T-cell Effector Function Research Articles

REGN5458, a BCMA x CD3 Bispecific Monoclonal Antibody, Induces Deep and Durable Responses in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

REGN5458, a BCMA x CD3 Bispecific Monoclonal Antibody, Induces Deep and Durable Responses in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

Multi-Omic Based Antigen Discovery for the Immunotherapy of Pediatric Acute T Cell Lymphoblastic Leukemia

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk disease due to treatment related complications and poor prognosis of patients with relapsed disease. Immunotherapy with monoclonal antibodies (MAbs) and/or chimeric antigen receptor (CAR) T-cells for T-ALL is limited by identification of tumor specific target antigens. Differential expression is necessary to prevent on-target/off-tumor toxicities and fratricide of activated T-cells. Targeting multiple antigens can bypass immune escape and result in improved T-cell effector function, since antigen density correlates with T-cell activation. Here we designed a pipeline (Figure 1) to identify unique surface antigens expressed in T-ALL using proteomic and transcriptomic analyses followed by flow cytometry validation, and functional studies with CAR T cells targeting the identified antigens. We generated an Illumina total stranded RNAseq library from healthy donor myeloid and lymphoid cells of bone marrow, peripheral blood and cord blood (N= 116). We compared data to 265 St. Jude pediatric T-ALL samples and against 53 normal tissue expression data from the GTEx (Genotype-Tissue Expression) project. To analyze the T-cell surface proteome, we isolated plasma membrane fractions from 11 samples including healthy T-cells and T-ALL cell lines using a differential centrifugation-based method. The purity of the plasma membrane fraction was confirmed by western blot. Na+/K+ ATPase and GAPDH were used as controls for the plasma membrane and cytosolic fractions respectively. Following plasma membrane enrichment, the membrane proteins were applied for proteomic analysis using an advanced TMT-L/LC-MS/MS pipeline, and the acquired proteomic data were further processed via the JUMP software suite. 997 unique proteins were quantified from the membrane fractions. Integrated analysis the transcriptomic and proteomic datasets showed significant correlation and yielded a list of candidate genes, which were validated by flow cytometry on a panel of T-ALL cell lines (CCRF, RPMI8402, and MOLT3) and resting and activated T-cells from healthy donors. We identified GRP78 as one of the differentially expressed cell surface antigens and further confirmed its expression on additional T-ALL cell lines (KE37, PF382, PEER, CEMC7) and 3 PDX samples. Finally, we generated GRP78-CAR T cells and demonstrate that GRP78-CAR T cells recognize and kill GRP78+ T-ALL cells and have potent antitumor activity in xenograft and PDX models. We have established an unbiased pipeline to identify differentially expressed antigens on the cell surface of T-ALL blasts and created a healthy tissue RNAseq library. The results from our analyses are encouraging and interrogation of our pipeline has yielded differentially expressed immunotherapy targets for the treatment of relapsed refractory T-ALL. Our results highlight the importance of integrated surface proteomics and transcriptomics analysis. Figure 1: Outline of strategy for target selection: Figure Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Gottschalk:Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; TESSA Therapeutics: Other: research collaboration; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Merck and ViraCyte: Consultancy. Mullighan:AbbVie, Inc.: Research Funding; Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties.

Impact of Itolizumab on the Expression of CD6 and the Function of Effector T Cells

Introduction: CD6 is a T-cell costimulatory receptor that has been implicated in the pathogenesis of multiple autoimmune and inflammatory (AI) diseases. In GVHD, CD6 is expressed on reconstituting T cells soon after transplant (Rambaldi et al., 2019), including Th1 and Th17 cells which are both implicated in the induction and pathogenesis of acute GVHD (aGVHD). CD6 is highly expressed on these cells and promotes immune synapse formation, T-cell activation, and T-cell migration via interaction with its ligand activated leukocyte cell adhesion molecule (ALCAM). Furthermore, studies have demonstrated that ex-vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998), highlighting the importance of CD6. While the contribution of CD6 to T cell activation has been well described, less is known regarding the expression levels and role of CD6 on effector and memory T cells (Teff) which are prominent in all diseases including aGVHD. Consequently, the aim of this study was to determine the role of CD6 specifically on effector T cells, and further illuminate the mechanism of itolizumab, an anti-CD6 monoclonal antibody. Methods: Naïve T cells were enriched from frozen PBMCs via a naïve T cell magnetic separation kit (Stemcell). Naïve T cells were polarized towards a Th1 phenotype for 6 days with a Th1 differentiation cocktail (Stemcell) and CD3/CD28 T cell activator (Stemcell) and rested overnight prior to entering restimulation conditions. Naïve T cells were polarized towards a Th17 phenotype for 8 days with IL-6, IL-1β, TGF-β, IL-23, anti-IL-4 and IFN-γ, the CD3/CD28 T cell activator was used to activate the T cells. To re-stimulate differentiated T cells, anti-CD3 mAb and ALCAM-Fc or anti-CD3 mAb alone were coated on 48-well plates overnight at 4oC. Th1 or Th17 T cells were labeled with CFSE and seeded with isotype control or itolizumab for 72hrs. Cells were collected for flow cytometry analysis and supernatant collected for relevant cytokine detection. To assess surface levels of CD6, cryopreserved PBMCs were thawed and incubated with itolizumab or isotype at 37oC for specific timepoints. Following incubation, cells were washed and stored at 4oC for subsequent staining. All samples were stained at the same time and surface levels of CD6 was detected using a monoclonal anti-CD6 antibody that does not compete with itolizumab. Results: Blockade of the CD6 pathway, using itolizumab during restimulation of differentiated Teff cells in the presence of ALCAM, inhibited multiple effector functions including proliferation and changes in cell size. An average of a 40% decrease in CFSE proliferation was observed across multiple donors. Furthermore, treatment of Teff cells with itolizumab resulted in a significant decrease in expression level of T cell markers of activation and exhaustion such as CD25, PD-1 and Tim3. This effect was exclusively in the presence of ALCAM, indicating that the effect was specific to blockade of the CD6-ALCAM pathway. When levels of CD6 were assessed, CD45RO+ (Teff/mem) cells expressed higher levels than CD45RA+CD45RO- (Tnaive). Itolizumab treatment of in vitro generated CD45RO+ T cells inhibited this stimulation-induced increase in CD6 in a dose-dependent manner, suggesting that the drug may modulate surface CD6 expression as a mechanism separate from physical blockade of CD6. Conclusions: These findings are the first to characterize the CD6-ALCAM pathway as a key regulator of differentiated effector T-cell function. Modulation of activation markers and CD6 itself by itolizumab, suggest modulation of Teff activity by both direct and indirect inhibition of CD6 signaling. These data further support targeting the CD6-ALCAM pathway to inhibit both naïve and effector T cell populations in aGVHD. Disclosures Ampudia: Equillium: Current Employment, Current equity holder in publicly-traded company. Chu:Equillium: Current Employment, Current equity holder in publicly-traded company. Doherty:Equillium Inc.: Research Funding. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company.

Co-inhibitory Receptor Signaling in T-Cell-Mediated Autoimmune Glomerulonephritis.

Autoimmune glomerulonephritis occurs as a consequence of autoantibodies and T-cell effector functions that target autoantigens. Co-signaling through cell surface receptors profoundly influences the optimal activation of T cells. The scope of this review is signaling mechanisms and the functional roles of representative T-cell co-inhibitory receptors in the regulation of autoimmune glomerulonephritis, along with current therapeutic challenges mainly on preclinical trials. Co-inhibitory receptors utilize both shared and unique signaling pathway, suggesting specialized functions that provide the rationale behind therapies for autoimmune glomerulonephritis by targeting these inhibitory receptors. These receptors largely suppress Th1 immunity, modify Th17 and Th2 immune response, and enhance Treg function. Anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) immunoglobulin (Ig), which is able to block both activating CD28 and inhibitory CTLA4 signaling, has been shown in preclinical and clinical investigations to have effects on glomerular disease. Other inhibitory receptors for treating glomerulonephritis have not been clinically tested, and efficacy of manipulating these pathways requires further preclinical investigation. While immune checkpoint inhibition using anti-CTLA4 antibodies and anti-programmed cell death 1 (PD-1)/PD-L1 antibodies has been approved for the treatment of several cancers, blockade of CTLA4 and PD-1/PD-L1 is associated with adverse effects that resemble autoimmune disorders, including systemic vasculitis. A renal autoimmune vasculitis model features an initial Th17 dominancy followed later by a Th1-dominant outcome and Treg cells that attenuate autoreactive T-cell function. Toward the development of effective therapies for T-cell-mediated autoimmune glomerulonephritis, it would be preferable to pay attention to the impact of the inhibitory pathways in immunological renal disease settings.

Abstract IA14: mTOR as a central regulator of T-cell activation, differentiation, and function

Abstract While the exquisite specificity of the T-cell receptor (TCR) MHC + peptide interaction heralds recognition, it is ultimately the cues from the immune microenvironment that dictate the outcome of this recognition. Our lab has proposed that mTOR plays a critical role in integrating signals and thus determining the outcome of antigen recognition. Indeed, we have demonstrated that mTORC1 and mTORC2 signaling can selectively guide CD4+ effector and regulatory T-cell differentiation and function as well as CD8+ effector and memory T-cell differentiation and function. Recent studies have revealed that mTOR performs this function in part by coordinating T-cell activation and function with metabolic reprogramming. Consequently, targeting downstream mTOR signaling and metabolism can promote T-cell effector function and persistence in order to enhance cancer immunotherapy. Likewise, targeting mTOR can inhibit T-effector function and promote the generation of regulatory T cells to treat autoimmune disease and prevent transplantation rejection. Citation Format: Jonathan Powell, Chirag Patel, Hong Sun, Michael Claiborne. mTOR as a central regulator of T-cell activation, differentiation, and function [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA14.

Translational and clinical advances in acute graft-versus-host disease.

Acute graft-versus-host disease (aGvHD) is induced by immunocompetent alloreactive T lymphocytes in the donor graft responding to polymorphic and non-polymorphic host antigens and causing inflammation in primarily the skin, gastrointestinal tract and liver. aGvHD remains an important toxicity of allogeneic transplantation, and the search for better prophylactic and therapeutic strategies is critical to improve transplant outcomes. In this review, we discuss the significant translational and clinical advances in the field which have evolved based on a better understanding of transplant immunology. Prophylactic advances have been primarily focused on the depletion of T lymphocytes and modulation of T-cell activation, proliferation, effector and regulatory functions. Therapeutic strategies beyond corticosteroids have focused on inhibiting key cytokine pathways, lymphocyte trafficking, and immunologic tolerance. We also briefly discuss important future trends in the field, the role of the intestinal microbiome and dysbiosis, as well as prognostic biomarkers for aGvHD which may improve stratification-based application of preventive and therapeutic strategies.

Radiation with STAT3 Blockade Triggers Dendritic Cell-T cell Interactions in the Glioma Microenvironment and Therapeutic Efficacy.

Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma. C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood-brain barrier-penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment. The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions. This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.

Defective CD19+CD24hiCD38hi transitional B-cell function in patients with relapsing-remitting MS

Background: Multiple sclerosis (MS) is characterized by central nervous system (CNS) infiltration of T and B cells, excess inflammatory cytokine and chemokine production and failure of immune regulation. CD19+CD24hiCD38hi transitional B cells producing interleukin (IL)-10 have been shown to suppress interferon-γ (IFNγ) and tumour necrosis factor-α (TNFα) production by CD4+ T cells and to be dysfunctional in autoimmune arthritis and systemic lupus erythematosus. Objective: We hypothesized that transitional B-cell-dependent immune regulation could be defective in MS and examined their function in healthy subjects and patients with relapsing-remitting multiple sclerosis (RRMS). Methods: A total of 62 healthy donors and 21 RRMS subjects donated peripheral blood for the study. IL-10-producing B cells, IFNγ and TNFα-producing T cells and proliferating T cells were quantified by flow cytometry. Results: In healthy individuals, CD19+CD24hiCD38hi transitional B cells produce more IL-10 than CD19+CD24+CD38+ naive and CD19+CD24hiCD38− memory B cells and are able to suppress CD4+ T-cell proliferation and IFNγ and TNFα-production. In subjects with RRMS, CD19+CD24hiCD38hi transitional B cells produce significantly less IL-10 and to fail to suppress effector T-cell function. Conclusion: CD19+CD24hiCD38hi transitional B cells physiologically represent the most potent regulatory B-cell subset and are functionally defective in patients with RRMS, an abnormality that may contribute to the immune pathological process.

Cellular and molecular profiling of T-cell subsets at the onset of human acute GVHD

AbstractThe cellular and molecular processes involved in acute graft-versus-host disease (aGVHD) development early after allogeneic hematopoietic cell transplantation (HCT) in humans remain largely unknown. We have performed multiparameter immunophenotyping and molecular profiling of CD4+and CD8+T cells in 2 independent cohorts of patients undergoing HCT, as well as in their HLA-identical sibling donors. Cellular profiling using spectral flow cytometry showed an incomplete reconstitution of the T-cell compartment in recipients without aGVHD early after transplantation, as well as a shift toward an effector memory phenotype, paralleled by depletion of the naive T-cell pool. Molecular profiling of T-cell populations in donors vs recipients without aGVHD revealed increased pathway activity of >40 gene modules in recipients. These pathways were associated in particular with T-cell activation, adhesion, migration, and effector functions. Cellular profiles from recipients developing aGVHD displayed an enrichment of cells with a T memory stem cell–like phenotype compared with recipients without aGVHD. Comparison of gene profiles from these recipients revealed that transforming growth factor-β (TGF-β) signaling was most significantly downregulated, whereas the pathway activity of NF-κB–associated transcription factors and signaling pathways were increased, at aGVHD onset. This study suggests that the integration of cellular and molecular profiles provides new insights into the development of aGVHD in humans.

TIGIT Can Exert Immunosuppressive Effects on CD8+ T Cells by the CD155/TIGIT Signaling Pathway for Hepatocellular Carcinoma In Vitro.

The efficacy of adoptive cellular immunotherapy against cancer cells is limited due to the presence of immunosuppressive cells within the solid tumor microenvironment. The upregulation of certain coinhibitory receptors may lead to exhaustion of the immune effector cells. T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an immune inhibitory receptor expressed by regulatory T cells and activated T cells and natural killer cells. The aim of this study was to determine the immunosuppressive effects of CD155/TIGIT signaling on CD8 T cells of adoptive cellular immunotherapy in hepatocellular carcinoma (HCC). Our studies found that CD155 was overexpressed in HCC, and CD155 HCC cells upregulated TIGIT on CD8 T cells, which decreased the secretion of interferon-γ, tumor necrosis factor-α, and interleukin-17A and increased that of interleukin-10 from the effector cells. However, TIGIT blockade or CD155-knockdown reversed the inhibitory effect of HCC cells on CD8 T-cell effector function. These results indicate that TIGIT can exert an immunosuppressive effect on CD8 T cells by modulating cytokine production through CD155, and is a promising target to optimize adoptive cellular immunotherapy against HCC.

Abstract 5573: Mechanism of resistance against T cell bispecific antibodies depends on targeted antigen

Abstract Immunotherapy has revolutionized the way several types of cancers are treated, including breast and gastric cancer. One of the most successful therapies against HER2 positive tumors, trastuzumab, is a monoclonal antibody originally designed to inhibit HER2, which has been shown to be effective because not only impairs cell cycle but also promotes an immune reaction. Despite that, lots of patients do not respond or relapse. T Cell Bispecific antibodies (TCBs) are a promising immunotherapeutic strategy designed to boost the immune response against tumors, as they redirect cytotoxic T cells against the tumor cells. TCBs are engineered molecules that include, within a single entity, binding sites to the T cell receptor and to a tumor-specific or a tumor-associated antigen. Clinical evidence shows that TCBs are an effective immunotherapy to treat cancer. However, little is known about the mechanisms of resistance against this promising therapy. Therefore, there is a need to anticipate the mechanism of response and resistance in order to improve the clinical outcome of patients. Indeed, as all TCBs behave with the same mechanism of action, the same mechanism of resistance, independent of the target, is expected. In this piece of work, we tried to address for the first time this question, by using as a tool a gastric HER2+/CEA+ cell line MKN45, and TCBs targeting these antigens. We generated resistant cells against both HER2-TCB and CEA-TCB in vitro. To generate these immunoresistant models we co-cultured peripheral blood mononuclear cells (PBMCs) with the TCBs for several months in order to obtain resistance. We corroborated the resistance in a organotypic 3D model and also in an in vivo humanized PBMC model. In addition, we assessed the impact on T-cell effector function, and resistant cells impair T-cell activity. We next evaluated the cause of resistance, and one plausible mechanism that also happen in hematological malignancies treated with Chimeric Antigen Receptor T-cells (CAR-Ts) is the loss of the antigen, in this case HER2 and CEA. Contrary to our expectations, we observed a dramatic loss of antigen in the case of CEA-TCB resistant cells, in contrast to HER2-TCB resistant cells, which maintain HER2 levels. Our results show for the first time that the mechanism of resistance against TCBs can be totally different depending on the target, and future studies and therapeutic approaches should take this into consideration. In addition, the HER2-TCB resistant MKN45 cells can be used as a tool to identify unknown mechanisms of resistance against the redirection of T-cells. Citation Format: Alex Martínez-Sabadell, Enrique J. Arenas, Irene Rius, Macarena Román, Beatriz Morancho, Cristina Bernado, Joaquin Arribas. Mechanism of resistance against T cell bispecific antibodies depends on targeted antigen [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5573.

Abstract 2288: Investigational CD25 x CTLA-4 bispecific DART® molecule for depletion of tumor infiltrating Tregs via an enhanced Fc-dependent effector mechanism

Abstract Background: Tregs are suppressive cells whose presence in the tumor microenvironment (TME) has been correlated with tumor progression and may be a major impediment in the effectiveness of tumor immunotherapy. Removal of Tregs has been shown to induce cancer regression in animal models; however, selective depletion of tumor-infiltrating Tregs (TI-Tregs) is complicated as they share phenotypic markers with Tregs in healthy tissues as well as with tumor-infiltrating effector cells. To overcome cross-reactivity and achieve enhanced targeting specificity, we employed a bispecific strategy to simultaneously target two cell surface antigens (CD25 and CTLA-4) with overlapping, high level expression on TI-Tregs. Furthermore, we incorporated Fc engineering to enable target-cell depletion via enhanced Fc-mediated effector function. Methods: CD25 x CTLA-4 DART proteins were constructed based on CD25 and CTLA-4 mAbs and incorporating an Fc domain with enhanced binding to the activating Fc-gamma RIIIa receptor (CD16A) and reduced binding to the inhibitory Fc-gamma RIIb receptor (CD32B). Depletion of FoxP3-positive cells ex vivo was monitored by flow cytometry. Immunomodulatory effects of the DART proteins were evaluated in cultured PBMC or dissociated tumor cells in various in vitro immune assays, including mixed lymphocytes reaction (MLR). Results: Consistent with their design, we observed increased binding of CD25 x CTLA-4 DART molecules to cells when both target antigens were engaged compared to single antigen engagement. The bispecific DART molecule depleted in vitro induced FoxP3+ CD4+ TI-like Tregs cells by an Fc-dependent mechanism, with minimal effect on the viability of FoxP3-negative T cells. A molecule bearing a null Fc was ineffective. The extent of FoxP3+ T-cell depletion by the Fc-enhanced CD25 x CTLA-4 DART molecule was greater than that exhibited by the combination of its monovalent components, with the depleting activity requiring both arms of the bispecific molecule. The DART molecule had only minor effect on the capacity of effector T cells to secrete IL-2 and interferon-gamma upon restimulation or to exert cytotoxic function. In contrast the combination of CD25 and CTLA-4 mAbs reduced the ability of effector T cells to produce cytokines in a restimulation assay. An increased response in the presence of the Fc-enhanced CD25 x CTLA-4 DART molecule was also observed in various immune assays, consistent with Treg suppression and preservation of T-cell effector function. Conclusions: A CD25 x CTLA-4 DART molecule was effective in depleting activated FoxP3+ CD4+ Tregs in vitro, with reduced effects on resting Tregs or activated FoxP3- effector cells. Targeting CD25 and CTLA-4 in a bispecific molecular format with enhanced Fc-dependent effector function may be suitable for specifically depleting TI-Tregs. Citation Format: Alexey Berezhnoy, Kalpana Shah, Daorong Liu, Rillema Jill, Claudia Fieger, Douglas Smith, Francine Chen, James Tamura, Ezio Bonvini, Gundo Diedrich, Paul A. Moore. Investigational CD25 x CTLA-4 bispecific DART® molecule for depletion of tumor infiltrating Tregs via an enhanced Fc-dependent effector mechanism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2288.

Abstract 6509: Hypofractionated radiotherapy as a vaccine in combination with the TLR7/8 agonist resiquimod leads to significant abscopal tumor response and persistence antitumor immunity

Abstract High doses of ionizing radiation (IR) as delivered with hypofractionated radiotherapy (HFRT) can generate immunogenic cell death and elicit adaptive anti-tumor immunity through cross priming of tumor associated antigens. Resiquimod (R-848), a TLR7/8 agonist, generates a systemic antitumor immunity resulting in tumor regression and enhanced T-cell effector function in cutaneous T-cell lymphomas clinically. We hypothesized that combining HFRT with immunotherapy would boost the antitumorigenic effects of either monotherapies, and if so, could lead to a readily translated clinical modality. B16F10 (murine melanoma) and SCC7 (murine squamous cell carcinoma) cells were used as to grow flank subcutaneous syngeneic tumor models in C57BL/6J and C3H/HeJ mice respectively. Our studies indicate that intratumoral administration of resiquimod combined with HFRT to those tumors leads to a significantly stronger antitumor response than either single treatment in both tumor models. The combination therapy in the primary tumor led to complete tumor regression of the SCC7 tumors in 86% of the treated mice to just 14% rejection in the HFRT group alone. Tumor re-challenge of those mice led to tumor rejection in 83% in the combination group compared to 0% of the HFRT group indicating a potent systemic effect of the combined therapy. Furthermore, the combination therapy led to significant reduction of the index (treated) but moreover in the abscopal (untreated) tumors compared to either monotherapy confirming a systemic antitumor effect in this dual-tumor in vivo model. RT-qPCR analysis revealed an increase expression of Th-1 signature (IFNγ, IL-12, Prf1, GzmB) in both primary and abscopal tumors and flowcytometric analysis of the abscopal B16F10 tumors revealed an increase in CD8+ infiltration. A more thorough flowcytometric analysis following staining with the gp100 tetramer revealed an increased cross-priming against gp100 (PMEL) and enhanced gp100-specific T-cell infiltration within the tumor. Moreover, splenocytes that were re-stimulated in vitro with the gp100 peptide in an IFNγ ELISPOT assay, they increased in mice treated with the dual therapy. Finally, an in vitro study of B16F10 cells, which express TLR7/8 receptors, revealed a substantial increase in IFNβ and CCL5 expression with the dual treatment. Knocking down TLR7 and Tmem173 (STING) in those cells with siRNAs completely reversed the phenotypic IFN type I expression signature. In conclusion, the combination of HFRT with Resiquimod leads to very potent antitumor effects in both primary and abscopal melanoma and squamous cell carcinoma tumors driven by an enhanced systemic immunologic response. Our findings provide the rational for translating therapies combining radiotherapy and intratumoral resiquimod into early-phase clinical trials. Citation Format: Ilias V. Karagounis, Stefano Pierini, Noëlle François, Christoforos Thomas, Xiaowei Xu, Constantinos Koumenis, Andrea Facciabene, Amit Maity. Hypofractionated radiotherapy as a vaccine in combination with the TLR7/8 agonist resiquimod leads to significant abscopal tumor response and persistence antitumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6509.

Cereblon harnesses Myc-dependent bioenergetics and activity of CD8+ T lymphocytes

AbstractImmunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known “neosubstrates,” such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.

Abstract 5570: Identification of novel mechanisms of resistance against the redirection of T-cell effector function for cancer therapy

Abstract In the recent times the way to tackle cancer has been broadly expanded to new opportunities, being the immunotherapy the spearhead of this revolution. Two of the different novel strategies to boost the immune response against tumor cells are the T Cell Bispecific antibodies (TCBs) and the Chimeric Antigen Receptors (CARs), both able to redirect the T cells of the host against the tumor cells. While the TCBs consist of an engineered antibody that binds both to a tumor associated antigen and a T cell receptor, activating it; the CARs consist of a modified T cell that exhibit the binding site to the antigen fused to the intracellular signaling motifs that activate the T cell. Clinical evidence shows that either TCBs or CARs are effective to treat cancer. These can be directed to a vast variety of tumor antigens. Some examples are the HER2-TCB, directed against the HER2 receptor tyrosine kinase overexpressed in 20% of the breast cancers. However, the therapeutic effectivity in tumors is lower than expected and the mechanisms of resistance to these promising immunotherapies have not been explored yet. Therefore, there is a need to anticipate them. In this project, we have developed a immunoresistant model against HER2-TCB, by using the HER2+ breast cancer cell line BT474 as a model. To generate immunoresistant cells, cells were exposed with the TCBs and peripheral blood mononuclear cells (PBMCs) for several months in order to obtain resistance. Indeed, these resistant cells were resistant also in a 3D organoid model and in an in vivo humanized PBMC model. Furthermore, these resistant cells were also resistant to a CAR expressing HER2. Interestingly, a cytokine array that assess more than 80 cytokines did not show a dramatic change in the secretome of resistant cells, in basal conditions and in presence of PBMCs and HER2-TCB, suggesting that the mechanism of resistant is intrinsic on the resistant cells. In addition, these resistant cells did not lose the expression of the antigen (HER2). Therefore, we aimed to identify intrinsic mechanisms of resistance by means of RNA-seq. Although we did not identify the cause of the resistance yet, transcriptomic analysis showed a dramatic change between sensitive and resistant cells, with more than 50 genes acutely up-or downregulated in resistant cells (≥4-fold; p &amp;lt; 10-5). Gene set enrichment analysis identified numerous differences in biological processes between both populations. In parallel, we have also generated the very same model in vivo, with serial passagues in humanized PBMC mice treated with HER2-TCB. The next step will be to identify the mechanism of resistance by gain and loss of function approaches and to validate it in a cohort of human samples and in our collection of Patient Derived Xenografts. In conclusion, we have generated an interesting resistant model to immunotherapies that can be used as a tool to identify unknown mechanisms of resistance and with potential clinical applications that will reinforce the use of immunotherapy in the clinics. Citation Format: Enrique Javier Arenas Lahuerta, Alex Martínez-Sabadell, Irene Rius Ruiz, Beatriz Morancho, Macarena Román, Cristina Bernadó, Joaquín Arribas. Identification of novel mechanisms of resistance against the redirection of T-cell effector function for cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5570.

Clinical Relevance of Polymorphonuclear Myeloid-Derived Suppressor Cells in Autoimmune-Blistering Disorders Pemphigus Vulgaris and Bullous Pemphigoid

Clinical Relevance of Polymorphonuclear Myeloid-Derived Suppressor Cells in Autoimmune-Blistering Disorders Pemphigus Vulgaris and Bullous Pemphigoid

Sirt2 Inhibition Enhances Metabolic Fitness and Effector Functions of Tumor-Reactive T Cells

Dysregulated metabolism is a key driver of maladaptive tumor-reactive T lymphocytes within the tumor microenvironment. Actionable targets that rescue the effector activity of antitumor Tcells remain elusive. Here, we report that the Sirtuin-2 (Sirt2) NAD+-dependent deacetylase inhibits Tcell metabolism and impairs Tcell effector functions. Remarkably, upregulation of Sirt2 in human tumor-infiltrating lymphocytes (TILs) negatively correlates with response to TIL therapy in advanced non-small-cell lung cancer. Mechanistically, Sirt2 suppresses Tcell metabolism by targeting key enzymes involved in glycolysis, tricarboxylic acid-cycle, fatty acid oxidation, and glutaminolysis. Accordingly, Sirt2-deficient murine Tcells exhibit increased glycolysis and oxidative phosphorylation, resulting in enhanced proliferation and effector functions and subsequently exhibiting superior antitumor activity. Importantly, pharmacologic inhibition of Sirt2 endows human TILs with these superior metabolic fitness and effector functions. Our findings unveil Sirt2 as an unexpected actionable target for reprogramming Tcell metabolism to augment a broad spectrum of cancer immunotherapies.

All-trans-retinoic acid shifts Th1 towards Th2 cell differentiation by targeting NFAT1 signalling to ameliorate immune-mediated aplastic anaemia.

Severe acquired aplastic anaemia (AA) is a serious disease characterised by autoreactive T cells attacking haematopoietic stem cells, leading to marrow hypoplasia and pancytopenia. Immunosuppressive therapy combined with antithymocyte globulin and ciclosporin can rescue most patients with AA. However, the relapse after ciclosporin withdrawal and the severe side effects of long-term ciclosporin administration remain unresolved. As such, new strategies should be developed to supplement current therapeutics and treat AA. In this study, the possibility of all-trans-retinoic acid (ATRA) as an alternative AA treatment was tested by using an immune-mediated mouse model of AA. Results revealed that ATRA inhibited T-cell proliferation, activation and effector function. It also restrained the Fas/Fasl pathway, shifted Th1 towards Th2 cell development, rebalanced T-cell subsets at a relatively high level and corrected the Th1/Th2 ratio by targeting NFAT1 signalling. In addition, ATRA inhibited Th17 cell differentiation and promoted regulatory T-cell development. Therefore, ATRA was an effective agent to improve AA treatment outcomes.

Genetically driven CD39 expression shapes human tumor-infiltrating CD8+ T-cell functions.

In our study, we investigated the role of CD39 on tumor-infiltrating CD8+ T lymphocytes (CD8+ TILs) in colorectal, head and neck and pancreatic cancers. Partially confirming recent observations correlating the CD39 expression with T-cell exhaustion, we demonstrated a divergent functional activity in CD39+ CD8+ TILs. On the one hand, CD39+ CD8+ TILs (as compared to their CD39- counterparts) produced significantly lower IFN-γ and IL-2 amounts, expressed higher PD-1, and inversely correlated with perforin and granzyme B expression. On the other, they displayed a significantly higher proliferative capacity ex vivo that was inversely correlated with the PD-1 expression. Therefore, CD39+ CD8+ TILs, including those co-expressing the CD103 (a marker of T resident memory [TRM] cells), were defined as partially dysfunctional T cells that correlate with tumor patients with initial progression stages. Interestingly, our results identified for the first time a single nucleotide polymorphism (SNP rs10748643 A>G), as a genetic factor associated with CD39 expression in CD8+ TILs. Finally, we demonstrated that compounds inhibiting CD39-related ATPases improved CD39+ CD8+ T-cell effector function ex vivo, and that CD39+ CD8+ TILs displayed effective suppression function in vitro. Overall these data suggest that the SNP analysis may represent a suitable predictor of CD39+ CD8+ T-cell expression in cancer patients, and propose the modulation of CD39 as a new strategy to restore partially exhausted CD8+ TILs.

LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells reveals ITSN 2 as a regulator of effector functions

T‐cell receptor (TCR) ligation‐mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time‐resolved high‐resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T‐cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast‐track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T‐cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T‐cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas.