses. CRISPR based gene editing of primary human NK cells has been technically challenging. In order to interrogate the role NKG2A may play in limiting ML NK cell responses, we optimized the MaxCyte GT electroporation system to introduce Cas9 and guide RNA into freshely isolated, purified human NK cells. As proof of principle, we introduced Cas9 and gRNA targeting CD56 into NK cells and assessed CD56 expression a week later. We observed a 96.5% ± 0.8% (SD) reduction in median CD56 expression as determined by flow cytometry, with little impact on cell viability (90.3% ± 2.7% live v 87.0% ± 3.1% live ΔCD56) after electroporation. Next we introduced Cas9 and gRNA against NKG2A into freshly isolated, purified human NK cells. After electroporation, cells were briefly incubated with IL-12/IL-15/IL-18, overnight. The cytokines are washed away and the cells incubated for 4 days in low-dose IL-15, which was required for their survival. NKG2A frequency was decreased 64.72% (ΔNKG2A v Control; 42.3-85.7% range, ± 13.18% SD) by 4 days post-electroporation. We compared the ability of these cells to respond to HLA-E+ K562 leukemia targets and observed a significantly enhanced ML NK cell response by ΔNKG2A ML NK cells compared to control ML NK cells (19.04 ± 5.9% IFN-γ+ v 34.9 ± 8.8% ΔNKG2A IFNγ+; Mean ± S.D.). Finally, we infused ΔNKG2A ML NK or control ML NK cells into NSG recipient mice and assessed the spleen at D7 and D14 for persistence and NKG2A expression. We were able to detect ΔNKG2A ML NK and control ML NK cells at both time points and the ΔNKG2A ML NK cells remain NKG2A-negative, post-transfer. Using gene-editing approaches, the data reveal an important inhibitory role for NKG2A on ML NK cell responses against HLA-E+ targets. Primary human NK cells are notoriously difficult to modify by virus or electroporation. Indeed, most reports utilize expanded NK cells or cord-blood differentiated NK cells which were edited in the stem cell stage. This report demonstrates that mature NK cells can be modified with little ex vivo manipulation with high efficiency and viability. This method has broad potential to expand our understanding of human NK cell biology using genetic loss or gain of function techniques, as exemplified by identification of NKG2A as a critical ML NK cell checkpoint. Figure Disclosures Cooper: Wugen: Consultancy, Equity Ownership, Patents & Royalties. Fehniger:Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse).
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de Witte, Moniek A., Anke Janssen, Klaartje Nijssen, Froso Karaiskaki, Luuk Swanenberg, Anna van Rhenen, Rick Admiraal i in. "αβ T-cell graft depletion for allogeneic HSCT in adults with hematological malignancies". Blood Advances 5, nr 1 (7.01.2021): 240–49. http://dx.doi.org/10.1182/bloodadvances.2020002444.
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Abstract We conducted a multicenter prospective single-arm phase 1/2 study that assesses the outcome of αβ T-cell depleted allogeneic hematopoietic stem cell transplantation (allo-HSCT) of peripheral blood derived stem cells from matched related, or unrelated donors (10/10 and 9/10) in adults, with the incidence of acute graft-versus-host disease (aGVHD) as the primary end point at day 100. Thirty-five adults (median age, 59; range, 19-69 years) were enrolled. Conditioning consisted of antithymocyte globulin, busulfan, and fludarabine, followed by 28 days of mycophenolic acid after allo-HSCT. The minimal follow-up time was 24 months. The median number of infused CD34+ cells and αβ T cells were 6.1 × 106 and 16.3 × 103 cells per kg, respectively. The cumulative incidence (CI) of aGVHD grades 2-4 and 3-4 at day 100 was 26% and 14%. One secondary graft failure was observed. A prophylactic donor lymphocyte infusion (DLI) (1 × 105 CD3+ T cells per kg) was administered to 54% of the subjects, resulting in a CI of aGVHD grades 2-4 and 3-4 to 37% and 17% at 2 years. Immune monitoring revealed an early reconstitution of natural killer (NK) and γδ T cells. Cytomegalovirus reactivation associated with expansion of memory-like NK cells. The CI of relapse was 29%, and the nonrelapse mortality 32% at 2 years. The 2-year CI of chronic GVHD (cGVHD) was 23%, of which 17% was moderate. We conclude that only 26% of patients developed aGVHD 2-4 after αβ T-cell–depleted allo-HSCT within 100 days and was associated with a low incidence of cGVHD after 2 years. This trial was registered at www.trialregister.nl as #NL4767.
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Guo, Yin, Liming Luan, Julia Bohannon, Naeem Patil, Benjamin Fensterheim, Antonio Hernandez, Jingbin Wang i Edward Sherwood. "The lack of NK and mCD8+ T cells in IL-15KO mice confers resistance to septic shock". Journal of Immunology 196, nr 1_Supplement (1.05.2016): 60.9. http://dx.doi.org/10.4049/jimmunol.196.supp.60.9.
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Abstract Septic shock remains one of the leading causes of death in the USA. However, the immunological mechanisms of septic shock are still not well understood. Interleukin (IL)-15 is an essential mediator for natural killer (NK) and memory (m) CD8+T cell survival, as indicated by the deficits of these cells in IL-15 knockout (KO) mice. As NK and CD8+T cells are implicated in the pathogenesis of septic shock, we hypothesized that IL-15KO mice will be resistant to septic shock due to their lack of NK and mCD8+T cells. IL-15KO mice showed a significant survival advantage over wild type (WT) control mice during septic shock caused by cecal ligation and puncture (CLP) or LPS challenge. IL-15KO mice displayed less sepsis-induced hypothermia and attenuated plasma IL-6 production in our sepsis models. Treatment with low-dose IL-15 superagonist (SA) regenerated NK and mCD8+T cells in IL-15KO mice and reestablished mortality in these mice during CLP- and LPS-induced septic shock. However, if NK and CD8+T cell regeneration was prevented by anti-asialoGM1 and anti-CD8α prior to IL-15 SA treatment, IL-15 SA failed to reestablish sepsis-induced mortality in IL-15 KO mice. When given to WT mice 4 days prior to CLP or LPS challenge, M96, an IL-15 neutralizing IgG, caused 80.8% depletion of splenic NK cells and conferred protection against septic shock. However, when given shortly prior to CLP or LPS challenge, M96 did not deplete splenic NK cells and failed to confer protection against septic shock. In conclusion, IL-15 contributes to the lethality of septic mice by maintaining NK and mCD8+ T cell populations, but does not play a direct role in septic shock. Thus, NK and mCD8+ T cells may represent effective therapeutic targets for septic shock.
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Lopes, Jared E., Jan L. Fisher, Heather L. Flick, Chunhua Wang, Lei Sun, Marc S. Ernstoff, Juan C. Alvarez i Heather C. Losey. "ALKS 4230: a novel engineered IL-2 fusion protein with an improved cellular selectivity profile for cancer immunotherapy". Journal for ImmunoTherapy of Cancer 8, nr 1 (kwiecień 2020): e000673. http://dx.doi.org/10.1136/jitc-2020-000673.
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BackgroundInterleukin-2 (IL-2) plays a pivotal role in immune homeostasis due to its ability to stimulate numerous lymphocyte subsets including natural killer (NK) cells, effector CD4+and CD8+T cells, and regulatory T cells (Tregs). Low concentrations of IL-2 induce signaling through the high-affinity IL-2 receptor (IL-2R) comprised of IL-2Rα, IL-2Rβ, and common γ chain (γc), preferentially expressed on Tregs. Higher concentrations of IL-2 are necessary to induce signaling through the intermediate-affinity IL-2R, composed of IL-2Rβ and γc, expressed on memory CD8+T cells and NK cells. Recombinant human IL-2 (rhIL-2) is approved for treatment of metastatic melanoma and renal cell carcinoma (RCC), but adverse events including capillary leak syndrome, potentially mediated through interaction with the high-affinity IL-2R, limit its therapeutic use. Furthermore, antitumor efficacy of IL-2 may also be limited by preferential expansion of immunosuppressive Tregs. ALKS 4230 is an engineered fusion protein comprised of a circularly-permuted IL-2 with the extracellular domain of IL-2Rα, designed to selectively activate effector lymphocytes bearing the intermediate-affinity IL-2R.ResultsALKS 4230 was equipotent to rhIL-2 in activating human cells bearing the intermediate-affinity IL-2R, and less potent than rhIL-2 on cells bearing the high-affinity IL-2R. As observed in vitro with primary human cells from healthy donors and advanced cancer patients, ALKS 4230 induced greater activation and expansion of NK cells with reduced expansion of Tregsrelative to rhIL-2. Similarly, in mice, ALKS 4230 treatment stimulated greater expansion of NK cells and memory-phenotype CD8+T cells at doses that did not expand or activate Tregs. ALKS 4230 treatment induced significantly lower levels of proinflammatory cytokines, including tumor necrosis factor alpha, interleukin-6, and interferon gamma relative to rhIL-2. Furthermore, ALKS 4230 exhibited superior antitumor efficacy in the mouse B16F10 lung tumor model, where ALKS 4230 could be administered via multiple routes of administration and dosing schedules while achieving equivalent antitumor efficacy.ConclusionsALKS 4230 exhibited enhanced pharmacokinetic and selective pharmacodynamic properties resulting in both improved antitumor efficacy and lower indices of toxicity relative to rhIL-2 in mice. These data highlight the potential of ALKS 4230 as a novel cancer immunotherapy, and as such, the molecule is being evaluated clinically.
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Pusceddu, Valeria, Clelia Donisi, Francesco Loi, Andrea Pretta, Stefano Mariani, Pina Ziranu, Marco Puzzoni i in. "Immunonutrition supplementation for resectable gastric cancer during standard perioperative chemotherapy (CT) FLOT: A pivotal study, I–SUPPLY." JCO Global Oncology 9, Supplement_1 (sierpień 2023): 57. http://dx.doi.org/10.1200/go.2023.9.supplement_1.57.
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57 Background: Gastric carcinomas (GC) can be divided into immunogenic and immune-resistant based on the infiltration of the tumour microenvironment (TME) from different immune cells (ICs) such as Natural Killer (NK), activated CD8+, CD3+ and CD4+ memory T cells. High ImmunoScore (IS) (positive cells/mm2 for each marker (CD8+, CD3+, Foxp3+) in the Core Tumour and Invasive Margins) is strongly related to better prognosis of GC patients in terms of Disease-Free Survival and Overall Survival. ICs activation and consequent immunogenic TME requires high nutrient absorption and synthesis accumulation of protein, lipid and nucleotides. However, tumour cells (TCs) promote their own proliferation by stealing the available micronutrients to ICs therefore leading to an immunosuppressive TME phenotype. Methods: This is a translational, prospective, non-randomized study. Primary endpoint is to assess if enteral immune nutrition (EIN) supplementation during the acme of the activity of cytotoxic of perioperative FLOT (docetaxel, oxaliplatin and 5-fluorouracil) in stage II-III GC can revert the TME in favour of ICs over TCs, witnessed by an increase of IS in surgical specimen over biopsy controls. A steady amount of EIN with Arginine, Omega-3-fatty acids, Glutamine and ribonucleic acid, will be added to patient’s diet from day 3 to 8 of each FLOT cycle, twice a day. We plan to use Image analysis software for automatic tumor detection and quantification of CD3+ and CD8+ stained cells whose densities will be converted into IS with pre-defined cutoffs. IS utilizes standardized percentile values (0 to 100%). We identify IS as Low, Intermediate (Int) and High with a mean percentile of 0-25%, >25-70%, >70-100% respectively. Two-group classifications are planned, corresponding to IS Low and Int-High. To better define the kind of tumor infiltrating cells (TILs) and assess the amount of immune suppressive cells, more IHC scoring will be evaluated: helper T-cells CD4+, CD8 T memory (CD 103+), T regulatory (both CD4+ and Foxp3+/CD105+), Macrophages M1 (CD 68+) and M2 (CD 163+). Results: Based on historical controls the rate of Int-High IS (>25-70%, >70% respectively) is about 64%, we expect to find a 20% increase of Int-High IS GC over biopsy control (internal validation cohort). To detect an increase in the percentage of high IS among patients treated with EIN (estimated around 85%) compared to biopsy controls (estimated around 64%) assuming a probability α and β of 0.2, the required sample size will be of 21 patients with resectable GC receiving EIN supplementation plus standard CT. To define the sample size a single proportion test was used. Conclusions: Our hypothesis is that EIN given during perioperative CT might prevent the establishment of an unfavourable TME for ICs in both auxotrophic and non-auxotrophic malignancies, thus leading to an increase of GC immunogenic pattern.
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Imataki, Osamu, Makito Tanaka, Alla Berezovskaya, Marcus O. Butler, Lee M. Nadler i Naoto Hirano. "Endogenous Ligands Selectively Stimulate Highly Avid Autoreactive Human Invariant Natural Killer T Cells with Distinctive T-Cell Receptor Vβ11 CDR3 Sequence Motifs". Blood 118, nr 21 (18.11.2011): 999. http://dx.doi.org/10.1182/blood.v118.21.999.999.
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Abstract Abstract 999 Human CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of innate T-cells that carry an invariant T-cell receptor (TCR) Vα24 chain paired with a TCR Vβ11 chain. They constitutively express a variety of activation and memory markers and possess the capacity to rapidly produce a variety of cytokines including IFN-γ and IL-4 upon TCR engagement. As an immune response launches, iNKT cells can induce innate immune responses and serve as a bridge between innate and adaptive immunity. In addition, it has also been shown that iNKT cells themselves have anti-tumor and anti-infectious effects in mice. However, to translate these findings and develop iNKT cell-mediated immunotherapy, iNKT cells must recognize target cells while sparing normal cells. Autoreactivity is primarily dictated by 1) the specificity and avidity of the TCR on iNKT cells; 2) the expression level of CD1d on target cells; and 3) the density and stability of endogenous ligand(s) presented by CD1d on target cells. In this study, we sought to determine the TCR Vβ11 CDR3 sequence motifs that distinguish high avidity and low avidity iNKT cells. We developed an artificial antigen-presenting cell (aAPC) to expand iNKT cells by transducing K562 with CD1d, CD80, and CD83. Using this CD1d+aAPC loaded with or without α-galactosylceramide (αGC), we expanded CD1d-restricted iNKT cells by stimulating primary CD3+ T cells expressing canonical iNKT TCR Vα24 chain. Expanded iNKT cells were stained with αGC-loaded CD1d tetramers and were found to express TCR Vβ11 chain in conjunction with transgenic canonical TCR Vα24 chain. Sequence analysis of cloned Vβ11 CDR3 revealed that the clonality of iNKT cells generated using unloaded aAPC was significantly lower than that of iNKT cells generated using loaded aAPC. Surprisingly, when cotransfected with canonical TCR Vα24 chain, some TCR Vβ11 chains isolated from iNKT cells generated using unloaded aAPC were stained with unloaded CD1d tetramers produced in HEK293 cells. This result suggests that these reconstituted iNKT TCR recognized endogenous ligand(s) derived from HEK293 cells in the context of CD1d. A comprehensive analysis of the structural avidity demonstrated that these TCR Vβ11 chains reconstituted TCR with significantly higher structural avidity than those cloned from NKT cells generated using loaded aAPC. However, this significant difference was only observed when the structural avidity was measured using unloaded tetramers but not αGC-loaded tetramers. A univariate analysis found that structural avidity was significantly higher when 1) Vβ11 CDR3 used J2-5; 2) Vβ11 CDR3 consisted of exactly 23 amino acids; and 3) Vβ11 CDR3 encoded 3 or more acidic amino acids (asparagic and glutamic acids). A multivariate analysis confirmed that all three variables were independent predictors of higher structural avidity. Furthermore, each variable is sufficient to increase the structural avidity of an iNKT TCR with low structural avidity. Intriguingly, unloaded mouse CD1d tetramers produced in HEK293 cells also stained a human iNKT TCR with high structural avidity reconstituted on both human and mouse T cells. This result suggests that the human iNKT TCR with high structural avidity possessed a cross-species reactivity to mouse CD1d in conjunction with endogenous ligand(s) derived from HEK293 cells. We next studied the autoreactivity of cloned human iNKT TCR with high structural avidity. A human T cell line, Jurkat, reconstituted with high avidity iNKT TCR, was able to recognize cells expressing CD1d endogenously (itself, SUP-T1, and primary human monocytes) and ectopically (K562, C1R, Hela). Furthermore, mouse T cells expressing human iNKT TCR with high structural avidity recognized mouse cell lines, B16, EL4, and 58, all endogenously expressing CD1d. Finally, human primary T cells transduced with the iNKT TCR with high structural avidity were autoreactive, secreting both IFN-γ and IL-4 in response to CD1d+ target cells. Using our CD1d+ aAPC-based system, we successfully isolated human iNKT cells with high structural avidity and identified the TCR Vβ11 CDR3 sequence motifs that dictate the structural avidity of iNKT cells. To develop clinically effective iNKT cell-mediated immunotherapy without unwanted autoimmunity, it will be critically important to define the range of iNKT TCR avidity that enables reactivity to pathologic but not normal cells. Disclosures: No relevant conflicts of interest to declare.