Adaptive Immunity: From Autoimmune Diseases to Neuroinflammation

2021 ◽  
Author(s):  
◽  
Wei Su ◽  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Neurodegenerative Diseases ◽  
Treg Cell ◽  
Immune Tolerance ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Intracellular Signaling ◽  
Protein Prenylation

Inflammation is essential for the clearance of pathogens and to facilitate healing of damaged tissue. However, this process must be controlled to limit immunopathology. Cell-intrinsic effects of inhibitory and signaling molecules are known to maintain quiescence and prevent effector differentiation and inflammation. Moreover, specific populations of immune cells exert cell-extrinsic effects for immunosuppression. Therefore, studies on the immunosuppressive functions of these cell populations will provide a better understanding of how inflammation is regulated and how its dysregulation causes human disease. Additional insights in this area may uncover novel targets to be manipulated for therapeutic benefit in autoimmune and inflammatory disorders, such as neurodegenerative diseases. Foxp3-expressing regulatory T (Treg) cells are specialized immunosuppressive cells that establish immune tolerance to prevent the development of autoimmune and other inflammatory diseases, with effector-Treg (eTreg) cells playing a pivotal role. Recently, cellular metabolism has emerged as a mediator to enforce Treg-cell function and heterogeneity. In Chapter 3, we used genetic and pharmacological tools to demonstrate that isoprenoid-dependent posttranslational lipid modifications dictate eTreg-cell accumulation and function by intersecting with T cell receptor (TCR)-induced intracellular signaling. We showed that isoprenoids are essential for activated Treg-cell suppressive activity, and Treg cell-specific deletion of the enzymes that mediate farnesylation and geranylgeranylation (encoded by Fntb and Pggt1b, respectively) leads to a reduction of eTreg cells and the development of fatal autoimmunity. In Chapter 4, we further explored the mechanistic role of protein prenylation in the regulation of eTreg cells by performing a comprehensive analysis of protein prenylation-dependent molecular signaling in eTreg-cells. Specifically, we found that Fntb drives eTreg-cell maintenance by promoting mTORC1 activity-dependent proliferation and ICOS-mediated cellular fitness. In contrast, Pggt1b orchestrated transcriptional programming by TCR stimulation and Rac signaling to establish eTreg-cell differentiation and immune tolerance. Therefore, our results reveal a bidirectional interplay between immune signals, metabolism-mediated posttranslational modifications, and intracellular signaling for the differentiation and maintenance of eTreg cells. Neuroinflammation is also a feature of neurogenerative diseases, but the underlying cellular mechanisms that limit inflammation in neurodegenerative diseases are largely unknown. In Chapter 5, using single cell RNA-sequencing (scRNA-seq) of immune cells in a mouse model of neurodegeneration (specifically, Alzheimer’s disease AD), we found that CD8 T cells accumulated in the brain parenchyma. These CD8 T cells had tissue resident memory-associated features and appeared to retain functionality. Importantly, T cell ablation was found to exacerbate disease-associated deposition of Beta- amyloid (A-Beta) and cognitive decline in a mouse model of AD. Moreover, in the absence of T cells, microglia acquired proinflammatory features and clustered in regions near A-Beta plaques, features associated with more severe disease. Collectively, these results suggest that T cells are critical to restrain microglia activation and limit neurodegeneration-associated pathologies in a murine model of AD.

scholarly journals Direct and Indirect endocrine-mediated suppression of human endometrial CD8+T cell cytotoxicity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Z. Shen ◽  
M. Rodriguez-Garcia ◽  
M. V. Patel ◽  
C. R. Wira
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cytotoxic Activity ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Cd8 T Cell ◽  
Gynecological Cancers ◽  
Protective Strategies ◽  
T Cell Cytotoxicity

AbstractRegulation of endometrial (EM) CD8+T cells is essential for successful reproduction and protection against pathogens. Suppression of CD8+T cells is necessary for a tolerogenic environment that promotes implantation and pregnancy. However, the mechanisms regulating this process remain unclear. Sex hormones are known to control immune responses directly on immune cells and indirectly through the tissue environment. When the actions of estradiol (E2), progesterone (P) and TGFβ on EM CD8+T cells were evaluated, cytotoxic activity, perforin and granzymes were directly suppressed by E2 and TGFβ but not P. Moreover, incubation of polarized EM epithelial cells with P, but not E2, increased TGFβ secretion. These findings suggest that E2 acts directly on CD8+T cell to suppress cytotoxic activity while P acts indirectly through induction of TGFβ production. Understanding the mechanisms involved in regulating endometrial CD8+T cells is essential for optimizing reproductive success and developing protective strategies against genital infections and gynecological cancers.

scholarly journals 708 Novel ways to exploit IL-21 to augment adoptive T cell transfer therapy against tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A737-A737
Author(s):  
Anna Cole ◽  
Guillermo Rangel RIvera ◽  
Aubrey Smith ◽  
Megan Wyatt ◽  
Brandon Ware ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Striking Difference ◽  
T Cell Therapy ◽  
Cell Immunity ◽  
Emory University

BackgroundIL-21 enhances the anti-tumor capacity of adoptively transferred CD8+ T cells, while IL-2 and IL-15 impair T cell immunity by driving their expansion to a more differentiated status. Yet, these cytokines can act on many different immune cells. Given the potency of IL-21, we tested if this cytokine directly augments T cells or rather if it enhances other immune cells in the culture that indirectly improves T cell therapy.MethodsTo test this question, splenocytes from pmel-1 transgenic mice were used, as all CD8+ T cells express a transgenic TCR specific for tumor-antigen gp10025–33 overexpressed on melanoma. We then peptide activated naïve CD8+ T cells enriched or not from the spleen of pmel-1 mice and expanded them in the presence of IL-21 or IL-2 (10 ng/mL) for four days. Expanded pmel-1 from these various cultures were then restimulated with irradiated splenocytes pulsed with gp10025–33 and grown an additional seven days with IL-2 (10 ng/mL), irrespective of their initial cytokine condition. The in vitro memory phenotype, exhaustion profile, and cytokine secretion of these cultures were then assayed. Furthermore, mice bearing B16KVP melanoma tumors were infused with pmel-1 T cells expanded via these various approaches and compared for their relative capacity to engraft, persist, and regress tumor in vivo.ResultsInterestingly, we discovered that IL-21-treated T cells generated from bulk splenocytes are phenotypically and functionally distinct from IL-21-treated isolated T cells. Upon restimulation, IL-21-treated T cells from bulk splenocytes exhibited an exhausted phenotype that was like anergic IL-2-treated T cells. Moreover, few cells expressed CD62L but expressed heightened markers of suppression, including TIM3, PD-1, and EOMES. Moreover, they produced more effector molecules, including granzyme B and IFN-gamma. In vivo IL-21-treated T cells expanded from bulk splenocytes engrafted and persisted poorly, in turn mediating suboptimal regression of melanoma. Conversely, IL-21 dramatically bolstered the engraftment and antitumor activity of T cells only if they were first isolated from the spleen prior to their expansion and infusion into the animal.ConclusionsCollectively, our data shows that IL-21 may improve ACT therapy best when used directly on antitumor CD8+ T cells. Further studies will illuminate the mechanism behind this striking difference and determine whether other cell subsets reactive to IL-21 cause T cell dysfunction and/or reduced bioavailability. These findings are important for defining the best culture conditions in which to use IL-21 for ACT.AcknowledgementsWe would like to acknowledge Emory University, The Winship Cancer Institute, and the Pediatrics/Winship Flow Cytometry Core.Ethics ApprovalAll animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University, protocol number 201900225.

Deciphering of CD4+ T Cell Subsets Involved in Maintenance and Break of Immune Tolerance in a Humanized Mouse Model That Is Immune Tolerant to Human FVII

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2188-2188
Author(s):  
Christine Lenk ◽  
Markus Pasztorek ◽  
Markus Weiller ◽  
Rafi Uddin Ahmad ◽  
Hans Peter Schwarz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Immune Tolerance ◽  
Neutralizing Antibodies ◽  
Subcutaneous Administration ◽  
T Cell Subsets ◽  
Regulatory Mechanisms ◽  
Clonal Deletion ◽  
Danger Signals

Abstract Abstract 2188 25%-30% of patients with hemophilia A develop neutralizing antibodies following replacement therapy with factor VIII (FVIII). These patients can be treated with factor VIIa (FVIIa) which triggers the extrinsic pathway of coagulation and thereby bypasses the requirement for FVIII. We developed a new mouse model that is transgenic for human FVII and expresses specific immune tolerance to native human FVIIa. We aim to investigate the immunological impact of modified FVIIa product candidates and to characterize their immunogenicity by analyzing emerging FVIIa-specific T cell responses. The new mouse model offers a unique opportunity to study central and peripheral immune regulatory mechanisms and the generation of immune responses by pro-inflammatory antigen-specific effector T cells (Teff). We hypothesized that FVIIa-specific Teff having escaped clonal deletion are present in the periphery and may be actively suppressed by FVIIa-specific regulatory T cells (Tregs). To study this hypothesis, we immunized mice with recombinant FVIIa (rFVIIa) with or without LPS, a well-described “danger signal” being able to break immune tolerance by stimulating the innate immune system. Intravenous or subcutaneous administration of rFVIIa alone did not elicit antibody responses and thus immune tolerance to rFVIIa was not broken. However, co-administration of rFVIIa and LPS resulted in a specific antibody response that was not isotypically restricted. To further analyze the mechanisms behind this break of specific immune tolerance, we characterized rFVIIa-specific T cells by the expression of CD154, a marker of antigen-specific T cells. Cytokine production and CD154 expression were assessed upon re-stimulation with rFVIIa. In contrast to mice that were immunized with rFVIIa only, we found increased numbers of rFVIIa-specific T cells in rFVIIa-LPS-treated mice displaying a stable, highly pro-inflammatory (IL-2+/IFN-g+) memory phenotype. These data could suggest that rFVIIa-specific Teff that escaped clonal deletion during induction of central immune tolerance, are present in the periphery of human FVII-transgenic mice. This would imply that rFVIIa-specific Teff could be actively suppressed by Tregs. This suppression could be overcome by danger signals like LPS. We currently study the regulatory mechanisms that maintain tolerance upon administration of FVIIa without LPS. We are approaching this question by correlating the characteristics of FVIIa-specific Teff and Treg responses under both tolerant and non-tolerant conditions. Ultimately, we aim to understand which danger signals have to be provided to break immune tolerance and how tolerance is regulated. Understanding these regulatory mechanisms will enable us to develop new therapeutic strategies and prevent conditions that lead to the induction of antibodies against drug products in patients. Disclosures: Lenk: Baxter BioScience: Employment. Pasztorek:Baxter BioScience: Employment. Weiller:Baxter BioScience: Employment. Ahmad:Baxter BioScience: Employment. Schwarz:Baxter BioScience: Employment. Scheiflinger:Baxter BioScience: Employment. Reipert:Baxter Innovations GmbH: Employment. de la Rosa:Baxter BioScience: Employment.

Induction and Break of Immune Tolerance to Human FVIII in a HLA-DRB1*1501 Humanized Hemophilic Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2280-2280
Author(s):  
Katharina Nora Steinitz ◽  
Brigitte Binder ◽  
Christian Lubich ◽  
Rafi Uddin Ahmad ◽  
Markus Weiller ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Mouse Model ◽  
Mhc Class Ii ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Hemophilia A ◽  
Class Ii ◽  
T Cell Epitopes

Abstract Abstract 2280 Development of neutralizing antibodies against FVIII is the major complication in the treatment of patients with hemophilia A. Although several genetic and environmental risk factors have been identified, it remains unclear why some patients develop antibodies while others do not. Understanding the underlying mechanisms that drive the decision of the immune system whether or not to make antibodies against FVIII would help to design novel therapeutics. We used a new humanized hemophilic mouse model that expresses the human MHC-class II molecule HLA-DRB1*1501 on the background of a complete knock out of all murine MHC-class II genes. Initial studies had indicated that only a fraction of these mice developed antibodies when intravenously (i.v.) treated with human FVIII. These findings which resemble the situation in patients with severe hemophilia A, evoked the question if the lack of antibody development in non-responder mice reflects the induction of specific immune tolerance after i.v. application of FVIII or represent non-responsiveness for other reasons. We addressed this question by choosing another application route (subcutaneous, s.c.) and by combining i.v. application with a concomitant activation of the innate immune system applying LPS, a well characterized ligand for toll-like receptor 4, together with FVIII. Both strategies resulted in the development of antibodies in all mice included in the study what suggested that non-responsiveness against i.v. FVIII does not reflect an inability to develop antibodies against FVIII. Next, we asked if i.v. FVIII does induce immune tolerance in non-responder mice. We pretreated mice with i.v. FVIII, selected non-responder mice and challenged them with s.c. FVIII. None of the mice developed antibodies what indicated that i.v. pretreatment had induced immune tolerance in non-responder mice. Currently, we test the hypothesis that immune tolerance after i.v. application is induced and maintained by FVIII-specific regulatory T cells. The differences in responder rates after i.v. and s.c. application of FVIII raised the question if there are differences in FVIII T-cell epitopes involved in the initial activation of FVIII-specific CD4+ T cells. We obtained spleen cells from mice treated with either i.v. or s.c. FVIII and generated CD4+ T-cell hybridoma libraries that were tested for peptide specificities. For this purpose we used a FVIII peptide library containing 15 mers with an offset of 3 amino acids. Our results indicate that the pattern of FVIII-specific T-cell epitopes involved in the activation of FVIII-specific CD4+ T cells after i.v. and s.c. application of FVIII is almost identical and represents a small set of FVIII peptides distributed over the A1, A2, B, A3 and C1 domains. Based on our results we conclude that the new HLA-DRB1*1501 hemophilic mouse model represents an interesting opportunity to uncover the mechanisms that drive the decision of the immune system whether or not to develop antibodies against FVIII. Disclosures: Steinitz: Baxter BioScience: Employment. Binder:Baxter BioScience: Employment. Lubich:Baxter BioScience: Employment. Ahmad:Baxter BioScience: Employment. Weiller:Baxter BioScience: Employment. de la Rosa:Baxter BioScience: Employment. Schwarz:Baxter BioScience: Employment. Scheiflinger:Baxter BioScience: Employment. Reipert:Baxter Innovations GmbH: Employment.

Separation of Gvhd from GVL Responses By Modulation of TCR Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3822-3822
Author(s):  
Mobin Karimi ◽  
Martha Jordon ◽  
Taku Kambayashi
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Leukemia Cells ◽  
Cell Phenotype ◽  
Tcr Signaling ◽  
Allogeneic Hsct

Abstract In allogeneic hematopoietic stem cell transplantation (HSCT), devising new strategies to separate GVHD and GVL responses is of critical importance. However, this is a difficult task, as GVHD and GVL rely on the same recognition of allogeneic MHC by donor-derived T cells. CD8+ T cells are key effector cells that mediate both GVHD and GVL. In mouse models of allogeneic HSCT, the infusion of donor-derived CD8+ T cells eliminates tumor growth but also causes severe GVHD. The activation of CD8+ T cells can be potentially manipulated by perturbing the signaling pathways downstream of the T cell receptor (TCR). TCR signaling depends on the formation of a proximal multimolecular complex, which is nucleated by adaptor proteins such as SLP-76. The phosphorylation of the Y145 residue of SLP-76 is critical for activation of the downstream enzyme PLCg1. As such, a YàF mutation at Y145 of SLP-76 (Y145F) causes decreased TCR-mediated signaling and attenuated T cell function. Here, we investigated how the SLP-76 Y145F mutation in CD8+T cells may impact GVHD and GVL responses in a mouse model of allogeneic HSCT. We employed a major MHC-mismatch mouse model of GVHD involving the transplantation of C57BL/6 (B6)-derived bone marrow (BM) into lethally irradiated Balb/c mice (B6àBalb/c). BM-transplanted mice were also injected with FACS-sorted CD8+ T cells either B6 wildtype (WT) mice or Y145F mice. Recipients of Y145F CD8+ T cells showed significantly (p<0.001) less weight loss, lower clinical score, and improved survival compared to mice injected with WT CD8+ T cells. Next, to determine whether the Y145F CD8+ T cells could mediate GVL effects, BM-transplanted Balb/c mice were additionally challenged intravenously with 1 x 105 luciferase-positive A20 leukemia cells. As expected, BM-transplanted Balb/c mice succumbed from A20 tumor growth, whereas mice injected with WT CD8+ T cells cleared the tumor but developed GVHD. Surprisingly, mice receiving Y145F CD8+ T cells eradicated the leukemic cells but did not develop GVHD. These data suggest that the Y145F mutation in CD8+T cells may be able to separate GVHD from GVL effects. In addition to defective TCR signaling observed in peripheral T cells of Y145F mice, a majority of Y145F KI CD8+ T cells adopt a memory-like CD44hi phenotype through exposure to high levels of IL-4 produced in the thymus of these mice. To test whether the CD44hi CD8+ T cell phenotype was necessary and/or sufficient for the separation of GVHD and GVL effects, BM-transplanted Balb/c mice were injected with FACS-sorted CD44hi or CD44lo CD8+ T cells from WT or Y145F KI mice and challenged with A20 leukemia cells. While BM-transplanted mice receiving CD44hi CD8+ T cells from Y145F mice displayed intact GVL responses without causing GVHD, mice injected with CD44lo CD8+ T cells from Y145F mice displayed impaired ability to clear the tumor cells. Moreover, recipients of CD44hi or CD44lo CD8+ T cells from WT mice cleared the tumor but exhibited severe GVHD. These findings were corroborated with data obtained with an inducible system, whereby CD8+ T cells are affected by the Y145F mutation only after full maturation and thus do not display a CD44hi phenotype (Y145F conditional knock-in mice). Bone marrow-transplanted recipients receiving Y145F conditional knock-in CD8+ T cells developed GVHD and exhibited an attenuated GVL response, suggesting that the Y145F mutation needed to be present during T cell development. Together, these data suggest that either the Y145F mutation or CD44hi phenotype alone in CD8+T cells is insufficient to separate GVHD from GVT. Our data demonstrate that perturbation of the TCR signaling pathway downstream of Y145 of SLP-76 in CD8+ T cells results in separation of GVHD from GVL effects. Experiments to mechanistically test how the Y145F signaling mutation synergizes with the CD44hi phenotype of CD8+ T cells to allow for the separation of the GVHD and GVL effects are currently underway. Our novel and unexpected finding could lead to a novel therapeutic strategy for treatment of acute GVHD after allogeneic HSCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Total CD3 T Cells Are Necessary and Sufficient to Induce Colitis in Immunodeficient Mice With Dendritic Cell–Specific Deletion of TGFbR2: A Novel IBD Model to Study CD4 and CD8 T-Cell Interaction

2019 ◽  
Vol 26 (2) ◽  
pp. 229-241
Author(s):  
Deepa Rana Jamwal ◽  
Raji V Marati ◽  
Christy A Harrison ◽  
Monica T Midura-Kiela ◽  
Vanessa R Figliuolo Paz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dendritic Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Immune Cell ◽  
Immunodeficient Mice ◽  
Cell Lineages ◽  
Adaptive Immune ◽  
Adaptive Immune Cells

Abstract Background Inflammatory bowel disease (IBD) is a multifactorial disorder, with the innate and adaptive immune cells contributing to disease initiation and progression. However, the intricate cross-talk between immune cell lineages remains incompletely understood. The role of CD8+ T cells in IBD pathogenesis has been understudied, largely due to the lack of appropriate models. Methods We previously reported spontaneous colitis in mice with impaired TGFβ signaling due to dendritic cell–specific knockout of TGFbR2 (TGFβR2ΔDC). Here, we demonstrate that crossing TGFβR2ΔDC mice with a Rag1-/- background eliminates all symptoms of colitis and that adoptive transfer of unfractionated CD3+ splenocytes is sufficient to induce progressive colitis in Rag1-/-TGFβR2ΔDC mice. Results Both CD4+ and CD8+ T cells are required for the induction of colitis accompanied by activation of both T-cell lineages and DCs, increased expression of mucosal IFNγ, TNFα, IL6, IL1β, and IL12, and decreased frequencies of CD4+FoxP3+ regulatory T cells. Development of colitis required CD40L expression in CD4+ T cells, and the disease was partially ameliorated by IFNγ neutralization. Conclusions This novel model provides an important tool for studying IBD pathogenesis, in particular the complex interactions among innate and adaptive immune cells in a controlled fashion, and represents a valuable tool for preclinical evaluation of novel therapeutics.

Transient NKG2D Blockade Attenuates Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia Effects

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3242-3242 ◽  
Author(s):  
Mobin Karimi ◽  
Theresa M Leichner ◽  
Atsushi Satake ◽  
David Raulet ◽  
Taku Kambayashi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cd8 T Cell ◽  
Allogeneic Hsct ◽  
Isotype Control ◽  
Isotype Control Antibody

Abstract In allogeneic hematopoietic stem cell transplantation (HSCT), identification of mechanisms to control GVHD yet maintain GVL responses is of critical importance. One key effector cell that mediates both GVHD and GVL is the CD8+ T cell, which expands in response to T cell receptor (TCR) stimulation by allogeneic MHC class I molecules during allogeneic HSCT. In addition, co-stimulatory molecules facilitate the TCR-mediated activation process and the effector function of CD8+ T cells. Recent data suggest that NKG2D may play a co-stimulatory role in activation and in augmenting anti-tumor cytotoxic responses of CD8+ T cells. NKG2D is an NK cell-associated receptor that is also expressed on all human CD8+ T cells and on activated/memory mouse CD8+ T cells. NKG2D recognizes a diverse array of MHC-related ligands that are expressed by many tumors and induced on cells under stress such as myeloablative conditioning during HSCT. As the role of NKG2D in allogeneic HSCT is unknown, we hereby investigated the role of NKG2D on CD8+ T cells in a mouse model of GVHD and GVL. Our results show that a large fraction (40-50%) of mouse CD8+ T cells inducibly express NKG2D upon activation by allogeneic MHC in vitro and in vivo. To test the role of NKG2D in GVHD pathogenesis, we employed a major MHC-mismatched mouse model of GVHD involving the transplantation of C57BL/6-derived CD8+ T cells and bone marrow (BM) into lethally irradiated Balb/c mice (B6→Balb/c). Using 3 different approaches to block NKG2D on CD8+ T cells (shRNA-mediated silencing, germline NKG2D deficiency, and antibody blockade), we found that weight loss, clinical score, and survival were significantly improved in transplanted mice with NKG2D blockade. The attenuation in GVHD correlated with a significant reduction in TNFα and IFNγ production, cytotoxicity, and proliferation (BrdU incorporation) by CD8+ T cells. Although CD4+ T cells did not express NKG2D, a protective effect of NKG2D blockade was still observed in GVHD induced by a mixture of CD8+ and CD4+ T cells, albeit to a lesser extent. We next tested the effects of NKG2D on CD8+ T cell-mediated GVL. To this end, irradiated Balb/c mice were transplanted with C57BL/6-derived CD8+ T cells and BM, challenged intravenously with luciferase-positive A20 leukemia cells, and followed by total body imaging of luciferase-expressing cells. Given that NKG2D ligands are constitutively expressed on many tumor cells and plays an important role in their eradication, we predicted that continuous NKG2D blockade would inhibit GVL effects. However, as NKGD ligands are upregulated only transiently on stressed normal tissue, we reasoned that transient NKG2D blockade might be sufficient to attenuate GVHD and allow CD8+ T cells to regain their GVL function. To test this hypothesis, we compared the effect of anti-NKG2D antibody as continuous treatment or as 5-day transient treatment to mice receiving isotype control antibody. As expected, mice that received isotype control antibody cleared the A20 cells but developed severe GVHD. Continuous anti-NKG2D antibody-mediated blockade improved GVHD but also blunted the GVL response leading to increased A20 growth. In contrast, a large proportion of mice transiently treated with anti-NKG2D antibody cleared the A20 cells, while maintaining the attenuated GVHD state. Together, these data support a positive role of NKG2D on CD8+ T cells in mediating GVHD and GVL. Given the transient nature of NKG2D ligand upregulation on stressed tissues, a window of opportunity may exist where transient NKG2D blockade could provide a novel therapeutic strategy for treatment of acute GVHD while preserving the GVL function of CD8+ T cells after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Bypassing the Constraint for Chimeric Antigen Receptor (CAR) Development in T-Cells Expressing the Targeted Antigen: Improvement of Anti-CS1 CAR Activity in Allogenic TCRa/CS1 Double Knockout T-Cells for the Treatment of Multiple Myeloma (MM)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 116-116 ◽  
Author(s):  
Roman Galetto ◽  
Isabelle Chion-Sotinel ◽  
Agnès Gouble ◽  
Julianne Smith
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
B Cell ◽  
Cd8 T Cells ◽  
Gene Editing ◽  
Transcription Activator ◽  
Car T Cell ◽  
Car T

Abstract Adoptive immunotherapy with autologous T-cells expressing chimeric antigen receptors (CARs) targeting CD19 has achieved long-term remissions in patients with B cell leukemia, pointing out that CAR technology may become a new alternative in cancer treatment. In this work we assessed the feasibility of targeting the CS1 antigen (SLAMF7) for the treatment of Multiple Myeloma (MM). MM is a B-cell neoplasia characterized by clonal expansion of malignant plasma cells in the bone marrow. Even if currently available therapies can improve overall survival, MM still remains incurable in most patients. Immunotherapy against MM is therefore an area in which extensive research is being made, with novel antigenic targets being considered. Among these is the CS1 glycoprotein, which is highly expressed on tumor cells from most patients with MM. However, CS1 is also expressed on normal CD8+ T-cells, which may be problematic for a CAR-based approach as antigen-expressing T cells will be targeted, impacting both the number and the phenotype of the final CAR T cell population. To circumvent this issue we have used our highly-efficient transcription activator-like effector nuclease (TALEN) gene-editing technology to inactivate CS1 in T-cells prior to transduction with a viral vector encoding an anti-CS1 CAR. Our results demonstrate that while non-gene-edited T-cells expressing an anti-CS1 CAR display limited cytolytic activity against MM cell lines, and resulted in a progressive loss of CD8+ T-cells. CS1-gene-edited CAR cells display significantly increased cytotoxic activity, with the percentage of CD8+ T-cells remaining unaffected. In addition, experiments in an orthotopic MM mouse model showed that CS1 disrupted T-cells were able to mediate an in vivo anti-tumoral activity. Subsequently, we have utilized this strategy for CS1 in the context of our allogeneic "off-the-shelf" engineered CAR+ T-cell platform. This allogenic platform utilizes TALEN gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating their potential to mediate Graft versus Host Disease (GvHD). We have previously shown that editing of the TRAC gene can be achieved at high frequencies, allowing efficient production of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. Our results also show that multiplex genome editing is possible and can lead to the production of double KO (TRAC and CS1) T-cells, allowing large scale manufacturing of allogeneic, non alloreactive CS1 specific T-cells with enhanced antitumor activity. Moreover, these allogenic T-cells could be easily available for administration to a large number of MM patients. Disclosures Galetto: Cellectis SA: Employment. Chion-Sotinel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.

scholarly journals The Inhibitory T Cell Receptors PD1 and 2B4 Are Differentially Regulated on CD4 and CD8 T Cells in a Mouse Model of Non-alcoholic Steatohepatitis

2019 ◽  
Vol 10 ◽  
Author(s):  
Cordula Hansel ◽  
Stephanie Erschfeld ◽  
Maike Baues ◽  
Twan Lammers ◽  
Ralf Weiskirchen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Cd8 T Cells ◽  
T Cell Receptors ◽  
Cell Receptors ◽  
Alcoholic Steatohepatitis

scholarly journals 661 Neoantigen-specific CD4+ T cells in human melanoma have diverse differentiation states and correlate with CD8+ T cell, macrophage, and B cell function

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A689-A689
Author(s):  
Naina Singhi ◽  
Carolyn Shasha ◽  
Sylvia Lee ◽  
Julia Szeto ◽  
Ata Moshiri ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Tumor Antigen ◽  
Cd4 T Cells ◽  
Viral Antigen ◽  
Tumor Antigens ◽  
Human Melanoma

BackgroundTumor-antigen specific CD4+ T cells are crucial for the efficacy of antibodies that block immune checkpoint proteins in mouse tumor models, but their activities in human tumor immunity are less clear. CD8+ T cells infiltrating human tumors, including those specific for tumor antigens, have been studied using single cell profiling techniques and exist in a variety of dysfunctional states. The transcriptional states of tumor-specific CD4+ T cells present in tumors and their potential contributions to the tumor microenvironment are less well understood.MethodsWe used targeted single cell RNA sequencing and matching of T cell receptor (TCR) sequences to identify phenotypic signatures that discriminated tumor antigen- and viral antigen-specific CD4+ T cells infiltrating human melanoma tumors in four patients. The presence of CD4+ T cells with these signatures was correlated with the number and phenotype of other immune cells in the tumor microenvironment in an extended cohort of 20 patients.ResultsWe identified 259 CD4+ T cells representing 40 different TCR clonotypes specific for 13 neoantigens and 108 cells representing 14 TCR clonotypes specific for self-antigens in four melanoma patients. High expression of CXCL13 defined conventional CD4+ T cells that recognize tumor associated neoantigens and self-antigens from bystander and viral antigen-specific CD4+ T cells. Tumor-reactive CD4+ T cells could be subdivided into clusters expressing memory and T follicular helper markers, and those expressing cytolytic markers and IFN-g. In an extended cohort of 20 patients with melanoma, the frequency of CXCL13+ CD4+ T cells in the tumor microenvironment correlated with the presence and proliferation of CD8+ T cells, the presence and maturation of B cells, the activation of interferon responsive genes in tumor associated macrophages, and patient survival. CD4+ T cells with similar transcriptional signatures were identified in data sets from breast and non-small cell lung cancer, suggesting these markers may enrich for tumor-reactive CD4+ T cells in many cancers.ConclusionsThese results identify a subset of tumor infiltrating conventional CD4+ T cells in melanoma that are enriched for reactivity to tumor antigens and exist in multiple phenotypic states. Correlations of the presence of these cells with the frequency and phenotype of other immune cells suggest roles for these tumor antigen-specific CD4+ T cells in providing CD8+ T cell help, driving recruitment and maturation of B cells, and activating macrophages. Isolating such cells based on their unique phenotype and utilizing them for adoptive therapy could alter the tumor microenvironment for therapeutic benefit.Ethics ApprovalAll Patient samples in this study were obtained from patients who signed informed consent in a study approved by the institutional review board of the Fred Hutchinson Cancer Research Center (protocol #2643).

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