Polyclonal CD4 Research Articles

Abstract P102: NTX-1088, A potent first-in-class, anti-PVR mAb, restores expression and function of DNAM1 for optimal DNAM1-mediated antitumor immunity

Abstract The poliovirus receptor (PVR, CD155) represents a resistance mechanism to approved immune checkpoint inhibitors (ICIs). It is a key regulator of immune activation, that modifies immune function through multiple mechanisms. Increased levels of PVR expression on tumor cells have been associated with resistance to anti-PD-(L)1 therapy in clinical settings, while loss of PVR led to reduced tumor growth in multiple pre-clinical models. Targeting PVR using blocking mAbs offers an attractive therapeutic approach for patients with advanced cancer. NTX-1088 is a first-in-class, potent, anti-PVR mAb being developed for the treatment of solid tumors. The antibody binds to PVR with high affinity, blocks its interactions with TIGIT and CD96, and thus interrupt their immunosuppressive signaling. However, NTX-1088 forte is manifested through its ability to block the critical interaction between PVR and the costimulatory receptor DNAM1 (CD226). This blockade prevents internalization of DNAM1, restores its expression on the surface of immune cells and results in a robust antitumor activation. NTX-1088 was tested using several tumor and immune cell co-culture systems. Various cancer cell lines were co-incubated with relevant immune effector cells from healthy human donors, in the presence of NTX-1088, as a single agent and in combination with anti-PD-1 mAb (pembrolizumab). NTX-1088 significantly increased immune cell activation, as measured by IFNg release from activated polyclonal CD8+ T cells, induction of CD137 and killing of tumor cells. When tested in combination with pembrolizumab, NTX-1088 further increased all measured activation parameters, suggesting a potential synergistic effect. When compared to anti-TIGIT mAb (tiragolumab), NTX-1088 demonstrated clear superiority in its ability to activate T and NK cells. Furthermore, NTX-1088 in combination with pembrolizumab was significantly superior to the combination of pembrolizumab with anti-TIGIT mAb. Interestingly, NTX-1088 as a single agent showed a comparable effect to that of the combined blockade of TIGIT and CD112R, and further synergized with anti-CD112R for maximal activity. NTX-1088 was the only intervention that significantly restored DNAM1 levels, whereas blockade of DNAM1 reduced the activity of NTX-1088 to levels comparable to that of anti-TIGIT mAb. Humanized murine models confirmed the above observations; NTX-1088 exhibited strong efficacy, inducing a robust tumor growth inhibition, accompanied by significantly higher prevalence of CD137+, DNAM1+, CD8+ tumor infiltrating cells, compared to control treated mice. This is the first report of drug-induced DNAM1 restoration and immune activation. NTX-1088 shows, for the first time, exclusive triple mechanism of action, whereby simultaneous and effective blockade of TIGIT and CD96 is complemented by the efficient restoration of DNAM1. This is a step change in antitumor immune activation, which will soon be tested in the clinic. Citation Format: Pini Tsukerman, Anas Atieh, Akram Obeidat, Keren Paz, Guy Cinamon, Tihana Lenac Roviš, Lea Hirsl, Paola Kucan Brilc, Stipan Jonjic, Ofer Mandelboim. NTX-1088, A potent first-in-class, anti-PVR mAb, restores expression and function of DNAM1 for optimal DNAM1-mediated antitumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P102.

Breadth of CD8 T-cell mediated inhibition of replication of diverse HIV-1 transmitted-founder isolates correlates with the breadth of recognition within a comprehensive HIV-1 Gag, Nef, Env and Pol potential T-cell epitope (PTE) peptide set.

Full characterisation of functional HIV-1-specific T-cell responses, including identification of recognised epitopes linked with functional antiviral responses, would aid development of effective vaccines but is hampered by HIV-1 sequence diversity. Typical approaches to identify T-cell epitopes utilising extensive peptide sets require subjects' cell numbers that exceed feasible sample volumes. To address this, CD8 T-cells were polyclonally expanded from PBMC from 13 anti-retroviral naïve subjects living with HIV using CD3/CD4 bi-specific antibody. Assessment of recognition of individual peptides within a set of 1408 HIV-1 Gag, Nef, Pol and Env potential T-cell epitope peptides was achieved by sequential IFNγ ELISpot assays using peptides pooled in 3-D matrices followed by confirmation with single peptides. A Renilla reniformis luciferase viral inhibition assay assessed CD8 T-cell-mediated inhibition of replication of a cross-clade panel of 10 HIV-1 isolates, including 9 transmitted-founder isolates. Polyclonal expansion from one frozen PBMC vial provided sufficient CD8 T-cells for both ELISpot steps in 12 of 13 subjects. A median of 33 peptides in 16 epitope regions were recognised including peptides located in previously characterised HIV-1 epitope-rich regions. There was no significant difference between ELISpot magnitudes for in vitro expanded CD8 T-cells and CD8 T-cells directly isolated from PBMCs. CD8 T-cells from all subjects inhibited a median of 7 HIV-1 isolates (range 4 to 10). The breadth of CD8 T-cell mediated HIV-1 inhibition was significantly positively correlated with CD8 T-cell breadth of peptide recognition. Polyclonal CD8 T-cell expansion allowed identification of HIV-1 isolates inhibited and peptides recognised within a large peptide set spanning the major HIV-1 proteins. This approach overcomes limitations associated with obtaining sufficient cell numbers to fully characterise HIV-1-specific CD8 T-cell responses by different functional readouts within the context of extreme HIV-1 diversity. Such an approach will have useful applications in clinical development for HIV-1 and other diseases.

21-Hydroxylase-Specific CD8+ T Cells in Autoimmune Addison's Disease Are Restricted by HLA-A2 and HLA-C7 Molecules.

ObjectivesCD8+ T cells targeting 21-hydroxylase (21OH) are presumed to play a central role in the destruction of adrenocortical cells in autoimmune Addison’s disease (AAD). Earlier reports have suggested two immunodominant CD8+ T cell epitopes within 21OH: LLNATIAEV (21OH342-350), restricted by HLA-A2, and EPLARLEL (21OH431-438), restricted by HLA-B8. We aimed to characterize polyclonal CD8+ T cell responses to the proposed epitopes in a larger patient cohort with AAD.MethodsRecombinant fluorescent HLA-peptide multimer reagents were used to quantify antigen-specific CD8+ T cells by flow cytometry. Interferon-gamma (IFNγ) Elispot and biochemical assays were used to functionally investigate the 21OH-specific T cells, and to map the exactly defined epitopes of 21OH.ResultsWe found a significantly higher frequency of HLA-A2 restricted LLNATIAEV-specific cells in patients with AAD than in controls. These cells could also be expanded in vitro in an antigen specific manner and displayed a robust antigen-specific IFNγ production. In contrast, only negligible frequencies of EPLARLEL-specific T cells were detected in both patients and controls with limited IFNγ response. However, significant IFNγ production was observed in response to a longer peptide encompassing EPLARLEL, 21OH430-447, suggesting alternative dominant epitopes. Accordingly, we discovered that the slightly offset ARLELFVVL (21OH434-442) peptide is a novel dominant epitope restricted by HLA-C7 and not by HLA-B8 as initially postulated.ConclusionWe have identified two dominant 21OH epitopes targeted by CD8+ T cells in AAD, restricted by HLA-A2 and HLA-C7, respectively. To our knowledge, this is the first HLA-C7 restricted epitope described for an autoimmune disease.

Coevolving JAK2V617F+relapsed AML and donor T cells with PD-1 blockade after stem cell transplantation: an index case

AbstractRelapse of myeloproliferative neoplasms (MPNs) after allogeneic hematopoietic stem cell transplantation (HSCT) is associated with poor outcomes, as therapeutic approaches to reinstate effective graft-versus-leukemia (GVL) responses remain suboptimal. Immune escape through overexpression of PD-L1 in JAK2V617F-mutated MPN provides a rationale for therapeutic PD-1 blockade, and indeed, clinical activity of nivolumab in relapsed MPN post-HSCT has been observed. Elucidation of the features of response following PD-1 blockade in such patients could inform novel therapeutic concepts that enhance GVL. Here, we report an integrated high-dimensional analysis using single-cell RNA sequencing, T-cell receptor sequencing, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), and assay for transposase-accessible chromatin using sequencing (scATAC-seq), together with mass cytometry, in peripheral blood mononuclear cells collected at 6 timepoints before, during, and after transient response to PD-1 blockade from an index case of relapsed MPN following HSCT. Before nivolumab infusion, acute myeloid leukemia (AML) blasts demonstrated high expression of chemokines, and T cells were characterized by expression of interferon-response genes. This baseline inflammatory signature disappeared after nivolumab infusion. Clinical response was characterized by transient expansion of a polyclonal CD4+ T-cell population and contraction of an AML subpopulation that exhibited megakaryocytic features and elevated PD-L1 expression. At relapse, the proportion of the AML subpopulation with progenitor-like features progressively increased, suggesting coevolution of AML blasts and donor-derived T cells. We thus demonstrate how single-cell technologies can provide complementary insight into cellular mechanisms underlying response to PD-1 blockade, motivating future longitudinal high-dimensional single-cell studies of GVL responses in relapsed myeloid disease.

Pre-clinical development and molecular characterization of an engineered type 1 regulatory T-cell product suitable for immunotherapy

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic approach for many hematological disorders. However, allo-HSCT is frequently accompanied by a serious side effect: graft-versus-host disease (GVHD). The clinical use of allo-HSCT is limited by the inability of current immunosuppressive regimens to adequately control GvHD without impairing the graft-versus-leukemia effect (GvL) conferred by transplanted healthy immune cells. To address this, the authors have developed an engineered type 1 regulatory T-cell product called CD4IL-10 cells. CD4IL-10 cells are obtained through lentiviral transduction, which delivers the human IL10 gene into purified polyclonal CD4+ T cells. CD4IL-10 cells may provide an advantage over standard-of-care immunosuppressants because of the ability to suppress GvHD through continuous secretion of IL-10 and enhance the GvL effect in myeloid malignancies through targeted killing of malignant myeloid cells. MethodsHere the authors established a production process aimed at current Good Manufacturing Practice (cGMP) production for CD4IL-10 cells. ResultsThe authors demonstrated that the CD4IL-10 cell product maintains the suppressive and cytotoxic functions of previously described CD4IL-10 cells. In addition, RNA sequencing analysis of CD4IL-10 identified novel transcriptome changes, indicating that CD4IL-10 cells primarily upregulate cytotoxicity-related genes. These include four molecules with described roles in CD8+ T and natural killer cell-mediated cytotoxicity: CD244, KLRD1, KLRC1 and FASLG. Finally, it was shown that CD4IL-10 cells upregulate IL-22, which mediates wound healing and tissue repair, particularly in the gut. ConclusionsCollectively, these results pave the way toward clinical translation of the cGMP-optimized CD4IL-10 cell product and uncover new molecules that have a role in the clinical application of CD4IL-10 cells.

Bim Regulates the Survival and Suppressive Capability of CD8+ Foxp3+ Regulatory T Cells during Graft Versus Host Disease

Graft versus host disease (GVHD) is a proinflammatory syndrome that is the major complication of allogeneic hematopoietic stem cell transplantation and can lead to significant pathological damage. The failure to reconstitute an effective regulatory T cell compartment is a defining characteristic of this disease and has been the genesis for strategies designed to augment reconstitution of regulatory T cells (Tregs) by either in vivo expansion or adoptive transfer of this population. While CD4+ Foxp3+ T cells have been the most carefully examined Treg population, CD8+ Foxp3+ Tregs also emerge early during GVHD, have suppressive function, and can attenuate disease severity. The precise role of these cells and the extent to which CD8+ Tregs possess functional and ontological similarities to CD4+ Tregs, however, have not been well delineated. Using established murine models of GVHD, we observed that adoptively transferred alloantigen-specific CD8+ Tregs had reduced in vivo survival in all target organs and were significantly less potent than CD4+ Tregs for the prevention of lethal GVHD. To define the mechanistic basis for their reduced efficacy, we first examined the T cell receptor (TCR) repertoire of in vivo-derived CD4+ and CD8+ Tregs to determine whether a restricted repertoire was a potential explanation for the diminished ability of CD8+ Tregs to prevent GVHD. We employed GVHD models in which both polyclonal CD8+ [B6→Balb/c] and alloantigen-specific [B6→Balb.B] CD8+ H60+ Treg populations could be assessed and used the Shannon entropy index to define TCR repertoire diversity which is critical for the maintenance of self-tolerance by Tregs. Amplicon sequencing of TCR-β rearrangements to analyze the TCR repertoires of in vivo-induced CD8+ and CD4+ Tregs, along with their conventional CD8+ and CD4+ T cell counterparts, revealed that polyclonal CD8+ and alloantigen-specific CD8+ H60+ Tregs had repertoire diversity that was similar to that of conventional CD8+ T cells, indicating that a more restricted repertoire was not the proximate cause of decreased suppressive capability. Subsequent transcriptional profiling and principal component analysis revealed that CD8+ Tregs arising during GVHD possessed a canonical Treg transcriptional signature that was similar to that observed in CD4+ Tregs, yet distinct from conventional CD8+ T cells. Gene set enrichment and Ingenuity pathway analyses, however, both demonstrated that CD8+ Tregs had differential gene expression in pathways involved in cell death and survival when compared to CD4+ Tregs. This was further confirmed by gene expression analysis and protein expression studies which revealed that CD8+ Tregs had increased expression of the pro-apoptotic proteins Bim, cleaved caspase 3, and cleaved caspase 7, as well as a reduction in the anti-apoptotic protein, Mcl-1. To determine whether Bim was functionally important for CD8+ Treg survival, we comparatively examined in vivo-derived CD4+ and CD8+ Treg induction using wild type Foxp3EGFP and Bim-/-Foxp3EGFP animals as donors in transplantation studies. The absence of Bim in the CD8+ Treg compartment resulted in a significant increase in absolute CD8+ Treg numbers in the liver, lung, and colon when examined on days 7, 14, and 21 post transplantation. Given that the absence of Bim augmented in vivo-generated CD8+ Treg survival, we then examined whether Bim also regulated the survival of adoptively transferred, alloantigen-specific CD8+ Tregs which we had observed were inferior to CD4+ Tregs for the suppression of GVHD. We noted an increased percentage and absolute number of adoptively transferred CD8+ Foxp3+ Bim-/- Tregs relative to wild type CD8+ Tregs in all tissue sites, indicating that the absence of Bim resulted in enhanced stabilization of Foxp3 expression and augmented in vivo survival. Finally, animals that were transplanted with in vitro-derived, alloantigen-specific CD8+ Tregs from Bim-/- Foxp3EGFP mice had significantly prolonged survival relative to mice reconstituted with wild type CD8+ Foxp3EGFP Tregs, demonstrating that the prolonged in vivo survival conferred by the absence of Bim resulted in an increased ability to prevent GVHD. Collectively, these studies demonstrate that Bim plays a critical role in the regulation of CD8+ Treg survival which may have clinical implications for the use of regulatory T cell therapy in the treatment and prevention of GVHD. DisclosuresVincent:Merck: Research Funding; Pharmacyclics: Research Funding; Nanostring: Membership on an entity's Board of Directors or advisory committees. Serody:Merck: Research Funding.

A subset of cytotoxic effector memory T cells enhances CAR T cell efficacy in a model of pancreatic ductal adenocarcinoma.

In humans, the natural killer (NK) cell marker CD161 identifies several subsets of T cells, including a polyclonal CD8 αβ T cell receptor-expressing subset with characteristic specificity for tissue-localized viruses. This subset also displays enhanced cytotoxic and memory phenotypes. Here, we characterized this unique T cell subset and determined its potential suitability for use in chimeric antigen receptor (CAR) T cell therapy. In mice, gene expression profiling among the CD161-equivalent CD8+ T cell populations (CD8+NK1.1+) revealed substantial up-regulation of granzymes, perforin, killer lectin-like receptors, and innate signaling molecules in comparison to CD8+NK1.1- T cells. Adoptive transfer of CD8+NK1.1+ cells from previously exposed animals offered substantially enhanced protection and improved survival against melanoma tumors and influenza infection compared to CD8+NK1.1- cells. Freshly isolated human CD8+CD61+ T cells exhibited heightened allogeneic killing activity in comparison to CD8+CD61- T cells or total peripheral blood mononuclear cells (PBMCs). To determine whether this subset might improve the antitumor efficacy of CAR T cell therapy against solid tumors, we compared bulk PBMCs, CD8+CD161-, and CD8+CD161+ T cells transduced with a human epidermal growth factor receptor-2 (HER2)-specific CAR construct. In vitro, CD8+CD161+ CAR-transduced T cells killed HER2+ targets faster and with greater efficiency. Similarly, these cells mediated enhanced in vivo antitumor efficacy in xenograft models of HER2+ pancreatic ductal adenocarcinoma, exhibiting elevated expression of granzymes and reduced expression of exhaustion markers. These data suggest that this T cell subset presents an opportunity to improve CAR T cell therapy for the treatment of solid tumors.

High-Throughput and High-Dimensional Single Cell Analysis of Antigen-Specific CD8+ T cells

Abstract Although critical to T cell function, antigen specificity is often omitted in high-throughput multi-omics based T cell profiling due to technical challenges. We describe a high-dimensional, tetramer-associated T cell receptor sequencing (TetTCR-SeqHD) method to simultaneously profile TCR sequences, cognate antigen specificities, targeted gene-expression, and surface-protein expression from tens of thousands of single cells. Using polyclonal CD8+ T cells with known antigen specificity and TCR sequences, we demonstrated over 98% precision for detecting the correct antigen specificity. We also evaluated gene-expression and phenotypic differences among antigen-specific CD8+ T cells and characterized phenotype signatures of influenza- and EBV-specific CD8+ T cells that are unique to their pathogen targets. Moreover, with the high-throughput capacity of profiling hundreds of antigens simultaneously, we applied TetTCR-SeqHD to identify antigens that preferentially enrich cognate CD8+ T cells in type 1 diabetes patients compared to healthy controls, and discovered a TCR that cross reacts between diabetic and microbiome antigens. TetTCR-SeqHD is a powerful approach for profiling T cell responses.

Epigenetic and transcriptional programs of CD4+ T cell anergy

Abstract T cell tolerance is critical to avoid autoimmunity and resolve inflammation. CD4+ T cells that recognize self-antigens in the periphery can exert active suppression as regulatory T cells (Treg) or enter an inactive state known as anergy. Recent evidence suggests that anergy occurs naturally within a subpopulation of polyclonal CD4+ T cells and can generate precursors to Foxp3-expressing Treg cells. We aimed to identify transcription factors underlying T cell anergy induction and its potential plasticity. Polyclonal anergic CD4+ T cells from untreated wildtype mice were sorted based on previously reported markers and compared to naïve, effector-memory, and Treg cell populations identified within the same samples. Paired epigenome and transcriptome analysis revealed a unique anergy signature driven by bZIP family (Batf, Nfil3), NFAT family (Nfatc1), and EGR family (Egr3, Egr4) transcription factors. Following tolerization by repeated peptide infusions, antigen-specific CD4+ T cells from Batf-deficient mice demonstrated exaggerated expansion and minimal Foxp3+ Treg cell differentiation. Our data suggest that a unique transcription factor network, including BATF, generates and maintains peripheral CD4+ T cell anergy.

Analysis of the structure and dynamics of synaptic interface revealed functional anomaly of naive CD8+ T cells during HIV infection

Abstract While it is thought that HIV infection leads to a global defect in T cells, effect of the infection on the T cells at various differentiation stages has not been studied. We have exposed freshly isolated polyclonal CD8 T cells at various differentiation stages to planar lipid bilayers that display anti-CD3 antibody and soluble ICAM-1 and analyzed morphology and dynamics of the synaptic interface formation by the T cells, parameters that are linked to their functional activities. We have found that a large fraction of purified naïve CD8 T cells from HIV-infected people developed mature synapses exhibiting atypical degranulation capacity. These data provide evidence that even at the earlier stages of differentiation CD8 T cells derived from HIV-infected patients reveal functional anomaly causing changes in the ability to degranulate. The latter mediated by aberrant functioning of integrins and actin cytoskeleton resulting from continuous inflammation during chronic HIV infection. Functional anomaly of naïve T cells further impairs functions of CD8 T cells at later differentiation stages contributing to a global defect in T cells and failure to control the infection.

Oral administration of a peptide antigen with the cholera toxin adjuvant induces Th17 rather than Th2 cells

Abstract Cholera toxin (CT) is widely used an as adjuvant to elicit mucosal immune responses to co-administered antigens. The observation that antigens given orally with CT induce IL-4-producing T cells and antigen-specific IgE has led to the concept that CT is a Th2 adjuvant. We revisited this issue by using a peptide:MHCII tetramer-based approach to follow the fate of polyclonal CD4+ T cells specific for an MHCII-bound peptide from Toxoplasma gondii CD4Ag28m in mice fed with this peptide and CT. We found that this regimen induced the clonal expansion of CD4Ag28m peptide-specific CD4+ T cells in the secondary lymphoid organs. Surprisingly, the expanded tetramer-binding population consisted of 20 percent RORgt+ Th17 cells but only 3 percent Th2 cells. Th17 cell generation induced by oral peptide plus CT immunization was lost in mice lacking IL-6 or the microbe sensor MyD88. The latter result raised the possibility that the adjuvanticity of CT depended on gut microbes. Treatment of B6 mice with antibiotics to eradicate commensal bacteria, however, only slightly reduced Th17 cell formation in response to oral administration of the peptide plus CT, raising the possibility that viral or fungal microbes are required. Collectively, our data demonstrate that CT induces a Th17 immune response via a pathway that depends on MyD88.

CD8+ T cell self-tolerance permits responsiveness but limits tissue damage.

Self-specific CD8+T cells can escape clonal deletion, but the properties and capabilities of such cells in a physiological setting are unclear. We characterized polyclonal CD8+ T cells specific for the melanocyte antigen tyrosinase-related protein 2 (Trp2) in mice expressing or lacking this enzyme (due to deficiency in Dct, which encodes Trp2). Phenotypic and gene expression profiles of pre-immune Trp2/Kb-specific cells were similar; the size of this population was only slightly reduced in wild-type (WT) compared to Dct-deficient (Dct-/-) mice. Despite comparable initial responses to Trp2 immunization, WT Trp2/Kb-specific cells showed blunted expansion and less readily differentiated into a CD25+proliferative population. Functional self-tolerance clearly emerged when assessing immunopathology: adoptively transferred WT Trp2/Kb-specific cells mediated vitiligo much less efficiently. Hence, CD8+ T cell self-specificity is poorly predicted by precursor frequency, phenotype, or even initial responsiveness, while deficient activation-induced CD25 expression and other gene expression characteristics may help to identify functionally tolerant cells.

The Immune Synapses Reveal Aberrant Functions of Cd8 T Cells During Chronic HIV Infection

It is well-established that chronic HIV infection causes persistent low-grade inflammation that induces premature aging of the immune system in HIV patient including senescence of memory and effector CD8 T cells. To uncover the reasons of gradually diminished potency of CD8 T cells from chronically HIV infected people, we have analyzed cellular morphology and dynamics of the synaptic interface established followed exposure of peripheral polyclonal CD8 T cells at various differentiation stages to planar lipid bilayers. The above parameters were linked to pattern of degranulation that determines efficiency of CD8 T cells cytolytic response. We found a large fraction of naive T cells from HIV infected people developing mature synapses and demonstrating focused degranulation, a signature of a differentiated T cells. Further differentiation of aberrant naive T cells leads to development of anomalous effector T cells undermining their capacity to control of many different viruses that otherwise could coexist in humans for a long time.

Protective efficacy of an attenuated Mtb ΔLprG vaccine in mice.

Bacille Calmette-Guerin (BCG), an attenuated whole cell vaccine based on Mycobacterium bovis, is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), but its efficacy is suboptimal and it fails to protect against pulmonary tuberculosis. We previously reported that Mtb lacking the virulence genes lprG and rv1410c (ΔLprG) was highly attenuated in immune deficient mice. In this study, we show that attenuated ΔLprG Mtb protects C57BL/6J, Balb/cJ, and C3HeB/FeJ mice against Mtb challenge and is as attenuated as BCG in SCID mice. In C3HeB/FeJ mice, ΔLprG vaccination resulted in innate peripheral cytokine production and induced high polyclonal PPD-specific cytokine-secreting CD4+ T lymphocytes in peripheral blood. The ΔLprG vaccine afforded protective efficacy in the lungs of C3H/FeJ mice following both H37Rv and Erdman aerosolized Mtb challenges. Vaccine efficacy correlated with antigen-specific PD-1-negative CD4+ T lymphocytes as well as with serum IL-17 levels after vaccination. We hypothesize that induction of Th17 cells in lung is critical for vaccine protection, and we show a serum cytokine biomarker for IL-17 shortly after vaccination may predict protective efficacy.

Preclinical Development of AT1412, a Patient Derived CD9 Antibody That Does Not Induce Thrombosis for Treatment of B ALL

Despite recent advances in treatment of B-acute lymphoblastic leukemia (B-ALL) there is still a need for novel targeted therapies. The tetraspanin CD9 is expressed in 60-80% of B-ALL and correlates with adverse prognosis. Recently, the mouse CD9 antibody ALB6 was shown to induce leukemia rejection in NOD/SCID mice. However, clinical development of ALB6 and other CD9-targeting antibodies was hampered by their CD9 mediated induction of platelet aggregation. It is known that CD9 is still expressed on tumor cells after treatment with chemotherapy or blinatumomab (Leung, 2019; Linder, 2016). AT1412 is a fully human antibody isolated from B cells of a patient that was cured from stage IV metastatic melanoma (Verdegaal, 2011). AT1412 targets CD9, without inducing platelet aggregation in vitro or thrombosis in cynomolgus monkeys after intravenous administration at therapeutic dose levels. By crystallography AT1412 was shown to bind a unique epitope preventing homodimerization of CD9, distinctly different from other CD9 antibodies. AT1412 binds a majority of patient B-ALL samples, but not T-ALL and induces ADCC and ADCP of CD9 positive B-ALL primary cells and the level of cytotoxicity significantly correlated with that of AT1412 binding. In both NSG and immunodeficient mice harboring a human immune system (HIS mice) AT1412 demonstrated a strong, dose-dependent tumor rejection of B-ALL, most pronounced in the extramedullary sites. In HIS mice AT1412 treatment led to an accumulation of T cells and CD14+ myeloid cells at the tumor sites. To support clinical development, pre-clinical safety of AT1412 was evaluated in cynomolgus monkeys. AT1412 demonstrated a half-life of 8.5 days, supporting 2-3 weekly administration in humans. Besides transient thrombocytopenia no other pathological deviations were observed. No effect on coagulation parameters, bruising or bleeding were observed macro- or microscopically. The thrombocytopenia is reversible, and its recovery accelerated in those animals developing anti-drug antibodies. Taken together, we demonstrate that CD9 on B-ALL cells can be successfully targeted by AT1412. AT1412 targets a unique epitope and does not induce thrombosis. Pre-clinical safety assessment is supporting that AT1412 can be safely administered. A First in Human clinical study is scheduled to start early 2021 in human solid tumors to determine safety and efficacy. AT1412 efficacy will be evaluated in B-ALL in expansion cohorts. Leung, K.T., Zhang, C., Chan, K.Y.Y., Li, K., Cheung, J.T.K., Ng, M.H.L., Zhang, X.-B., Sit, T., Lee, W.Y.W., Kang, W., et al. (2019). CD9 blockade suppresses disease progression of high-risk pediatric B-cell precursor acute lymphoblastic leukemia and enhances chemosensitivity. Leukemia. Linder, K., Gandhiraj, D., Hanmantgad, M., Seiter, K., and Liu, D. (2016). Complete remission after single agent blinatumomab in a patient with pre-B acute lymphoid leukemia relapsed and refractory to three prior regimens: HyperCVAD, high dose cytarabine mitoxantrone and CLAG. Exp. Hematol. Oncol. 5, 5-8. Verdegaal, E.M.E., Visser, M., Ramwadhdoebé, T.H., van der Minne, C.E., van Steijn, J. a Q.M.J., Kapiteijn, E., Haanen, J.B. a G., van der Burg, S.H., Nortier, J.W.R., and Osanto, S. (2011). Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha. Cancer Immunol. Immunother. 60, 953-963. Disclosures Schotte: AIMM Therapeutics: Current Employment, Current equity holder in private company. Villaudy:AIMM Therapeutics: Current Employment, Current equity holder in private company. Levie:AIMM Therapeutics: Current Employment, Current equity holder in private company. Go:AIMM Therapeutics: Current Employment, Current equity holder in private company. Yasuda:AIMM Therapeutics: Current Employment, Current equity holder in private company. Frankin:AIMM Therapeutics: Current Employment, Current equity holder in private company. Cercel:AIMM Therapeutics: Current Employment, Current equity holder in private company. van Hal-van Veen:AIMM Therapeutics: Current Employment, Current equity holder in private company. van de Berg:AIMM Therapeutics: Current Employment, Current equity holder in private company. Fatmawati:AIMM Therapeutics: Current Employment, Current equity holder in private company. Kedde:AIMM Therapeutics: Current Employment, Current equity holder in private company. Claassen:AIMM Therapeutics: Current Employment, Current equity holder in private company. Rijneveld:Amgen: Research Funding; Servier: Research Funding. Spits:AIMM Therapeutics: Current equity holder in private company, Ended employment in the past 24 months, Membership on an entity's Board of Directors or advisory committees. van Eenennaam:AIMM Therapeutics: Current Employment, Current equity holder in private company.

Type I interferons induce peripheral T regulatory cell differentiation under tolerogenic conditions.

The type I interferons are central to a vast array of immunological functions. The production of these immune-modulatory molecules is initiated at the early stages of the innate immune responses and, therefore, plays a dominant role in shaping downstream events in both innate and adaptive immunity. Indeed, the major role of IFN-α/β is the induction of priming states, relevant for the functional differentiation of T lymphocyte subsets. Among T-cell subtypes, the CD4+CD25+Foxp3+ T regulatory cells (Tregs) represent a specialized subset of CD4+ T cells with a critical role in maintaining peripheral tolerance and immune homeostasis. Although the role of type I interferons in maintaining the function of thymus-derived Tregs has been previously described, the direct contribution of these innate factors to peripheral Treg (pTreg) and induced Treg (iTreg) differentiation and suppressive function is still unclear. We now show that, under tolerogenic conditions, IFN-α/β play a critical role in antigen-specific and also polyclonal naive CD4+ T-cell conversion into peripheral antigen-specific CD4+CD25+Foxp3+ Tregs and inhibit CD4+ T helper (Th) cell expansion in mice. While type I interferons sustain the expression and the activation of the transcription master regulators Foxp3, Stat3 and Stat5, these innate molecules reciprocally inhibit Th17 cell differentiation. Altogether, these results indicate a new pivotal role of IFN-α/β on pTreg differentiation and induction of peripheral tolerance, which may have important implications in the therapeutic control of inflammatory disorders, such as of autoimmune diseases.

Abstract 4554: Functionally distinct T cell subsets contribute to ImmTAC-mediated anti-tumor response

Abstract Introduction: Bispecific T cell engagers hold promise for effective cancer treatment. The anti-tumor activity of these therapeutics molecules predominantly relies on their capacity to induce direct killing of tumor cells by engaging the cytotoxic machinery of polyclonal CD8+ T cells. However, the impact of additional functionally different T cell populations that can be engaged alongside CD8+ T cells remains unclear. ImmTAC molecules are a novel class of bispecific fusion proteins that use an affinity-enhanced monoclonal TCR to target tumor antigen-derived peptides presented by HLA, and an anti-CD3 monomeric antibody to engage T cells. Tebentafusp, our lead clinical candidate, targets a gp100-derived peptide and is under clinical investigation in metastatic melanoma. Preliminary data revealed T cell activation, trafficking and infiltration into the tumor bed in patients responding to treatment. Importantly not only CD8+ T cells, but also different subsets of CD4+ T cells appeared to be activated, mobilized and redirected to the tumor site following tebentafusp treatment, suggesting a potential contribution of these T cell subsets to the anti-tumor response. The aim of this study was to investigate in vitro the functional capacity of different T cell populations, besides CD8+ T cells, following ImmTAC-mediated redirection toward cancer cells. Methods: Th1, Th2, Th17 CD4+ T cells, γδ T cells and Mucosal-Associated Invariant T cells (MAIT) were isolated from healthy volunteers and their functional response following exposure to ImmTAC in the presence of target cells was analysed. Results: In addition to CD8+ T cells, other T cell populations, were activated in vitro by ImmTAC and elicited a range of effector functions that may differentially contribute to anti-tumor responses. For example, we found that γδ T cells were able to mount rapid and potent cytokine and cytotoxic responses against cancer cells upon redirection with ImmTAC. The mechanisms that underpin our in vitro observations and the potential correlations in patients treated with tebentafusp are under investigation. Conclusions: Multiple T cells populations display functionally distinct responses following ImmTAC-mediated redirection. These results will add valuable insight to our understanding of bispecific T cell engager mechanism of action. In addition, they will help guide rationale for improving the design of these therapeutic molecules and select appropriate combination strategies. Citation Format: Adel Benlahrech, David K. Cole, Christopher J. Holland, Rupert Kenefeck, Rahul C. Khanolkar, Cheryl McAlpine, Sion Lewis, Angela Valerio-Fernandes, Mariantonella Vardeu, David Berman, Marco Lepore. Functionally distinct T cell subsets contribute to ImmTAC-mediated anti-tumor response [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 4554.

The Impact of MHC Class I Dose on Development and Maintenance of the Polyclonal Naive CD8+ T Cell Repertoire.

Naive CD8+ T cell survival in the periphery is critically dependent on tonic TCR signaling through peptide + MHC class I (MHCI) recognition; however, little is known about how natural variation in MHCI levels impacts the naive CD8+ T cell repertoire. Using mice that are hemizygous or homozygous for a single MHCI allele, we showed that despite a reduction in peripheral CD8+ T cell numbers of ∼50% in MHCI hemizygous mice, MHCI levels had no notable impact on the rate of thymic generation or emigration of CD8 single-positive T cells. Moreover, the peripheral T cell repertoire in hemizygous mice showed selective retention of T cell clonotypes with a greater competitive advantage as evidenced by increased expression of CD5 and IL-7Rα. The qualitative superiority of CD8+ T cells retained in hemizygous mice was also seen during influenza A virus infection, in which epitope-specific CD8+ T cells from hemizygous mice had a higher avidity for pMHCI and increased cytokine polyfunctionality, despite a reduced response magnitude. Collectively, this study suggests that natural variation in MHCI expression levels has a notable and biologically relevant impact on the maintenance, but not generation, of the naive CD8+ T cell repertoire.

Oral administration of antigen mainly induces T helper lineage-negative CD200+ CTLA4+ anergic CD4+ T cells which tend to become regulatory T cells

Abstract CD4+ T cells do not normally undergo productive immune responses to orally administered foreign antigens. It has been proposed that this oral tolerance phenomenon is explained by CD4+ T cells with T cell receptors (TCRs) specific for MHCII-bound peptides from ingested antigens becoming unresponsive (anergic) or differentiating into T regulatory (Treg) cells. Not much is known, however, about the phenotype and function of cells undergoing this process within the polyclonal CD4+ T cell repertoire. We addressed this issue by using I-Ab tetramers containing a foreign peptide called 2W to track 2W:I-Ab-specific CD4+ T cells after oral gavage of C57BL/6 mice with 2W peptide. These CD4+ T cells underwent weak clonal expansion when exposed orally to 2W peptide and generated a population consisting of 10% Foxp3+ Treg cells, 10% CXCR5+ follicular helper cells (Tfh), and ~3 % TBET+ Th1 or RORgt+ Th17 cells. The dominant subpopulation, however, did not express any of the known T helper cell lineage markers. Analysis of 80 surface proteins including CD73, FR4, CD200, PD-1, P2X7, and CTLA-4 revealed that these T helper lineage-negative (Thlin−) cells were more similar to anergic cells than to effector or Treg cells. In addition, adoptive transfer of sorted 2W:I-Ab-specific Thlin− cells followed by oral gavage of the peptide showed that these Foxp3− cells can become Foxp3+ Treg cells much more efficiently than naïve T cells. Collectively, our data demonstrate that the majority of CD4+ T cells exposed to oral antigen become Foxp3− anergic cells with a tendency to become Treg cells.

Abstract A36: Single-cell characterization of tumor-infiltrating T cells from renal cell carcinoma

Abstract Purpose: T-cell infiltration of renal cell carcinoma (RCC) is commonly viewed as evidence for antitumor immunity that may be harnessed by T cell-directed immunotherapies. We hypothesize that bona fide tumor-reactive T cells constitute only a fraction of tumor-infiltrating lymphocytes (TIL) that may be composed largely of nonspecific passenger cells. This study deployed TRB-CDR3 repertoire characterization and targeted single-cell RNAseq of RCC versus normal tissue and peripheral blood mononuclear cells (PBMC)-derived T cells to analyze phenotypes that may associate with tumor-antigen specificity. Experimental Procedures: RCC tumor, normal adjacent kidney tissue (NAT), skin, and PBMC were collected from 9 RCC patients at the time of nephrectomy surgery. Tissue samples were digested to single-cell suspensions and CD45+CD8+CD3+ T cells were sorted. Sequencing of TCR-β CDR3 regions (TRB-CDR3) was carried out on polyclonal CD8+ T-cell populations. Targeted single-cell RNAseq was then applied to sorted CD8+ T cells from the same samples focusing on TRB-CDR3 and TRA-CDR3 regions and 25 T-cell phenotype genes. Full-length T-cell receptor (TCR) genes from selected, clonally expanded, tumor-associated CD8+ T cells were cloned into lentiviral expression vectors for re-expression in CD8+ T cells from healthy donors to facilitate analysis of TCR specificity. Results: TCR-β CDR3 sequencing demonstrated the frequency of clonally expanded TRB was higher in tumors (mean 45%; range 27%-65%) compared with PBMCs (mean 14%; range 4%-22%) or NATs (mean 15%; range 2%-26%) for all 9 cases. Expanded T-cell clonotypes present in the tumor and not detected in NAT or PBMC samples were present in every case. Dimensionality reduction of targeted single-cell RNAseq analysis identified phenotypic differences between clonally expanded and nonexpanded T cells in 3 of 9 tumors. Overall, IFN-γ, perforin, and granzymeB were upregulated in clonally expanded T cells, while IL-2, IL-13, and CTLA-4 were upregulated in nonexpanded cells. A total of 116 TRB-CDR3 and TRA-CDR3 sequence pairs were identified from clonally expanded RCC TIL, among which 49 were only found in tumor tissue. Based on effector gene expression, as well as the availability of an autologous RCC tumor line, a total of 12 expanded TRAB pairs were cloned into lentiviral expression vectors. Analysis of TCR specificity for autologous tumor as well as HLA-matched RCC cell lines will be updated. Conclusions: T-cell clonal expansions associated with the microenvironment of RCC tumors and may represent antigen engagement. Phenotypic differences in effector gene expression between clonally expanded and nonexpanded CD8+ T cells from RCC TIL are heterogeneous between tumors. The association of such phenotypic differences with clinical outcomes is a priority for further investigation. Evaluation of alternate 10x platform and analysis of tumor antigen recognition by TCRs from clonally expanded T cells with cytotoxic phenotype unique to the RCC microenvironment is ongoing. Citation Format: Yuexin Xu, Alicia J. Morales, Andrea M.H. Towlerton, Edus H. Warren, Scott S. Tykodi. Single-cell characterization of tumor-infiltrating T cells from renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A36.