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McCarty, Todd M., Zhiwei Yu, Xiping Liu, Don J. Diamond y Joshua D. I. Ellenhorn. "An HLA-restricted, p53 specific immune response from HLA transgenic p53 knockout mice". Annals of Surgical Oncology 5, n.º 1 (enero de 1998): 93–99. http://dx.doi.org/10.1007/bf02303770.
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Suzuki, Daisuke, Naoshi Sugimoto, Norihide Yoshikawa, Hiroshi Endo, Sou Nakamura, Akitsu Hotta y Koji Eto. "Natural Killer Cell Activities Against iPSCs-Derived HLA-Knockout Platelets and Megakaryocytes Reveal Perfect Rejection Profiles for Allotransfusion". Blood 128, n.º 22 (2 de diciembre de 2016): 3841. http://dx.doi.org/10.1182/blood.v128.22.3841.3841.
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Abstract Background Platelet transfusion refractoriness (PTR) due to immune factors occurs in 5-15% of thrombocytopenic patients who have received transfusions. The dominant cause of immune PTR is the production of allo-antibodies to human leukocyte antigen (HLA) class I, which is expressed on platelets. In current clinical settings, transfusion of HLA-compatible platelets is the only practical strategy, but their supply is weak due to limited donor source, gives excessive burden on specific donors, and requires increased efforts and costs. To overcome these issues, we plan to produce HLA-knockout platelets from iPSCs-derived megakaryocytes (MKs) as an alternative solution, applicable to all HLA types. However, whether they would be attacked by natural killer (NK) cells has not been well-studied. NK cells are known to show cytotoxic activity against cells downregulated for HLA class I ("missing self" theory). Therefore we assessed the interaction between HLA-knockout platelets derived from induced pluripotent stem cells (iPSCs) and NK cells in allogeneic settings. Methods and Results Immortalized megakaryocyte progenitor cell lines (imMKCLs) were previously established from iPSCs as a source of platelet production with a robust proliferation potential (Nakamura, 2014). Beta 2-microglobulin gene was knocked-out by CRISPR/Cas9 system to obtain HLA-knockout imMKCLs and platelets. NK cells were prepared from peripheral blood of eleven healthy donors. After co-cultures of NK cells and target cells for 6 hours with IL-2, we examined the NK cell cytolytic activity marker CD107, and target cell damage marker Annexin V using flow cytometry. Positive rates of both markers were not enhanced by co-culture with either HLA-expressed or HLA-knockout platelets for all donors. Furthermore, addition of platelets showed minimal effect on high cytotoxic activity of NK cells against K562 cells. In contrast, coculture of imMKCLs with NK cells resulted in higher detection of CD107 and Annexin V staining in some NK cell donors. These data suggested that platelets are immunologically inert for NK cells irrespective of class I HLA expression, while imMKCLs can be potentially attacked. Accordingly, platelets did not express NK cell activating ligands, which were expressed on imMKCLs and K562 cells. To confirm the above-mentioned results in vivo, mice were transfused with NK cells and platelets and MKs together. In our preliminary data, the circulation of platelets was not different between HLA-expressed or HLA-knockout type. In contrast, MKs were shown to be attacked in some cases. Conclusion HLA-knockout platelets evaded attacked from NK cells, while imMKCLs possessed immunogenicity to NK cells. This study provides extended experimental evidence that HLA-knockout platelets produced from a single imMKCL clone are immunologically applicable to all HLA types including majority of patients with PTR. On the other hand, contaminating imMKCLs in imMKCL-derived platelet products can be rejected by NK cells, contributing to their enhanced safety profiles. Taken together, stage of HLA-deficiency in imMKCLs as a starting material of platelet supply shall lead to industrial production of HLA universal platelets. Disclosures No relevant conflicts of interest to declare.
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Kwon, Yoo-Wook, Hyo-Suk Ahn, Jin-Woo Lee, Han-Mo Yang, Hyun-Jai Cho, Seok Joong Kim, Shin-Hyae Lee et al. "HLA DR Genome Editing with TALENs in Human iPSCs Produced Immune-Tolerant Dendritic Cells". Stem Cells International 2021 (20 de mayo de 2021): 1–14. http://dx.doi.org/10.1155/2021/8873383.
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Although human induced pluripotent stem cells (iPSCs) can serve as a universal cell source for regenerative medicine, the use of iPSCs in clinical applications is limited by prohibitive costs and prolonged generation time. Moreover, allogeneic iPSC transplantation requires preclusion of mismatches between the donor and recipient human leukocyte antigen (HLA). We, therefore, generated universally compatible immune nonresponsive human iPSCs by gene editing. Transcription activator-like effector nucleases (TALENs) were designed for selective elimination of HLA DR expression. The engineered nucleases completely disrupted the expression of HLA DR on human dermal fibroblast cells (HDF) that did not express HLA DR even after stimulation with IFN-γ. Teratomas formed by HLA DR knockout iPSCs did not express HLA DR, and dendritic cells differentiated from HLA DR knockout iPSCs reduced CD4+ T cell activation. These engineered iPSCs might provide a novel translational approach to treat multiple recipients from a limited number of cell donors.
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Zha, Shijun, Johan Chin-Kang Tay, Sumin Zhu, Zhendong Li, Zhicheng Du y Shu Wang. "Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived β2 Microglobulin Knockout Induced Pluripotent Stem Cells". Cell Transplantation 29 (1 de enero de 2020): 096368972096552. http://dx.doi.org/10.1177/0963689720965529.
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Mesenchymal stromal cells (MSCs) are viewed as immune-privileged cells and have been broadly applied in allogeneic adoptive cell transfer for regenerative medicine or immune-suppressing purpose. However, the surface expression of human leukocyte antigen (HLA) class I molecules on MSCs could still possibly induce the rejection of allogeneic MSCs from the recipients. Here, we disrupted the β2 microglobulin ( B2M) gene in human peripheral blood mononuclear cell-derived induced pluripotent stem cells (iPSCs) with two clustered regulatory interspaced short palindromic repeat (CRISPR)-associated Cas9 endonuclease-based methods. The B2M knockout iPSCs did not express HLA class I molecules but maintained their pluripotency and genome stability. Subsequently, MSCs were derived from the HLA-negative iPSCs (iMSCs). We demonstrated that B2M knockout did not affect iMSC phenotype, multipotency, and immune suppressive characteristics and, most importantly, reduced iMSC immunogenicity to allogeneic peripheral blood mononuclear cells. Thus, B2M knockout iPSCs could serve as unlimited off-the-shelf cell resources in adoptive cell transfer, while the derived iMSCs hold great potential as universal grafts in allogeneic MSC transplantation.
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Karkischenko, V. N., A. G. Berzina, I. A. Pomytkin, E. S. Glotova, M. A. Savina, D. V. Petrov, L. A. Taboyakova, L. А. Bolotskih y I. A. Vasil’eva. "Immune Response in HLA-A*02:01 Transgenic Humanized Mice to the Introduction of Horse IgG Antigen". Journal Biomed 20, n.º 2 (23 de julio de 2024): 45–52. http://dx.doi.org/10.33647/2074-5982-20-2-45-52.
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The introduction of a transgene can impact negatively the functioning of vital systems in biomodels. We carried out a comparative analysis of the immune response of mice of the HLA-A*02:01 humanized transgenic line, mice with mouse β2-microglobulin gene knockout, and wild-type mice to the introduction of horse immunoglobulin as an antigen. The biomodel lines were created at the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia. The maximum immune response was achieved on the 30th day from the onset of immunization in animals of the HLA-A*02:01 line and wild-type mice. Antibody titers in these groups increased sharply and approached 1:8,000,000 and 1:4,000,000, respectively. This indicates that genome modification in HLA-A*02:01 transgenic humanized mice did not affect functioning of the immune system. No similar dynamics of the increase in antibody titers was observed in the mice line with mouse β2-microglobulin gene knockout. On the 7th and 30th day, the antibody titer in this group increased to a value of 1:400 and 1:6,400, respectively. The weak immune response in mice with mouse β2-microglobulin gene knockout confirms the undeniably important role of this protein in immune response formation.
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Rivera González, Lorena, Yaritza Inostroza-Nieves, Alexandra Lozano, Pablo J. López, Jamie Rosado Alicea, Gregory N. Prado, Jose R. Romero y Alicia Rivera. "Endothelin-1 Regulates Molecules of the Major Histocompatibility Complex: Role in Sickle Cell Disease". Blood 128, n.º 22 (2 de diciembre de 2016): 3638. http://dx.doi.org/10.1182/blood.v128.22.3638.3638.
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Abstract Molecules of the Major Histocompatibility Complex (MHC), and in particular specific human leukocyte antigen (HLA) alleles, have been proposed in the pathophysiology of immune and vascular alterations leading to vasoocclusive crises (VOC) and stroke in Sickle Cell Disease (SCD). Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion and excess levels of the potent mitogen, endothelin-1 (ET-1). ET-1 activates endothelial cells, induces oxidative stress and inflammation in the vascular wall and regulates erythrocyte homeostasis. However, the role of ET-1 on MHC regulation in SCD is not clear. We first characterized the effect of ET-1 on MHC expression in the human endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p<0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p<0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p<0.05 n=4) in EA.hy926 cells. ET-1-stimulated expression of HLA-A2, HLA-DR and CIITA were significantly blocked by pre-incubation of cells with 10 µM BQ788, a selective blocker of ET-1 type B receptors (p<0.01, n=4). Chromatin immunoprecipitation (ChIP) studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the promoter region within MHC molecules (HLA-A2 62% ± 5%, HLA-DRB 33% ± 18%, p<0.01, n=4); an event that was likewise blocked by BQ788. We then studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βSAntilles and Berkeley (BERK) mice, respectively. We observed significant increases in MHC molecule, H2-Aa mRNA levels (n=7; p<0.01) in spleens from sickle transgenic mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). We then treated BERK, βSAntilles and HbA mice for 14 days with ET-1 receptor antagonists and observed significant reductions in H2-Aa mRNA levels in spleen tissue from sickle transgenic mice but not in HbA mice (n=7; p<0.05). These results implicate ET-1 as a novel regulator of MHC molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD. Disclosures No relevant conflicts of interest to declare.
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Veldman, Johanna, Lydia Visser, Magdalena Huberts-Kregel, Natasja Muller, Bouke Hepkema, Anke van den Berg y Arjan Diepstra. "Rosetting T cells in Hodgkin lymphoma are activated by immunological synapse components HLA class II and CD58". Blood 136, n.º 21 (19 de noviembre de 2020): 2437–41. http://dx.doi.org/10.1182/blood.2020005546.
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Abstract A unique feature of Hodgkin lymphoma (HL) is the presence of CD4+ T cells that surround, protect, and promote survival of tumor cells. The adhesion molecules involved in this so-called T-cell rosetting are important components of the immunological synapse (IS). However, it is unknown whether this synapse is fully assembled and leads to T-cell activation by enabling interaction between the T-cell receptor (TCR) and human leukocyte antigen class II (HLA-II). We established a novel rosetting model by coculturing HLA-II–matched peripheral blood mononuclear cells with HL cell lines and showed IS formation with activation of rosetting T cells. HLA-II downregulation by class II transactivator knockout did not affect the extent of rosetting, but almost completely abrogated T-cell activation. Intriguingly, the level of CD58 expression correlated with the extent of rosette formation, and CD58 knockout or CD2 blockade reduced both rosette formation and T-cell activation. The extension of our findings to primary HL tissue by immunohistochemistry and proximity ligation assays showed interaction of CD2 with CD58 and of TCR-associated CD4 with HLA-II. In conclusion, T-cell rosetting in HL is established by formation of the IS, and activation of rosetting T cells critically depends on the interaction of both TCR-HLA-II and CD2-CD58.
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Chen, Liye, Hui Shi, Jack Yuan y Paul Bowness. "Position 97 of HLA-B, a residue implicated in pathogenesis of ankylosing spondylitis, plays a key role in cell surface free heavy chain expression". Annals of the Rheumatic Diseases 76, n.º 3 (11 de agosto de 2016): 593–601. http://dx.doi.org/10.1136/annrheumdis-2016-209512.
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ObjectiveAssociation of position 97 (P97) residue polymorphisms in human leucocyte antigen (HLA)-B, including HLA-B*27, with ankylosing spondylitis (AS) has recently been reported. We studied the effect of P97 variations on cell surface expression of the AS-associated HLA-B*27 and HLA-B*51, and the AS-protective HLA-B*7.MethodsFlow cytometry was used to measure surface expression of HLA-B*27 in C1R/HeLa cells expressing HLA-B*27 (N97) and six mutants at P97 (N97T, N97S, N97V, N97R, N97W and N97D). Transporter associated with antigen processing-deficient T2, tapasin-deficient 220, β2m-deficient HCT15 and endoplasmic reticulum aminopeptidase 1 or β2m-clustered regularly interspaced short palindromic repeats/Cas9-knockout HeLa cells were used to provide evidence for specific protein interactions. Surface expression of HLA-B*7/HLA-B*51 P97 mutants was also studied.ResultsMutation of HLA-B*27 P97 to the AS risk residue threonine increased cell surface free heavy chain (FHC) expression. Protective residues (serine or valine) and non-AS-associated residues (arginine or tryptophan) did not alter FHC expression. The N97D mutation reduced expression of conventional and FHC forms of HLA-B*27. Differences in FHC expression levels between HLA-B*27, HLA-B*27-N97T and HLA-B*27-N97D were dependent on the presence of functional β2m. HLA-B*7, which has an AS-protective serine at P97, expressed lower levels of FHC than HLA-B*27 or HLA-B*51. Introduction of asparagine at P97 of both HLA-B*7 and HLA-B*51 increased FHC expression.ConclusionsThe nature of P97 residue affects surface expression of HLA-B*27, B*7 and B*51, with AS-associated residues giving rise to higher FHC expression levels. The association of P97 amino acid polymorphisms with AS could be, at least in part, explained by its effect on HLA-B*27 FHC cell surface expression.
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Torikai, Hiroki, Andreas Reik, Carrie Yuen, Yuanyue Zhou, Denise Kellar, Helen Huls, Edus H. Warren et al. "HLA and TCR Knockout by Zinc Finger Nucleases: Toward “off-the-Shelf” Allogeneic T-Cell Therapy for CD19+ Malignancies." Blood 116, n.º 21 (19 de noviembre de 2010): 3766. http://dx.doi.org/10.1182/blood.v116.21.3766.3766.
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Abstract Abstract 3766 Cell therapy by infusion of T cells can reconstitute immunity to combat pathogens and malignancies. However, the time required to manufacture T cells with the desired properties and in sufficient numbers ex vivo is often incompatible with the treatment window for patients. Furthermore, autologous T cells from patients with advanced disease may have compromised function and be tolerant to desired antigens. A potential solution would be an approach to infuse allogeneic T cells that avoids immune-mediated rejection caused by host T cells recognizing disparate major or minor histocompatibility antigens on the infused cells. To broaden the application of T cell therapy, we investigated whether HLA gene expression can be disrupted by designer zinc-finger nucleases (ZFNs). ZFNs comprise a zinc finger DNA binding domain designed to bind a specific DNA sequence fused to the cleavage domain of Fok I endonuclease. Since FokI dimerization is required to introduce a double strand break (DSB), we generated ZFN pairs that flank the intended DNA target sequences in the required spatial conformation. Cellular repair of the DSB by error-prone non-homologous end joining allows disruption of HLA gene expression. As an initial proof of concept experiment, transfection of ZFN pairs designed to target exon 3 of the HLA-A locus into the human kidney cell line HEK293 resulted in 10% genetic modification of the HLA-A loci. We generated clones of HEK293 cells that showed deletion or insertion mutations within the ZFN binding site of one or both HLA-A alleles leading to early termination of translation. These HLA-Anull HEK293 clones evaded HLA-A-restricted lysis by T cell clones, even after interferon-γ and TNF-α treatment was used to upregulate HLA expression. Since only transient expression of ZFNs is needed to disrupt a target gene, we tested the ability to disrupt HLA-A gene expression by electro-transfer of in vitro-transcribed ZFN mRNA into primary T cells. We show that a single administration of the mRNA encoding the ZFNs targeting HLA-A could render over 40% of primary T cells HLA-A negative. We enriched the HLA-Anull population by paramagnetic bead separation to obtain a pool of T cells >90% of which lack HLA-A expression. An attractive potential clinical application for HLAnull allogeneic T cells is to redirect their specificity independent of HLA via expression of a chimeric antigen receptor (CAR) targeting CD19. Thus, we eliminated HLA-A expression from CD19-specific CAR+ T cells and demonstrated that they (i) evade HLA-A-restricted lysis by T cell clones, and (ii) specifically lysed CD19+ tumor targets. Finally, to further improve this T cell product and eliminate potential deleterious immune mediated recognition by the endogenous T cell receptor (TCR) on allogeneic CAR+ T cells, we used ZFN pairs targeting the TCR α or the TCR β locus. Transient expression of these ZFNs resulted in permanent disruption of endogenous TCR expression and a highly enriched αβ TCRnull cell population could be generated by paramagnetic bead selection. These data support our plans to develop allogeneic T cells as “off-the-shelf” biologics that can be infused on demand as “drugs”. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment.
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Legut, Mateusz, Garry Dolton, Afsar Ali Mian, Oliver G. Ottmann y Andrew K. Sewell. "CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells". Blood 131, n.º 3 (18 de enero de 2018): 311–22. http://dx.doi.org/10.1182/blood-2017-05-787598.
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Key Points Endogenous TCR knockout increases the expression and functional activity of simultaneously transduced TCR (TCR replacement). TCR replacement results in superior targeting of hematological malignancies by T cells transduced with a non–HLA-restricted γδ TCR.
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Croom-Perez, Tayler J., Liza D. Robles-Carrillo, Md Faqrul Hasan y Alicja J. Copik. "Abstract 2910: NKG2A suppression enhances the function of primary human Natural Killer cells". Cancer Research 83, n.º 7_Supplement (4 de abril de 2023): 2910. http://dx.doi.org/10.1158/1538-7445.am2023-2910.
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Abstract In this study we propose inhibition of NKG2A signaling enhances the anti-tumor response of primary human NK cells. One mechanism employed by tumor cells to evade immunosurveillance is through induction of the surface expression of unconventional HLA ligands that agonize inhibitory receptors on immune cells. Specifically on some tumors, HLA-E is either expressed or its expression is known to be induced by IFNγ and is indicative of resistance to immunotherapy. HLA-E agonizes the CD94/NKG2A inhibitory complex on NK cells and some T cells to lessen their cytotoxicity, potentially decreasing the efficacy of cellular therapy with these immune cells. In this study ex vivo-expanded primary human NK cells were shown to have significantly elevated expression of NKG2A. These expanded NK cells also secreted high levels of IFNγ and could induce expression of HLA-E on tumor cells. Suppression of NKG2A by either antibody blockade or gene deletion increased the cytotoxicity of NK cells against the lung cancer cell line A549 stably expressing HLA-E. NKG2A knockout cells had an almost 10-fold decrease in EC50 compared to wild-type NK cells (EC50 0.15 vs 0.94 at 48 h), meaning nearly log-fold fewer NKG2A knockout cells are needed to kill the same number of tumor cells. The effect of NKG2A knockout on cytotoxicity of NK cells against other cancer cell lines stably expressing HLA-E will be presented along with effects of NKG2A suppression on NK cell phenotype, cytokine secretion and degranulation. These initial data suggest that blockade of the CD94/NKG2A inhibitory complex can improve the function of ex vivo expanded human NK cells and could provide an effector population with the potential for enhanced therapeutic efficacy. Citation Format: Tayler J. Croom-Perez, Liza D. Robles-Carrillo, Md Faqrul Hasan, Alicja J. Copik. NKG2A suppression enhances the function of primary human Natural Killer cells [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 2910.
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Krco, Christopher J., Shohei Watanabe, Jerry Harders, Marie M. Griffths, Harvinder Luthra y Chella S. David. "Identification of T Cell Determinants on Human Type II Collagen Recognized by HLA-DQ8 and HLA-DQ6 Transgenic Mice". Journal of Immunology 163, n.º 3 (1 de agosto de 1999): 1661–65. http://dx.doi.org/10.4049/jimmunol.163.3.1661.
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Abstract HLA-DQA1*0301 and HLA-DQB1*0302 genes encoding the HLA-DQ8 molecule and HLA-DQA1*0103 and HLA-DQB1*0601 genes encoding the HLA-DQ6 molecule were introduced into H-2Aβo knockout mice. Three lines of transgenic mice were established: HLA-DQ8, HLA-DQ6, and HLA-DQ8β6α. HLA-DQ8 mice are susceptible to collagen-induced arthritis, while HLA-DQ6 mice are resistant. HLA-DQ8β6α mice develop polychrondritis in addition to arthritis. Transgenic mice were primed and challenged with individual synthetic peptides representing human type II collagen. A total of 101 synthetic peptides were tested in each transgenic line of mice. HLA-DQ8 mice responded to 15 synthetic peptides representing all cyanogen bromide fragments. In contrast, HLA-DQ6 mice responded to a subset of the peptides recognized by HLA-DQ8 T cells. HLA-DQ8β6α mice, although exhibiting diminished responses to the majority of HLA-DQ8-restricted determinants, elicited enhanced responses to two peptides. In addition, HLA-DQ8β6α mice respond to two unique peptide determinants contained within cyanogen bromide fragments CB10 and CB11 showing the significance of mixed isotype dimers in the immune response. The determinants recognized by the HLA-DQ transgenic mice are distinct from those previously identified using conventional laboratory mice. These results suggest that human class II transgenic mice offer a means of identifying human class II-restricted epitopes associated with potential human autoantigens.
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Neeno, T., C. J. Krco, J. Harders, J. Baisch, S. Cheng y C. S. David. "HLA-DQ8 transgenic mice lacking endogenous class II molecules respond to house dust allergens: identification of antigenic epitopes." Journal of Immunology 156, n.º 9 (1 de mayo de 1996): 3191–95. http://dx.doi.org/10.4049/jimmunol.156.9.3191.
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Abstract We have introduced HLA-DQ8 (HLADQB*0302 and HLA-DQA*0301) genes into A beta 0 knockout mice. Transgenic animals were immunized with a whole body extract of Dermatophagoides pteronyssinus (Der p), one of the causative agents of house dust mite allergy. Transgenic mice expressing HLA-DQ8 genes elicited HLA-DQ8-restricted responses driven by CD4+ T cells. Synthetic-overlapping peptides representing a major allergen of house dust mite (Der p 2) were synthesized and used as immunogens. HLA-DQ8+ mice responded to three peptides: 8 (residues 61-80), 11 (residues 91-110), and 13 (residues 111-129). The mice produced IL-2, IL-4, and IL-6 response to Der p challenge, suggesting a mixed Th1/Th2 response. These mice represent a new model for studies of the immune basis of allergy.
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Karkischenko, V. N., V. A. Ezerskiy, E. M. Koloskova y M. S. Nesterov. "Preparation of Differentiated Recombinant Human β2-Microglobulin and Mouse β2-Microglobulin Proteins for its Detection in Class I HLA Chimeric Molecules". Journal Biomed 20, n.º 2 (23 de julio de 2024): 21–31. http://dx.doi.org/10.33647/2074-5982-20-2-21-31.
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Transgenic humanized animals are increasingly in demand for biomedical research and pharmacological testing. More and more lines of transgenic animals are being created, including those with knockout of their own genes. There is an urgent need for an evidence base for the integration of a transgene, its expression, determination of the knockout state of its own gene at the molecular genetic level, detection of translation of the target protein in different organs and tissues, proof for the absence of protein synthesis (or its non-functionality), the gene of which has been modified. This requires highly specific reagents, proteins and antibodies to them in particular, the vast majority of which are presented by foreign manufacturers. The task was set to identify mouse and human β2-microglobulin in protein fractions of organs and tissues of transgenic and knockout mice of several HLA lines created in recent years at the Scientific Center of Biomedical Technologies, Russia. At the first stage of our research, recombinant E. coli producing strains were obtained.
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Ureta-Vidal, Abel, Hüseyin Firat, Béatrice Pérarnau y François A. Lemonnier. "Phenotypical and Functional Characterization of the CD8+ T Cell Repertoire of HLA-A2.1 Transgenic, H-2K b °D b ° Double Knockout Mice". Journal of Immunology 163, n.º 5 (1 de septiembre de 1999): 2555–60. http://dx.doi.org/10.4049/jimmunol.163.5.2555.
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Abstract Homozygous HLA-A2.1 transgenic H-2Kb°Db° double knockout (KO) mice were created. Their potential to develop HLA-A2.1-restricted cytolytic responses was compared with that of their classical transgenic counterparts, which still express H-2Kb, Db molecules. On cell surfaces, both strains express similar amounts of chimeric (α1α2 domains of human, α3 cytoplasmic domains of mouse) HLA-A2.1 molecules in noncovalent association with mouse β2-microglobulin. Compared with mice that are totally deprived of histocompatibility class Ia molecules (H-2Kb°Db° double KO), the expression of HLA-A2.1 in transgenic/double KO mice resulted in sizeable increase in the periphery of CD8+ T cells with a normally diversified TCR repertoire. A biased education in favor of HLA-A2.1, ascribable to the absence of H-2 class Ia molecules, was evidenced in these transgenic/double KO mice by their improved capacity to mount HLA-restricted cytolytic responses, regardless of whether they were virally infected or injected with synthetic epitopic peptide. HLA class I transgenic, H-2 class Ia KO mice should represent useful animal models for the preclinical evaluation of vaccine formulations aiming at the induction of HLA class I-restricted CTL responses.
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Nalawade, Saisha A., Niannian Ji, Ellen Kraig y Thomas Forsthuber. "Aire is not essential for regulating autoimmune pathology in mice transgenic for human autoimmune-disease associated MHC class II genes HLA-DR2b and HLA-DR4." Journal of Immunology 200, n.º 1_Supplement (1 de mayo de 2018): 167.8. http://dx.doi.org/10.4049/jimmunol.200.supp.167.8.
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Abstract The human MHC class II molecules HLA-DR2b (DRB1*1501) and HLA-DR4 (DRB1*0401) are strongly linked to increased susceptibility to autoimmune diseases, such as multiple sclerosis (MS) and rheumatoid arthritis (RA). However, their contribution to disease pathogenesis is not completely understood. Conceivably, autoimmune disease-associated HLA alleles could shape the repertoire of pathogenic T cells via central or peripheral tolerance. The ectopic expression of tissue-specific antigens promoted by the autoimmune regulator (AIRE) is thought to play a critical role in central tolerance. Aire knockout mice (Aire−/−) develop spontaneous autoimmune pathology characterized by multi-organ lymphocytic infiltrates. Aire deficiency in humans causes multi-organ autoimmune conditions observed in autoimmune polyendocrinopathy syndrome type 1 (APS I). We wanted to investigate whether lack of Aire expression promoted spontaneous MS- or RA-like autoimmune pathology in HLA-DR2b or HLA-DR4 transgenic (tg) mice. We observed that Aire-deficiency modestly enhanced experimental autoimmune encephalomyelitis (EAE) in HLA-DR tg mice, but did not lead to spontaneous neuroinflammation or arthritis. Our results suggest that Aire has a mild and non-essential effect in restraining the autoimmune response in the context of autoimmune disease-associated human HLA alleles.
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Santos, M., M. W. Schilham, L. H. Rademakers, J. J. Marx, M. de Sousa y H. Clevers. "Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man." Journal of Experimental Medicine 184, n.º 5 (1 de noviembre de 1996): 1975–85. http://dx.doi.org/10.1084/jem.184.5.1975.
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Previously, hepatic iron overload resembling that in hereditary hemachromatosis (HH) has been found in beta 2-microglobulin knockout (beta 2m-/-) mice. We have now characterized iron metabolism in beta 2m-/- mice. The mutant mice fail to limit the transfer of iron from mucosal cells into the plasma. Transferrin saturation is abnormally high. Pathologic iron depositions occur predominantly in liver parenchymal cells. Reconstitution with normal hematopoietic cells redistributes the iron from parenchymal to Kupffer cells, but does not correct the mucosal defect. We conclude that (a) iron metabolism is defective in the gut mucosa as well as the liver of beta 2m-/- mice; and (b) a beta 2m-dependent gene product is involved in iron homeostasis. Recently, a novel gene of the major histocompatibility complex class I family, HLA-H, has been found to be mutated in a large proportion of HH patients. Our data provide functional support for the proposed causative role of HLA-H mutations in HH.
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Pascolo, Steve, Nathalie Bervas, Jan M. Ure, Austin G. Smith, François A. Lemonnier y Béatrice Pérarnau. "HLA-A2.1–restricted Education and Cytolytic Activity of CD8+ T Lymphocytes from β2 Microglobulin (β2m) HLA-A2.1 Monochain Transgenic H-2Db β2m Double Knockout Mice". Journal of Experimental Medicine 185, n.º 12 (16 de junio de 1997): 2043–51. http://dx.doi.org/10.1084/jem.185.12.2043.
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Three different HLA-A2.1 monochains were engineered in which either the human or mouse β2-microglobulin (β2m) is covalently linked to the NH2 terminus of the heavy chain by a 15– amino acid long peptide: HHH, entirely human, HHD, with the mouse H-2Db α3, transmembrane, and cytoplasmic domains, and MHD, homologous to HHD but linked to the mouse β2mb. The cell surface expression and immunological capacities of the three monochains were compared with transfected cells, and the selected HHD construct was introduced by transgenesis in H-2Db−/− β2m−/− double knockout mice. Expression of this monochain restores a sizable peripheral CD8+ T cell repertoire essentially educated on the transgenic human molecule. Consequently, infected HHD, H-2Db−/− β2m−/− mice generate only HLA-A2.1–restricted CD8+ CTL responses against influenza A and vaccinia viruses. Interestingly, the CTL response to influenza A virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1–restricted immunodominant epitope in humans. Such mice might constitute a versatile animal model for the study of HLA-A2.1–restricted CTL responses of vaccine interest.
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Kushniarova, Lizaveta V., Alexandr A. Migas, Hanna V. Klych, Yauheni A. Lasiukov y Alexander N. Meleshko. "Knockout of the T-cell receptor and HLA class I genes in human cells using the CRISPR /Cas9 system". Experimental Biology and Biotechnology, n.º 2 (6 de julio de 2022): 19–26. http://dx.doi.org/10.33581/2957-5060-2022-2-19-26.
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The CRISPR /Cas9 system has found a wide application in cell biology as a tool for gene knockout. In particular, the CRISPR /Cas9 system is used to create allogeneic CAR-T lymphocytes by knocking out the genes TRAC, TRBC1, TRBC2 and B2M. To obtain a large number of cells of the desired phenotype, it is necessary to optimise the process of genomic editing, the effectiveness of which is determined by the sgRNA used. In this work, we experimentally determined the most effective sequences that allow to obtain up to 60.3 % of cells negative for the expression of the B2M protein and up to 71.8 % of cells negative for the expression of the T-cell receptor. It has also been shown that the simultaneous use of two sgRNAs for gene knockout demonstrates a lower efficiency compared to using these sgRNAs separately.
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Rajagopalan, Govindarajan, Ashenafi Tilahun y Vaidehi Chowdhary. "Chronic activation with a staphylococcal superantigen drives the expansion of CD4, CD8 double negative T cells and promotes multiorgan inflammation mimicking systemic lupus erythematosus in HLA class II transgenic mice. (HUM7P.306)". Journal of Immunology 192, n.º 1_Supplement (1 de mayo de 2014): 184.15. http://dx.doi.org/10.4049/jimmunol.192.supp.184.15.
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Abstract CD4 and CD8 coreceptors play an important role in the development and maturation of αβ TCR+ T cells. However, T cells lacking CD4 and CD8 (double negative T or DNT cells) are present in the periphery and are significantly expanded in systemic lupus erythematosus (SLE) and Sjogren’s syndrome. The antigens that cause expansion of these DNT cells are unknown. As T cell activation by superantigens does not require coreceptors, we hypothesized that superantigens could activate DNT cells and contribute to autoimmunity. HLA-DQ8 and HLA-DR3 transgenic mice lacking both CD4 and CD8 were generated by intercrossing CD4 and CD8 single knockout mice. The CD4.CD8 double knockout (DKO) mice harbored a diverse repertoire of DNT cells in the periphery, albeit at reduced numbers compared to wild type (WT) mice. DNT cells from DKO mice responded robustly to the superantigen, staphylococcal enterotoxin B, but not to HLA restricted conventional antigens. Implantation of mini-osmotic pumps delivering 10 or 50 μg of SEB continuously over 7 or 28 days induced a lupus-like disease in WT as well as DKO DR3 and DQ8 mice. However, the immunopathology was significantly more pronounced in DKO mice. Immunostaining revealed extensive infiltration of multiple organs including lungs, liver and kidneys with CD3+ T cells in DKO mice. Overall, our study unraveled the role of CD4 and CD8 coreceptors in T cell development/function and the role of superantigens and DNT cells in the pathogenesis of lupus.
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Elliott, J. F., J. Liu, Z. N. Yuan, N. Bautista-Lopez, S. L. Wallbank, K. Suzuki, D. Rayner et al. "Autoimmune cardiomyopathy and heart block develop spontaneously in HLA-DQ8 transgenic IA knockout NOD mice". Proceedings of the National Academy of Sciences 100, n.º 23 (21 de octubre de 2003): 13447–52. http://dx.doi.org/10.1073/pnas.2235552100.
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Chen, Liye, Hui Shi, Danai Koftori, Takuya Sekine, Annalisa Nicastri, Nicola Ternette y Paul Bowness. "Identification of an Unconventional Subpeptidome Bound to the Behçet's Disease-associated HLA-B*51:01 that is Regulated by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1)". Molecular & Cellular Proteomics 19, n.º 5 (11 de marzo de 2020): 871–83. http://dx.doi.org/10.1074/mcp.ra119.001617.
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Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behçet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by mass spectrometry. The characteristics of non-Pro/Ala2, Pro2, and Ala2 peptides and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Pro or Ala at P2. This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared with the Pro2 and Ala2 subpeptidomes and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant leucine at position Ω). Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to ∼40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell-type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 subpeptidome. It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.
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Venkatasubramaniam, Arundhathi, Tulasikumari Kanipakala, Nader Ganjbaksh, Rana Mehr, Ipsita Mukherjee, Subramaniam Krishnan, Taeok Bae, M. Aman y Rajan Adhikari. "A Critical Role for HlgA in Staphylococcus aureus Pathogenesis Revealed by A Switch in the SaeRS Two-Component Regulatory System". Toxins 10, n.º 9 (18 de septiembre de 2018): 377. http://dx.doi.org/10.3390/toxins10090377.
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Cytolytic pore-forming toxins including alpha hemolysin (Hla) and bicomponent leukotoxins play an important role in the pathogenesis of Staphylococcus aureus. These toxins kill the polymorphonuclear phagocytes (PMNs), disrupt epithelial and endothelial barriers, and lyse erythrocytes to provide iron for bacterial growth. The expression of these toxins is regulated by the two-component sensing systems Sae and Agr. Here, we report that a point mutation (L18P) in SaeS, the histidine kinase sensor of the Sae system, renders the S. aureus Newman hemolytic activity fully independent of Hla and drastically increases the PMN lytic activity. Furthermore, this Hla-independent activity, unlike Hla itself, can lyse human erythrocytes. The Hla-independent activity towards human erythrocytes was also evident in USA300, however, under strict agr control. Gene knockout studies revealed that this Hla-independent Sae-regulated activity was entirely dependent on gamma hemolysin A subunit (HlgA). In contrast, hemolytic activity of Newman towards human erythrocytes from HlgAB resistant donors was completely dependent on agr. The culture supernatant from Newman S. aureus could be neutralized by antisera against two vaccine candidates based on LukS and LukF subunits of Panton-Valentine leukocidin but not by an anti-Hla neutralizing antibody. These findings display the complex involvement of Sae and Agr systems in regulating the virulence of S. aureus and have important implications for vaccine and immunotherapeutics development for S. aureus disease in humans.
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Karkischenko, N. N., V. N. Lazarev, V. A. Manuvera, P. A. Bobrovsky, N. V. Petrova, E. M. Koloskova y E. S. Glotova. "Principles of Creation of a Genetic Engineering Construction for Obtaining Humanized Transgenic Mice with <i>HLA-C*07:02:01:01</i>, as a Promote of Innovative Transgenic and Knockout Biomodels". Journal Biomed 20, n.º 1 (5 de abril de 2024): 8–20. http://dx.doi.org/10.33647/2074-5982-20-1-8-20.
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Genetic differences in different populations influence the mechanism and efficacy of drugs. Biomodels that take into account the peculiarities of genetic polymorphism in different individuals allow to more fully investigate the molecular-genetic mechanisms of action of pharmacological agents, including immunobiological ones. Recombinant DNA encoding a hybrid MHC class I protein containing human ß2-microglobulin fused with antigen-presenting domains (α1 and α2 domains) of the HLA-C*07:02:01:01 molecule and α3 domain of the mouse H2-complex was created. The purified linearized DNA fragment containing the target construct flanked by regulatory fragments ensuring its stable transcription was used to obtain a new line of humanized transgenic mice. The principles of designing humanized transgenic mice by encoding a chimeric MHC class I protein containing antigen-presenting domains HLA-C*07:02:01:01 are similar to those for obtaining mice of the HLA-А*02:01:01 and HLA-B*18:01:01:02 humanized transgenic lines. These transgenic lines of laboratory mice are independent biomodels, and also be used as baselines for obtaining corresponding transgenic and knockout lines.
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Chapoval, Svetlana P., Teresa Neeno, Christopher J. Krco, Eric V. Marietta, Jerry Harders y Chella S. David. "HLA-DQ6 and HLA-DQ8 Transgenic Mice Respond to Ragweed Allergens and Recognize a Distinct Set of Epitopes on Short and Giant Ragweed Group 5 Antigens". Journal of Immunology 161, n.º 4 (15 de agosto de 1998): 2032–37. http://dx.doi.org/10.4049/jimmunol.161.4.2032.
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Abstract We have investigated the genetic and molecular basis of immune responsiveness to short ragweed (SRW) (Ambrosia artemisiifolia) extract, and group 5 allergens from short and giant (Ambrosia trifida) ragweed using transgenic mice expressing DQ6 (HLA-DQA1*0103, HLA-DQB1*0601) and DQ8 (HLA-DQA1*0301, HLA-DQB1*0302) genes in class II knockout (Aβ0) mice. Panels of overlapping peptides spanning the Amb a 5 and Amb t 5 Ags were synthesized. Mice were immunized with whole SRW extract or individual peptides s.c. and lymph node cells (LNC) were challenged in vitro. Strong T cell responses to SRW extract were measured in both HLA-DQ transgenic mice, while control, HLA-DQ6−/DQ8−/H-2Aβ0, mice were unresponsive. IL-5 and IL-10 were the primary cytokines produced by in vitro challenged LNC of SRW-primed transgenic mice. HLA-DQ6-restricted T cell responses were detected to all three peptides of Amb t 5 and two determinants (residues 1–20 and 11–30) on Amb a 5. In contrast, LNC of HLA-DQ8 mice did not recognize peptide 11–30 of Amb t 5 Ag, but recognized several Amb a 5 determinants. The immune response in transgenic mice was dependent upon CD4+ T cells and was HLA-DQ restricted. Primed with purified Amb t 5, both transgenics recognized peptide 21–40, and an additional DQ6-restricted epitope was found within residue 1–20. SRW-immunized HLA-DQ6 mice respond to peptide 11–30 of Amb a 5, while HLA-DQ8 mice strongly recognize peptide 1–20. These results demonstrate the specificity of HLA class II polymorphism in allergen sensitivity and pave the way for developing antagonistic peptides for desensitization.
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Catelli, Lucas Ferioli, Marcus Alexandre Finzi Corat, Nádia Ghinelli Amôr, Irene Santos, Fernanda Soares Niemann, Adriana da Silva Santos Duarte y Sara Teresinha Olalla Saad. "Knockout of the Beta-2 Microglobulin Gene in Adipose Tissue-Derived Cells Using CRISPR/CAS9 System for the Generation of Universal HLA Class I Platelets". Blood 144, Supplement 1 (5 de noviembre de 2024): 1266. https://doi.org/10.1182/blood-2024-205233.
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Platelet refractoriness is a recurring problem in the treatment of thrombocytopenic patients, hindering the restoration of normal circulating platelet levels after platelet transfusion therapy. The primary cause is the incompatibility between the transfused platelets' HLA Class I antigens and the patient's antibodies. The production of platelets in vitro from genetically edited stem cells may become a future source of HLA Class I universal platelets that could help mitigate this problem. Adipose tissue-derived mesenchymal stem/stromal cells line (ASCL), generated from the dedifferentiation of adipocytes using the ceiling culture method, can be obtained in abundance and differentiated into megakaryocytes and platelets under suitable conditions. HLA Class I antigens are fixed on the surface of human cells through the binding to Beta-2 Microglobulin (B2M), expressed by the B2M gene. In this study, we performed gene editing on ASCL cells to obtain cell populations without HLA Class I antigens by knocking out the B2M gene using the CRISPR/Cas9 tool. Three guide RNAs (sgRNA) were designed and synthesized specifically to target DNA breaks in exons 1 or 2 of the B2M gene. The different sgRNAs were cloned into the pL-CRISPR.EFS.GFP plasmid, which was then sequenced by the Sanger automatic sequencing method for confirmation. Each cloned plasmid, along with two others (second generation) for lentiviral particle assembly, was transfected into HEK 293T cells, and each resulting lentiviral variety was collected and stored at -80°C until use. Different batches of each produced lentivirus variety were pooled, and a small sample was used for K562 cell transductions to estimate lentiviral production by titration, quantified by detecting green fluorescent protein (GFP) via flow cytometry. HEK 293T cells were also used for initial transduction tests and knockout efficiency using each of the three lentiviral varieties, either individually or in combination. The lentivirus carrying the cloned plasmid with the sgRNA guide#2 (LV-CRISPR-B2M-G2) was the most effective in reducing HLA Class I levels on the surface of HEK 293T cells, compared to the control (LV-CRISPR-B2M-EMPTY), and was then chosen for further study. Transductions of ASCL cells using the lentivirus LV-CRISPR-B2M-G2, with an MOI of approximately 13.5, resulted in viable, GFP-positive cell populations lacking B2M protein and HLA Class I antigens on their surface. The culture of transduced ASCL cells proliferated in vitro for more than 42 days, with at least 90% remaining negative for B2M and HLA Class I. Transduced and non-transduced ASCL cells were induced to megakaryocytic and platelet differentiation in vitro for 12 days, and the resulting cells were characterized for the expression of specific lineage markers and ploidy. The ASCL cells transduced with LV-CRISPR-B2M-G2 generated megakaryocyte and platelets populations with reduced expression of B2M and HLA Class I. The functionality of the platelets was verified through the augmented expression of p-selectin (CD62p) after stimulation with thrombin. The efficiency achieved in the transduction and knockout of the B2M gene using CRISPR/Cas9 technology, along with the significant reduction in HLA Class I antigen levels detected on the surface of ASCLs, megakaryocytes and platelets, demonstrates the potential of this approach for generating functional platelets lacking HLA Class I antigens. This suggests that these modified cells may be stored and later induced to differentiate into HLA Class I negative platelets, offering a promising strategy for developing alternative platelet sources for transfusions in refractory patients.
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Dufva, Olli, Jay Klievink, Khalid Saeed, Matti Kankainen, Mette Ilander, Tiina Hannunen, Sonja Lagström, Pekka Ellonen, Dean Anthony Lee y Satu Mustjoki. "Genome-Scale CRISPR Screens Identify Essential Genes for Sensitivity to Natural Killer Cells in Hematological Malignancies". Blood 132, Supplement 1 (29 de noviembre de 2018): 732. http://dx.doi.org/10.1182/blood-2018-99-117985.
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Abstract Immunotherapy is a promising approach to improve treatment responses in hematological malignancies. Accumulating evidence suggests a role for natural killer (NK) cells in controlling hematological malignancies. However, mechanisms regulating sensitivity or resistance of hematologic cancer cells to the effector function of NK cells are incompletely understood. Here, we performed genome-scale CRISPR-Cas9 loss-of-function screens to systematically map genes that regulate sensitivity of hematologic malignancies to NK cells. To screen for genes involved in the interaction between NK and cancer cells, we infected human cancer cells expressing Cas9 with a genome-scale lentiviral guide RNA library (Figure). The resulting pool of knockout cells was exposed to NK cells expanded from peripheral blood of healthy donors. Enriched and depleted knockouts were detected by next-generation sequencing of the integrated sgRNA cassettes, enabling identification of genes conferring resistance or susceptibility to NK cell-mediated lysis. The screens were performed in cell lines from diverse hematological malignancies, including chronic myeloid leukemia (CML), B cell acute lymphoblastic leukemia, diffuse large B cell lymphoma (DLBCL), and multiple myeloma. We recovered several known mechanisms of NK cell/cancer cell interactions, demonstrating feasibility of the screening approach. Loss of genes encoding components of the MHC class I complex (B2M, HLA-A, HLA-C, HLA-E) sensitized multiple cancer cell lines to NK cell lysis, consistent with missing-self recognition. Furthermore, knockout of IFN-JAK-STAT signaling mediators led to increased tumor cell lysis, suggesting that MHC class I induction in response to NK cell-derived IFN gamma enables NK cell evasion by tumor cells. We also identified genes essential for effective NK cell-mediated lysis. NCR3LG1, encoding the B7-H6 ligand for the NKp30 activating NK cell receptor, was essential for NK cell lysis of CML cells. In contrast, knockout of apoptotic mediators and TRAIL pathway components conferred resistance to NK cell cytotoxicity in DLBCL cells, indicating heterogeneity in NK cell/cancer cell interactions between cancer types. Our data support a view that distinct mechanisms regulate sensitivity to NK cell cytotoxicity in different hematologic cancers. Importantly, our results indicate that loss-of-function mutations in the antigen-presenting machinery and the IFN-JAK-STAT pathway sensitize tumors to NK cell effector function. As alterations in these genes are associated with resistance to T cell immunotherapies such as PD-1 blockade, NK cell-based therapies could be employed to overcome resistance in these patients. In summary, we suggest that systematic identification of mechanisms governing tumor immune susceptibility has the potential to uncover novel immunotherapy targets. Figure. Figure. Disclosures Kankainen: Medix Biochemica: Consultancy. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria; Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.
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Zaia, John A., Xiuli Li, Anne E. Franck, Xiwei Wu, Lia Thao y Ghislaine Gallez-Hawkins. "Biologic and Immunologic Effects of Knockout of Human Cytomegalovirus pp65 Nuclear Localization Signal". Clinical and Vaccine Immunology 16, n.º 6 (15 de abril de 2009): 935–43. http://dx.doi.org/10.1128/cvi.00011-09.
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ABSTRACT The human cytomegalovirus (CMV) pp65 protein contains two bipartite nuclear localization signals (NLSs) at amino acids (aa) 415 to 438 and aa 537 to 561 near the carboxy terminus of CMV pp65 and a phosphate binding site related to kinase activity at lysine-436. A mutation of pp65 with K436N (CMV pp65mII) and further deletion of aa 537 to 561 resulted in a novel protein (pp65mIINLSKO, where NLSKO indicate NLS knockout) that is kinaseless and that has markedly reduced nuclear localization. The purpose of this study was to biologically characterize this protein and its immunogenicity compared to that of native pp65. Unlike the native CMV pp65, following either DNA- or recombinant adeno-associated virus-based transduction of CMV pp65mIINLSKO into cells in vitro, the first observation of pp65mIINLSKO expression was in the cytoplasm and pp65mIINLSKO was expressed at higher levels than the native protein. The CMV pp65mIINLSKO mRNA was more abundant earlier than CMV pp65 mRNA (at 4 h and 8 h, respectively), but the half-lives of the proteins were the same. This modification altered the antigenic processing of CMV pp65 in vitro, as measured by the improved efficiency of cytotoxic killing in a pp65mIINLSKO-transduced human HLA A*0201 target cell line. In HHDII mice expressing HLA A*0201, pp65mIINLSKO was as immunogenic as CMV pp65. By RNA microarray analysis, expression of the CMV pp65mIINLSKO had less of an effect on cell cycle pathways than the native CMV pp65 did and a greater effect on cell surface signaling pathways involving immune activity. It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and should be considered in the design of a vaccine.
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Chandrasekaran, Siddarth, Vignesh Janardhanam, Ian Cardle, Justin Yoo, Yue Zhang-Wong, Julia Bershadsky, Elisa Martinez et al. "A Layered Cloaking Strategy to Generate Allogeneic iPSC-Derived CD8 T-Cells That Evade NK Clearance". Blood 144, Supplement 1 (5 de noviembre de 2024): 2045. https://doi.org/10.1182/blood-2024-210003.
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Gene-editing approaches have paved the way to manipulate allogeneic CAR-T cells for longer persistence by escaping host immune cell mediated rejection. While the design concepts for evading patient T-cell mediated rejection have been widely agreed upon, a lack of consensus remains around the optimal design for avoiding missing-self natural killer (NK) responses resulting from HLA Class I knockout. The most common approach for NK cell evasion includes knock in of HLA-E, a non-classical class I molecule with limited polymorphism that inhibits NK cells via binding to an inhibitory receptor NKG2A. HLA-E can also stimulate NK subsets expressing the activating receptor NKG2C, leading to accelerated allogeneic clearance of HLA-E engineered cells. The prevalence of NKG2C+ adaptive NK cells can increase after cytomegalovirus reactivation in patients who have undergone hematopoietic stem cell transplantation and lymphodepletion, common features of CAR-T cell eligible patients. Taking cues from the biology of adaptive NK cells, we designed a layered approach to cloak allogeneic iPSC-derived CD8 T-cells for protection against a broader NK cell phenotype. It has been long known that CD2 and CD58 interactions are pivotal for the activation and function of adaptive NK cells via formation of a stable immune synapse. We demonstrate that NKG2C+ adaptive NK cells express higher levels of the co-activating receptor CD2, and we also observe that CD58 is upregulated on iPSC-derived CD8 T-cells. To avoid rejection of class I deficient iPSC CD8 T cells by NK cells, we evaluated use of HLA-E in combination with disruption of NK-iPSC CD8-T cell synapse. To ensure effective inhibition of NK cells by engagement of NKG2A while minimizing activating NK cells via NKG2C, HLA-E expression levels were optimized by regulating expression of HLA-E by site-specific integration under the control endogenous and exogenous promoters. We show that surface expression level of HLA-E is critical to balance engagement of NKG2A while minimizing activation of NK cells via NKG2C. We also show that additional layering of synapse disruption by knockout of adhesion molecules such as CD58 and ICAM3 (CD50) leads to best-in-class NK cell protection against NKG2C+ NK donors. Our in vitro clearance assays demonstrate that HLA-E plus NK-T cell synapse disruption together are required for comprehensive NK evasion in iPSC derived CD8 T cells. Individually these approaches are ineffective for providing protection against all NK donors. We also show that incorporation of these allogeneic edits does not interfere with the ability of these iPSCs to differentiate into CD8 T-cells and function against disease relevant tumor cells in vitro. In summary, rejection by host NK cells is a critical consideration for allogeneic cell therapies incorporating HLA class I disruption. Our layered approach of incorporating HLA-E and synapse disruption allows for universal protection against broader subsets of NK cells. After decades of development, the promise of off-the-shelf allogeneic CAR-T cells from iPSCs is closer to becoming a clinical reality. With our scalable manufacturing platform, we are making best-in-class cytotoxic T-cells in stirred-tank reactors and with gene-edited starting material incorporating a cloaking strategy we will be able to rapidly bring affordable allogeneic T-cells for clinical application against a wide range of disease indications.
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Firat, H. "Comparative analysis of the CD8+ T cell repertoires of H-2 class I wild-type/HLA-A2.1 and H-2 class I knockout/HLA-A2.1 transgenic mice". International Immunology 14, n.º 8 (1 de agosto de 2002): 925–34. http://dx.doi.org/10.1093/intimm/dxf056.
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Steinitz, Katharina N., Pauline M. van Helden, Brigitte Binder, David C. Wraith, Sabine Unterthurner, Corinna Hermann, Maria Schuster et al. "CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice". Blood 119, n.º 17 (26 de abril de 2012): 4073–82. http://dx.doi.org/10.1182/blood-2011-08-374645.
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Abstract Today it is generally accepted that B cells require cognate interactions with CD4+ T cells to develop high-affinity antibodies against proteins. CD4+ T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+ T cells that can bind to the MHC class II–peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+ T-cell epitopes presented by HLA-DRB1*1501 to CD4+ T cells during immune responses against FVIII. CD4+ T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.
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Rohrlich, P. S. "HLA-B*0702 transgenic, H-2KbDb double-knockout mice: phenotypical and functional characterization in response to influenza virus". International Immunology 15, n.º 6 (1 de junio de 2003): 765–72. http://dx.doi.org/10.1093/intimm/dxg073.
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Black, Kay E., Joseph A. Murray y Chella S. David. "HLA-DQ Determines the Response to Exogenous Wheat Proteins: A Model of Gluten Sensitivity in Transgenic Knockout Mice". Journal of Immunology 169, n.º 10 (15 de noviembre de 2002): 5595–600. http://dx.doi.org/10.4049/jimmunol.169.10.5595.
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Guo, Chao, Yanying Fan, Alexander Aronov, Luxuan Buren, Ming-Hong Xie, Ivan Chan, Sasha Lazetic y James Trager. "113 CISH gene-knockout anti-CD70-CAR NK cells demonstrate potent anti-tumor activity against solid tumor cell lines and provide partial resistance to tumor microenvironment inhibition". Journal for ImmunoTherapy of Cancer 9, Suppl 2 (noviembre de 2021): A123. http://dx.doi.org/10.1136/jitc-2021-sitc2021.113.
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BackgroundPeripheral blood natural killer (NK) cells are attractive candidates for adoptive cell therapy. NK cells possess innate ability for tumor cell killing and are also amenable to genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for allogeneic, off-the-shelf therapy since, unlike T cells, they are neither HLA-restricted nor known to cause graft-versus-host disease. Cytokine inducible SH2-containing protein (CISH) is a negative regulator of interleukin 15 (IL-15) signaling in natural killer (NK) cells. Here we show the potential application of CISH gene-knockout CAR NK cells targeting CD70 and expressing a membrane-bound form of IL-15. CD70 is an antigen that is aberrantly expressed in a variety of malignant settings, including renal cell carcinoma (RCC), while its expression in normal tissues is restricted to a subset of lymphoid cell types.MethodsTo target CD70 on RCC cells, we generated CD70-CAR NK cells with CISH deletion. Using the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from healthy donors. Since CD70 expression is present on activated NK cells, we also targeted CD70 for CRISPR knockout to avoid fratricide. We then expanded these edited NK cells by using IL-2 and stimulation using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD70-targeted CAR and mbIL-15. We assessed CAR expression, NK cell persistence, and NK cell activity against RCC target cells using end-point cytotoxicity assays and IncuCyte.ResultsCISH gene-knockout CD70-CAR NK cells could be produced efficiently and exhibited extended persistence in culture. After engineering and expansion, CD70-CAR transduction efficiency was 60–80%. CD70-CAR NK cells displayed potent cytotoxicity against CD70-expressing renal cancer derived cell lines. Interestingly, cytotoxicity assays demonstrated that CISH gene-knockout CD70-CAR NK cells were partially resistant to TGFß and adenosine inhibition of cytotoxicity. Furthermore, CISH gene-knockout CD70-CAR NK cells maintained their activity during prolonged culture.ConclusionsIn summary, we show CISH gene-knockout CD70-CAR NK cells demonstrate potent anti-tumor activity against relevant solid tumor cell lines and partially provide resistance to tumor microenvironment inhibition. These data support the further exploration of CISH gene-knockout CD70 CAR NK cells for clinical application.
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Gu, Xiaorong, Songa Bae, Yahan Zhang, Nakisha D. Williams, Dongxu Jiang, Simon Schlanger, Valeria Visconte, Jaroslaw Maciejewski y Babal K. Jha. "Loss of TET2 Increases MHC Class I Expression in Acute Myeloid Leukemia". Blood 144, Supplement 1 (5 de noviembre de 2024): 4159. https://doi.org/10.1182/blood-2024-208348.
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Background: One of the major challenges in the treatment of acute myeloid leukemia (AML) is the elimination of undifferentiated immature blood cells, often referred to as leukemia stem and progenitor cells (LSPC). LSPCs persist after treatment and are considered a major cause of relapse and refractory disease. Despite initially high remission rates with recent advances in treatment strategies, relapse occurs in a large proportion of patients, leading to high mortality in AML. Myeloid neoplasia (MN) is driven by somatic mutations, with loss of function TET2 mutations (TET2MT) being one of the most common somatic lesions in MN. TET2, a member of the TET-family of DNA dioxygenases, is the major methyl-cytosine oxidase in hematopoietic cells, including T-cells. TET2 (along with TET1/3) are Fe2+ and αKG-dependent dioxygenases that utilize molecular oxygen to progressively oxidize 5-methyl cytosine (mC) in promoters, enhancers and silencer-associated mCpG segments of the genome, leading to their demethylation. This process is essential for mounting an accurate and efficient gene transcription profile that determines cell lineage fate, differentiation and proliferation. Here we report, while loss of TET2 creates a proliferative advantage to the LSPCs, it may also make them immunogenic and susceptible to immune surveillance due to higher expression of MHC class I/II molecules. Methods: We generated isogenic TET2 knockout (TET2KO) leukemic cell models in THP1 and K562 cell lines using CRISPR-Cas9 gene editing. We also utilized natural TET2-deficient cell models, including OCI-AML5 (TET2+/-) and SIMG5 (TET2-/-). Western blot (WB) analysis, bicolor immunofluorescent (IF) imaging and confocal microscope were employed to assess protein expression and protein subcellular localization in these models. To validate our findings, we performed flow cytometry. In addition, we analyzed the HLA expression in TET2MT mutant associated MN patients and compared it with WT. Results: Our study revealed that HLA- A/-B/-C (pan-HLA) expression in THP1 cells (MHC Class I positive) increased by more than 2-fold as a consequence of TET2 deletion compared to their isogenic wildtype counterparts, as observed in both WB and IF analyses. Additionally, pan-HLA protein levels were also significantly higher in cells with naturally TET2 deficiency (OCI-AML5 and SIGM5) compared to TET2 wildtype cells (THP1). The increased HLA protein levels was further confirmed by treatment with small molecule inhibitor of TET2 (TETi). Our data demonstrated that TETi treatment significantly increased pan-HLA expression in TET2WT cells but not in TET2KO cells. Furthermore, treatment with interferon gamma (INFg), a known inducer for HLA expression, significantly increased pan-HLA protein levels in TET2WT but not in TET2KO, suggesting that HLA expression is epigenetically regulated by TET2 in interferon mechanism of action. Interestingly, K562, an erythroid leukemia cell line known to be negative for surface expression of HLA- A/-B/-C had a significant pan-HLA upregulation upon genetic deletion or small molecule inhibition of TET2 dioxygenase activity. This observation was further confirmed in our analysis of human myeloid neoplasia (MDS and AML) patient cohort. Analysis of the RNAseq data from a cohort of 157 MN patients, including 38 with TET2MT and 64 healthy bone marrow samples, demonstrated that both MHC class I and II were upregulated in TET2 mutant MN patients. Conclusion: Our findings demonstrate that the loss of TET2 function significantly increased the surface expression of MHC class I/II proteins in both isogenic and natural TET2-deficient leukemic cell models, as well as in MDS and AML patients, compared to TET2WT and healthy control. The use of TET family inhibitors to chemically mimic TET2 loss further substantiated the increased pan-HLA expression, specifically in TET2 wildtype cells. Moreover, the differential response to INFγ treatment between TET2 wildtype and TET2 knockout cells highlights the critical role of TET2 in regulating HLA protein expression. These results suggest that targeting TET2 could enhance anti-cancer immune responses by increasing MHC class I/II expression, thereby improving the efficacy of immunotherapy therapies. This study provides a promising avenue for advancing cancer immunotherapy through epigenetic modulation.
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Garner, Elizabeth, Erin Kelly, Sai Namburi, Cian Colgan, Tristan Fowler, Devin Mutha, Art Aviles et al. "Abstract 3201: CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy engineered with next-generation CRISPR technology to resist both the immunosuppressive tumor microenvironment and immune cell-mediated rejection, for patients with relapsed or refractory acute myeloid leukemia". Cancer Research 83, n.º 7_Supplement (4 de abril de 2023): 3201. http://dx.doi.org/10.1158/1538-7445.am2023-3201.
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Abstract Background: CB-012 is an allogeneic anti-CLL-1 CAR-T cell therapy in development for evaluation in relapsed or refractory acute myeloid leukemia (r/r AML). CB-012 is engineered with a next-generation CRISPR genome-editing technology to leverage both checkpoint disruption and immune cloaking to enhance persistence of CAR-T cell antitumor activity. Methods: Caribou’s CRISPR hybrid RNA-DNA (chRDNA) guides in combination with Cas12a were used to make five edits in the manufacture of CB-012. A fully human anti-CLL-1 CAR transgene was site-specifically inserted into the TRAC gene, thereby eliminating TCR expression to prevent graft-versus-host disease. A B2M-HLA-E fusion transgene was inserted into the native B2M gene, preventing expression of all HLA class I antigens except HLA-E, to blunt both T and NK cell-mediated allograft rejection of the CAR-T cells. A knockout of the PDCD1 gene prevented PD-1 receptor expression and thus PD-L1 ligand binding, thereby prolonging antitumor activity. These multiplex genome-editing strategies were designed to enhance persistence of CB-012 antitumor activity and potentially achieve durable therapeutic responses. Results: CB-012 CAR-T cells express a potent, fully human anti-CLL-1 scFv CAR construct and demonstrate CLL-1-dependent cytotoxicity, proliferation, and cytokine secretion in vitro using AML cell line models. In AML xenografts, robust tumor control and prolongation of survival was observed following a single dose of CB-012. In serial rechallenge assays, CB-012 cells maintained increased cytolytic potential when repeatedly challenged with CLL-1-expressing tumor cells compared to similarly engineered CAR-T cells without a PD-1 knockout, demonstrating a functional benefit of PD-1 abrogation. This advantage was modelled in vivo, conferring superior prolongation of survival with CB-012 treatment compared to similarly engineered CAR-T cells without a PD-1 knockout. Additionally, CB-012 cells expressing the B2M-HLA-E fusion demonstrated resistance to NK cell-mediated cytotoxicity in vitro. Conclusions: CLL-1 is a compelling therapeutic target as it is highly expressed on AML tumor cells and leukemic stem cells, but not expressed on hematopoietic stem cells. It has also been established as a target in human proof-of-concept studies. CB-012 demonstrated potent and specific CLL-1-targeted cytolytic activity, and the genome-editing strategy employed to manufacture and armor CB-012 conferred a functional advantage in the context of the potentially immunosuppressive tumor microenvironment associated with r/r AML. Citation Format: Elizabeth Garner, Erin Kelly, Sai Namburi, Cian Colgan, Tristan Fowler, Devin Mutha, Art Aviles, Morena Stanaway, Raymond Guo, Zili An, Emilie Degagne, George Kwong, Leslie Edwards, Emma Jakes, McKay Shaw, Benjamin Schilling, Jeremy Huynh, Ricky Luu, Max Sidorov, Rhonda Mousali, Mikk Otsmaa, Justin Skoble, Steven Kanner. CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy engineered with next-generation CRISPR technology to resist both the immunosuppressive tumor microenvironment and immune cell-mediated rejection, for patients with relapsed or refractory acute myeloid leukemia [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 3201.
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Pajot, Anthony, Marie-Louise Michel, Nicolas Fazilleau, Véronique Pancré, Claude Auriault, David M Ojcius, François A. Lemonnier y Yu-Chun Lone. "A mouse model of human adaptive immune functions:HLA-A2.1-/HLA-DR1-transgenicH-2 class I-/class II-knockout mice". European Journal of Immunology 34, n.º 11 (30 de septiembre de 2004): 3060–69. http://dx.doi.org/10.1002/eji.200425463.
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Dufva, Olli, Khalid Saeed, Sara Gandolfi, Michal Sheffer, Jay Klievink, Petri Pölönen, Tiina Hannunen et al. "CRISPR Screens Identify Mechanisms of Natural Killer Cell Evasion across Blood Cancers". Blood 134, Supplement_1 (13 de noviembre de 2019): 3597. http://dx.doi.org/10.1182/blood-2019-129837.
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Natural killer (NK) cells have been suggested to control progression and relapse in several hematological malignancies. Enhancing NK cell reactivity represents an attractive approach to improve treatment responses. However, mechanisms enabling evasion of hematologic cancer cells from NK cells are incompletely understood. To identify cancer cell-intrinsic factors enabling resistance to NK cell cytotoxicity, we conducted genome-wide CRISPR screens in a range of hematological malignancies. Cas9-expressing cancer cells from diverse hematological malignancies, including acute and chronic myeloid leukemia (AML and CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), and B cell acute lymphoblastic leukemia were infected with a genome-scale lentiviral sgRNA library and exposed to primary human NK cells. Genes essential for NK cell cytotoxicity were discovered from surviving cancer cells which were enriched with gene knockouts inducing reduced sensitivity to NK cell killing. Additional data from a gain-of-function screen using a genome-scale CRISPR activation system were generated using the MM.1S myeloma cell line. Results from the functional genomic screens were integrated with transcriptomic data from > 9,000 patients across hematological cancers, as well as multi-omics data from AML and DLBCL, and both public and in-house single-cell RNA-sequencing data from AML patients. Knockout of genes encoding components involved in antigen presentation (B2M, HLA-A, HLA-C, HLA-E, TAP1, TAP2) sensitized multiple blood cancer cell lines to NK cell cytotoxicity, consistent with the inhibitory effect of MHC I on NK cells. Furthermore, knockout of interferon and JAK-STAT signaling components sensitized cancer cells to NK cell lysis, suggesting that MHC class I induction in response to interferon promotes NK cell resistance across cancer types. Interestingly, genes and pathways whose disruption conferred resistance for NK cell-mediated lysis exhibited partial overlap but also substantial variability across blood cancer types. Knockout of NCR3LG1 (B7-H6, ligand for the NKp30 activating NK cell receptor), resulted in resistance of K562 CML cells to NK cell cytotoxicity. In contrast, disruption of genes encoding apoptotic mediators (FADD, PMAIP1, BID) and TRAIL receptors (TNFRSF10B) conferred resistance to NK cell cytotoxicity in SUDHL4 DLBCL cells. The same pathways were identified in the MM cell line MM.1S, in which knockout of FAS, CASP2, and CASP8 as well as the TRAIL receptor TNFRSF10A induced NK cell resistance. Furthermore, loss of CD48, a ligand of the non-MHC binding receptor CD244 on NK cell surface, resulted in resistance and a genome-scale CRISPR gain-of-function screen in the same cell line showed sensitization upon CD48 overexpression. A CRISPR screen in the AML cell line MOLM14 identified disruption of TNFRSF1B encoding TNFR2 as strongly conferring NK cell resistance. Interestingly, TNFRSF1B overexpression sensitized the MM cell line MM.1S to NK cell treatment in the gain-of-function screen. Integration with genomic data from patients with hematological malignancies revealed selective expression of TNFRSF1B in AML. Within AML, TNFRSF1B expression was enriched in myelomonocytic and monocytic subtypes as well as in MLL-rearranged cases represented by the MOLM14 cell line. Further dissection at the single-cell level revealed increased expression of TNFRSF1B with differentiation of AML cells along the monocytic lineage. Consistently, the less differentiated MOLM13 cell line established from the same patient as MOLM14 was resistant to NK cell killing, suggesting that a less differentiated phenotype of AML cells confers resistance to NK cell cytotoxicity through lack of TNFRSF1B expression. Our data suggest that different lineages of hematological malignancies may be susceptible to NK cells through distinct mechanisms. In some cases, this heterogeneity is governed by lineage-specific expression of susceptibility genes, such as TNFRSF1B in monocytic AML. Particularly, our data identify a mechanism of NK cell evasion in less differentiated AML cells, suggesting potential for enhancing immune clearance of AML cells through differentiating therapies. figure Disclosures Lee: Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding.
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Triolo, Taylor M., J. Quinn Matuschek, Roberto Castro-Gutierrez, Ali H. Shilleh, Shane P. M. Williams, Maria S. Hansen, Kristen McDaniel, Jessie M. Barra, Aaron Michels y Holger A. Russ. "Stem-Cell-Derived β-Like Cells with a Functional PTPN2 Knockout Display Increased Immunogenicity". Cells 11, n.º 23 (30 de noviembre de 2022): 3845. http://dx.doi.org/10.3390/cells11233845.
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Type 1 diabetes is a polygenic disease that results in an autoimmune response directed against insulin-producing beta cells. PTPN2 is a known high-risk type 1 diabetes associated gene expressed in both immune- and pancreatic beta cells, but how genes affect the development of autoimmune diabetes is largely unknown. We employed CRISPR/Cas9 technology to generate a functional knockout of PTPN2 in human pluripotent stem cells (hPSC) followed by differentiating stem-cell-derived beta-like cells (sBC) and detailed phenotypical analyses. The differentiation efficiency of PTPN2 knockout (PTPN2 KO) sBC is comparable to wild-type (WT) control sBC. Global transcriptomics and protein assays revealed the increased expression of HLA Class I molecules in PTPN2 KO sBC at a steady state and upon exposure to proinflammatory culture conditions, indicating a potential for the increased immune recognition of human beta cells upon differential PTPN2 expression. sBC co-culture with autoreactive preproinsulin-reactive T cell transductants confirmed increased immune stimulations by PTPN2 KO sBC compared to WT sBC. Taken together, our results suggest that the dysregulation of PTPN2 expression in human beta cell may prime autoimmune T cell reactivity and thereby contribute to the development of type 1 diabetes.
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Karkischenko, N. N., V. A. Ezerskiy, O. B. Zhukova, E. M. Koloskova y N. V. Petrova. "Increasing, the Specificity of Polyclonal Antibodies to Human and Mouse β2-Microglobulin as an Alternative to the Use of Monoclonal Antibodies in Immunological Analysis". Journal Biomed 20, n.º 2 (23 de julio de 2024): 53–65. http://dx.doi.org/10.33647/2074-5982-20-2-53-65.
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Highly specific reagents, i.e., proteins and antibodies to them, are the necessary components of systems for verifying the effectiveness of transgenic/knockout animal biomodels. In particular, the identification of mouse and human β2-microglobulin in the protein fractions of organs and tissues of transgenic and β2m knockout mice of several HLA lines, which have been created in recent years at the Scientific Center of Biomedical Technologies of the FMBA of Russia, is the most important stage of their certification. At the first stage of our research, E. coli producing strains of recombinant mouse and human β2-microglobulin (mβ2mg and hβ2mg) were obtained, the proteins were isolated and purified. At the next stage of the work, affine sorbents with immobilized mβ2mg and hβ2mg were obtained. To increase the species specificity of the serum, “rabbit-anti-hβ2mg” were depleted against the recombinant protein mβ2mg, and, conversely, “rabbit-anti-mβ2mg” were depleted against the recombinant protein hβ2mg. Highly specific antibodies were purified from depleted sera using affinity sorbents. Using dot- and western-blotting methods оn the example of depleted and affinity-purified rabbit-anti-hβ2mg antibodies, a significant increase in their specificity relative to hβ2mg was shown.
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Khare, Sanjay D., Michael J. Bull, Julie Hanson, Harvinder S. Luthra y Chella S. David. "Spontaneous Inflammatory Disease in HLA-B27 Transgenic Mice Is Independent of MHC Class II Molecules: A Direct Role for B27 Heavy Chains and Not B27-Derived Peptides". Journal of Immunology 160, n.º 1 (1 de enero de 1998): 101–6. http://dx.doi.org/10.4049/jimmunol.160.1.101.
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Abstract Although association of HLA-B27 with human spondyloarthropathies has been known for several years, its role in disease pathogenesis is not understood. Recently, a few investigators have proposed that presentation of B27-derived peptides by MHC class II molecules may be the underlying mechanism. HLA-B27 transgenic rat and mouse models have provided a new tool for understanding the exact role of B27 in disease pathogenesis. HLA-B27 mice lacking endogenous β2-microglobulin (B27+β2mo) develop disease after they are transferred from the barrier facility to the conventional colony. This model was utilized to test the hypothesis that B27-derived peptide presented by MHC class II molecules is the cause of the disease. The MHC class II knockout gene, Aβo, was bred into our B27+β2mo mice, and disease manifestation was monitored. These mice develop spontaneous disease, demonstrating that MHC class II molecules do not play a major role in B27-related disease. Thus, the disease is not manifested by presentation of B27-derived peptides by class II molecules, since these mice are devoid of H2-A and H2-E molecules. Furthermore, in vivo treatment with mAb against the heavy chain of B27 reduced the incidence of disease in B27+β2mo mice. Our results clearly demonstrate that B27 heavy chains are directly involved in the disease process.
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Crivello, Pietro, Müberra Ahci, Fabienne Maaßen, Natalie Wossidlo, Esteban Arrieta-Bolaños, Andreas Heinold, Vinzenz Lange et al. "Multiple Knockout of Classical HLA Class II β-Chains by CRISPR/Cas9 Genome Editing Driven by a Single Guide RNA". Journal of Immunology 202, n.º 6 (30 de enero de 2019): 1895–903. http://dx.doi.org/10.4049/jimmunol.1800257.
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Robinson, Philip C., Eugene Lau, Patricia Keith, Max C. Lau, Gethin P. Thomas, Linda A. Bradbury, Matthew A. Brown y Tony J. Kenna. "ERAP2functional knockout in humans does not alter surface heavy chains or HLA-B27, inflammatory cytokines or endoplasmic reticulum stress markers". Annals of the Rheumatic Diseases 74, n.º 11 (18 de junio de 2015): 2092–95. http://dx.doi.org/10.1136/annrheumdis-2015-207467.
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Firat, Hüseyin, Francisco Garcia-Pons, Sophie Tourdot, Steve Pascolo, Antonio Scardino, Zacarias Garcia, Marie-Louise Michel et al. "H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies". European Journal of Immunology 29, n.º 10 (octubre de 1999): 3112–21. http://dx.doi.org/10.1002/(sici)1521-4141(199910)29:10<3112::aid-immu3112>3.0.co;2-q.
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Yang, Hongyun, Wen Jiang, Emma E. Furth, Xiaoming Wen, Jonathan P. Katz, Rance K. Sellon, Debra G. Silberg, Toni M. Antalis, Clifford W. Schweinfest y Gary D. Wu. "Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea". American Journal of Physiology-Gastrointestinal and Liver Physiology 275, n.º 6 (1 de diciembre de 1998): G1445—G1453. http://dx.doi.org/10.1152/ajpgi.1998.275.6.g1445.
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The pathogenesis of diarrhea in intestinal inflammatory states is a multifactorial process involving the effects of inflammatory mediators on epithelial transport function. The effect of colonic inflammation on the gene expression of DRA (downregulated in adenoma), a chloride-sulfate anion transporter that is mutated in patients with congenital chloridorrhea, was examined in vivo as well as in an intestinal epithelial cell line. DRA mRNA expression was diminished five- to sevenfold in the HLA-B27/β2m transgenic rat compared with control. In situ hybridization showed that DRA, which is normally expressed in the upper crypt and surface epithelium of the colon, was dramatically reduced in the surface epithelium of the HLA-B27/β2m transgenic rat, the interleukin-10 (IL-10) knockout mouse with spontaneous colitis, and in patients with ulcerative colitis. Immunohistochemistry demonstrated that mRNA expression of DRA reflected that of protein expression in vivo. IL-1β reduced DRA mRNA expression in vitro by inhibiting gene transcription. The loss of transport function in the surface epithelium of the colon by attenuation of transporter gene expression, perhaps inhibited at the level of gene transcription by proinflammatory cytokines, may play a role in the pathogenesis of diarrhea in colitis.
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Hahn, Cynthia K., Gavin E. Hooper, Alexandra Forman, Gabriela Brunsting Hoffmann, Sam Sadigh, Kun Huang, Erin M. Parry et al. "SEC62 Regulates HLA-E Expression in Diffuse Large B-Cell Lymphoma to Function As a Mechanism of Immune Evasion". Blood 144, Supplement 1 (5 de noviembre de 2024): 335. https://doi.org/10.1182/blood-2024-210613.
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The expression of HLA-E, a non-classical HLA class I molecule, is an emerging mechanism of immune escape in cancer. Increased HLA-E is associated with poor prognosis in multiple malignancies and is a mechanism of resistance to immune-based therapies via an inhibitory interaction with the NKG2A/CD94 receptor on NK and T cells. We identified high expression of HLA-E in diffuse large B-cell lymphoma (DLBCL) and hypothesized that this may constitute an immune evasion mechanism that could be therapeutically targetable in this disease. We found that DLBCL has the highest median gene expression of HLA-E when compared to 32 solid tumors and leukemias profiled in The Cancer Genome Atlas. In existing datasets, we observed that HLA-E is also upregulated in DLBCL relative to its normal cell of origin, germinal center B cells (P = 0.0001). By immunohistochemistry in DLBCL cases, HLA-E was expressed in 37 of 48 cases (77%) and present even in some cases with low or absent classical HLA class I (HLA-I). HLA-E is also abundant in transformed DLBCL and we confirmed presence of NKG2A+ cells in the tumor microenvironment in four excisional biopsies using highly multiplexed immunofluorescence. Finally, there is existing evidence that DLBCL patients have higher frequencies of NKG2A+ NK cells than healthy donors. Altogether, these findings support the notion that the HLA-E/NKG2A checkpoint may function as a mechanism of immune evasion in DLBCL. Given that HLA-E expression can be retained when classical HLA-I is downregulated, we questioned if there may be distinct mechanisms of regulation. First, to comprehensively define the mechanisms of HLA-E regulation, we completed two genome-wide CRISPR knockout (KO) screens in germinal center B (GCB) and activated B cell (ABC) DLBCL cell lines, SU-DHL-8 and TMD8, respectively. The top and bottom 5% of HLA-E expressing cells were isolated by fluorescence activated cell sorting. Genes were ranked based on the enrichment of their corresponding sgRNAs in the top vs. bottom HLA-E expression, which identified 379 candidate regulators. As validation, we then performed a secondary screen using a custom library consisting of eight newly designed sgRNAs per gene and identified HLA-E specific regulators through parallel assessment of HLA-I. The custom library was screened in SU-DHL-8 and TMD8, plus an additional GCB (SU-DHL-4) and ABC DLBCL line (HBL1). Of the 379 primary screen hits, 337 (88.9%) validated in the original SU-DHL-8 and TMD8 cells and 284 (74.9%) validated in all cell lines (P < 0.0005). Confirming the robustness of our system, top hits enriched in HLA-E and HLA-I low populations included known antigen presentation pathway genes, while HLA-E was the top hit in the HLA-E low population without altering HLA-I expression. Most positive and negative regulators of HLA-E were shared with HLA-I, but nine genes decreased HLA-E to a greater degree than HLA-I in three of four cell lines. These genes were all involved in protein processing and trafficking pathways, including aminopeptidases (ERAP1, HM13, and SPCS3) and regulators of transport through the endoplasmic reticulum (ER) and Golgi apparatus (SEC62 and USO1). The only shared HLA-E specific regulator in all cell lines screened was SEC62, an ER membrane protein that facilitates the post-translational import of a subset of proteins into the ER. To further validate SEC62 as a positive regulator of HLA-E, we generated isogenic SEC62 KOs in SU-DHL-4, HBL1, and TMD8 DLBCL cell lines. We found that SEC62 KO reduced surface and whole cell HLA-E protein expression without decreasing HLA-I in flow cytometry, western blotting and immunofluorescence assays. However, HLA-E mRNA levels were unchanged, consistent with regulation via a post-transcriptional mechanism. By co-culturing isogenic SEC62 KO DLBCL cell lines with a Jurkat-NKG2A reporter system, we functionally confirmed that SEC62 KO reduced engagement with the NKG2A/CD94 receptor. Together, our findings reveal a potential role for HLA-E in DLBCL as a mechanism of immune escape and identify SEC62 as a distinct regulator of HLA-E in this malignancy.
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Moise, Leonard, Jonathan Skupsky, Ryan Tassone, Julie A. McMurry, William D. Martin, Anne S. De Groot y David W. Scott. "De-Immunization of Human Factor VIII: Identification of Epitopes in the C2 Domain". Blood 112, n.º 11 (16 de noviembre de 2008): 1030. http://dx.doi.org/10.1182/blood.v112.11.1030.1030.
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Abstract Most immune responses to protein antigens are dependent on T-cell recognition of discrete peptide epitopes presented in an MHC groove. The pattern of peptide recognition can be predicted from the primary structure of a given protein based on residues that bind (anchor) to a given HLA phenotype. Algorithms such as EpiMatrix can be applied to identify such epitopes and measure the potential immunogenicity of proteins based on epitope content. One approach to reduce immunogenicity is to generate recombinant proteins whose constituent epitopes have been modified so as to reduce their HLA binding. Modification of the sequence can be performed in silico using algorithms such as OptiMatrix. This “de-immunization” method has been used effectively with a number of therapeutic proteins already in use in clinical trials. It may therefore also be possible to target those residues of fVIII that, while contributing to HLA binding, can be mutated without altering the functional ability of fVIII to initiate clotting. We have begun this de-immunization process with the fVIII, starting with C2 domain because C2 has been confirmed to be a major target of both T cells and inhibitory antibodies. Using EpiMatrix, we selected 10 peptides in human fVIII that would be predicted to bind to eight class II HLA DR molecules that encompass over 95% of the U.S. population. These epitopes were synthesized and eight of the ten were assayed in vitro and found to bind to HLA at IC50 <100μM. Immunization of fVIII knockout mice with whole fVIII or with constituent fVIII peptide epitopes resulted in significant T cell proliferation to the peptide epitopes as measured by thymidine incorporation assays. By contrast, homologous peptide epitopes modified by 1–2 residues elicited significantly lower levels of proliferation. These preliminary results point to the feasibility of generating a de-immunized fVIII molecule with reduced immunogenicity; validation of these results is planned. If such proteins retain even partial activity to initiate clotting, then they would become useful in treating hemophilia A patients to avoid inhibitor formation. (Supported by NIH R43 HL088834-01)
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Song, Nianbin, Yuri Poluektov, Robin Welsh y Scheherazade Sadegh-Nasseri. "MHC class II antigen-processing chaperone H2-O shapes CD4 T cell receptor repertoire". Journal of Immunology 196, n.º 1_Supplement (1 de mayo de 2016): 46.11. http://dx.doi.org/10.4049/jimmunol.196.supp.46.11.
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Abstract HLA-DO (DO) is a MHC class II-encoded, nonpolymorphic heterodimeric protein in humans (known as H2-O in mice). It is expressed only in thymic medulla, B lymphocytes and some subsets of dendritic cells. Although DO is found to form a stable complex with HLA—DM (DM), its physical function remains unknown. Some studies have shown that it inhibits the activity of DM to edit peptide presentation on MHC II molecules while others reported that the DO can up- or downmodulate presentation of certain peptides in association with DM on HLA-DR4 molecules. To solve this controversy, our lab recently proposed a model suggesting that the DO facilitates the binding of DM-resistant peptides and inhibits the binding of DM-sensitive peptides to the HLA-DR1 molecules in vitro. Based on this model, we expected to see lower density of pMHC expression on mTEC cells in thymus of H2-O-KO mice, which will lead to faulty negative selection and different CD4 TCR repertoires as compared to H2-O-WT mice. By developing a “Prime-Restim” strategy based on the principle of mixed lymphocyte reaction, we observed the proliferation of primed H2-O-WT CD4 T cells in responding to repeated in vitro stimulations by antigen presenting cells from H2-O-KO mice and vice versa. The results demonstrate that in the absence of H2-O, the peptide repertoire of MHC II in the H2-O knockout mice is different from the peptide repertoire of MHC II in the H2-O-WT mice. It establishes a role for DO in regulation of epitope selection for presentation by MHC class II molecules in vivo. These findings support the idea that the expression of DO in thymic medulla might lead to better thymic deletion of self-reactive CD4 T cells.
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Chen, Huanhuan, Keqing Yang, Lingxiao Pang, Jing Fei, Yongliang Zhu y Jianwei Zhou. "ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer". Journal for ImmunoTherapy of Cancer 11, n.º 2 (febrero de 2023): e005527. http://dx.doi.org/10.1136/jitc-2022-005527.
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BackgroundOvarian cancer is the deadliest type of malignant gynecological tumor. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are involved ovarian cancer and are closely related to adverse outcomes. However, the immunosuppressive mechanism of PMN-MDSCs remains elusive.MethodsThe types and numbers of ANKRD22-expressing cells were investigated by bioinformatics analysis and immunohistochemical staining.Ankrd22-/-C57BL/6 mice were constructed with CRISPR-Cas9 technology. Mouse PMN-MDSCs were obtained from bone marrow (BM)-derived CD11b+Ly6G+Ly6Clowcells sorted by fluorescence-activated cell sorting with treatment of GM-CSF and IL-6, and the immunosuppressive activity of PMN-MDSCs was evaluated by flow cytometry (FCM) and ELISA. The expression level of CCR2 and the exogenous glucose uptake capacity were determined by FCM. RT-qPCR was used to detectANKRD22expression in CD11b+HLA-DR-CD14-CD15+cells from human ovarian cancer tissues, and the correlations ofANKRD22expression with the clinical characteristics and prognosis of patients were evaluated by the χ2test.ResultsWe identified a novel protein involved in regulating the immunosuppressive ability of PMN-MDSCs, ANKRD22.Ankrd22expression was high in mouse CD11b+Ly6G+Ly6Clowcells and could be significantly downregulated after exposure to a simulated microenvironmental stimulus. Knockout ofAnkrd22increased the expression level of CCR2 of CD11b+Ly6G+Ly6Clowcells and the immunosuppressive activity of PMN-MDSCs. BM-derived CD11b+Ly6G+Ly6Clowcells ofAnkrd22-/-mice significantly promoted the proliferation of ovarian cancer cells in tumor xenograft mouse models. Mechanistically, RNA sequencing showed thatWdfy1expression was obviously increased inAnkrd22-knockout BM-derived CD11b+Ly6G+Ly6Clowcells and that ectopic expression ofWdfy1increased the levels ofArg1,Inos,IdoandPdl1inAnkrd22+/+PMN-MDSCs derived from BM-derived CD11b+Ly6G+Ly6Clowcells. Surprisingly, an ANKRD22-activating candidate small-molecule compound attenuated the immunosuppressive activity ofAnkrd22+/+PMN-MDSCs. Finally, we found that lowANKRD22levels in CD11b+HLA-DR-CD14-CD15+cells derived from primary ovarian tissues were associated with a more advanced International Federation of Gynecology and Obstetrics stage, a higher recurrence rate, and a higher neutrophil-to-lymphocyte ratio.ConclusionsThese results suggest that ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs.
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Liu, Fuguo, Mubin Tarannum, Yingjie Zhao, Yiming J. Zhang, James Dongjoo Ham, Kewen Lei, Yuhao Qiang et al. "One-Step Construction of Allogeneic CAR-NK Cells Preventing Rejection and Mediating Enhanced Anti-Tumor Responses". Blood 144, Supplement 1 (5 de noviembre de 2024): 915. https://doi.org/10.1182/blood-2024-198167.
Texto completoResumen
Chimeric antigen receptor (CAR)-armed immune cells have demonstrated promising efficacy in several hematologic malignancies. There is a great interest in developing allogeneic “off-the-shelf” cellular therapies to reduce manufacturing time and costs, allow multiple dosing, and increase accessibility. To mitigate the problem of rejection of allogeneic products, common approaches include abrogation of MHC I expression by deleting b2-microglobulin (B2M) or TAP genes to prevent host T cell killing, and expression of HLA-E, HLA-G or CD47 to inhibit host NK killing. Typically, B2M or TAP knockout is first carried out using CRISPR/CAS-9 and then, CAR is introduced by lentiviral transduction and thus requiring at least two steps of genetic manipulation. Here, we describe the development of a single novel lentiviral gene construct incorporating a novel shRNA that selectively targets prevalent HLA-A, -B, -C (referred to as HLA-ABC) alleles while maintaining endogenous HLA-E and simultaneously expressing a CAR and a single chain HLA-E (SCE) or PD-L1. Our screening of shRNAs helped identify a novel shRNA (shRNA #1) targeting a unique sequence common to 121 prevalent HLA-ABC alleles but has two mismatches with HLA-E alleles. The data revealed that shRNA #1 universally decreased surface HLA-ABC in HEK293T, Jurkat T, and primary NK cells isolated from twelve unrelated donors. Furthermore, it did not decrease HLA-E expression in IFNγ pre-treated Jurkat T cells. To investigate the effect of PD-1/PD-L1 interaction in the control of T cell responses to allogeneic NK cells. NK cells were transduced with a lentivector expressing PD-L1, and PD-L1+ NK cells were sorted and expanded. PD-L1+ NK cells were then co-cultured with HLA mismatched CD3+ T cells. The data revealed that PD-L1 could moderately inhibit allogeneic T cell responses, as indicated by lower percentages of CD69+, CD25+, and proliferated T cells after co-culture compared with the controls. To inhibit allogeneic NK cell killing of HLA-ABC reduced cells, we tested three single-chain HLA-E (SCE) variants: SCE WT, SCE Y84A, and SCE Y84C. The SCE WT incorporates peptide derived from the HLA-G leader sequence (VMAPRTLFL), B2M, and HLA-E*01:03 heavy chain, whereas SCE Y84A and SCE Y84C have further mutations in HLA-E*01:03 heavy chain and/or the linker region. The data revealed that all three SCE variants supported surface HLA-E expression, with SCE Y84C showing the highest expression level. We also found that SCE Y84C was able to inhibit third party NK cells, as indicated by the lowest lysis of SCE Y84C+ target cells and decreased degranulation and IFNγ production upon coculture. We show that combining HLA-ABC knockdown and PD-L1 or SCE expression conferred resistance to allogeneic immune cell killing in vitro and in vivo. The combination of HLA-ABC knockdown, CAR, and PD-L1 or SCE overexpression was achieved by one-step lentiviral transduction of NK cells with a novel CAR construct incorporating shRNA#1, CAR, and PDL-1 or SCE. To test the CAR-mediated target cell killing, we assayed the killing of Raji cells by CD19-CAR NK cells and OVCAR8 cells by MSLN-CAR NK cells in vitro. The CAR NK cells killed target cells efficiently, and unexpectedly, PD-L1 and SCE overexpression significantly enhanced CAR NK cell cytotoxicity, likely related to the upregulation of cytotoxicity-related genes and better proliferation fitness. In a xenograft model (NSG mice), the allogeneic CD19-CAR NK cells with HLA-ABC knockdown and PD-L1 or SCE overexpression escaped immune rejection (with third party PBMCs) while maintaining potent anti-tumor responses against Raji cells (as assessed by BLI). In summary, we identified a potent shRNA that can specifically downregulate surface HLA-ABC while maintaining surface HLA-E expression, and showed that PD-L1 can moderately inhibit allogeneic T-cell responses, and SCE Y84C is potent in inhibiting host NK cell cytotoxicity. Transduction of NK cells with our gene construct that combines a CAR, shRNA#1, and PD-L1 or SCE expression allows one-step construction of allogeneic CAR-NK cells that escape host immune cell rejection in vitro and in vivo while mediating enhanced anti-tumor responses. Our approach could also be applied to other allogeneic immune cell-based therapies and therefore represents a significant advancement in enabling “off-the-shelf” allogeneic immunotherapies.