Abstract Background: Death receptor 5 (DR5), a member of the tumor necrosis factor (TNF) receptor superfamily that binds to TNF-related apoptosis-inducing ligand and activates the extrinsic apoptotic pathway, is an attractive target for cancer therapies given its upregulated expression in multiple cancers and ability to induce apoptosis. However, key limitations of therapies targeting DR5 are a lack of potency due to an inability to multimerize DR5, and/or clinically significant hepatoxicity. IGM-8444 is an IgM-based DR5 agonist with 10 binding sites that allow multimerization and subsequent activation of DR5. Preclinical studies demonstrate IGM-8444 cytotoxicity as a single agent and in combination in a range of tumors. We discuss our ongoing randomized study of IGM-8444 in combination with FOLFIRI + bevacizumab compared to FOLFIRI + bevacizumab alone in second line metastatic colorectal cancer (mCRC) (NCT04553692). Methods: This study is a Phase 1b, multicenter, randomized, open-label clinical trial of IGM-8444 in combination with FOLFIRI + bevacizumab compared to FOLFIRI + bevacizumab alone in patients with second line mCRC who have not received prior irinotecan in either the adjuvant, locally advanced, or metastatic setting. Patients will be randomized 1:1 either to Treatment Arm 1: IGM-8444 in combination with FOLFIRI + bevacizumab or Treatment Arm 2: FOLFIRI + bevacizumab. Patients will be stratified by the presence of liver metastases and KRAS mutational status. Patients randomized in Treatment Arm 2 will have the opportunity to crossover to Treatment Arm 1 after confirmed radiographic progression. Patients will receive IGM-8444 on Days 1 and 15 of each 28-day cycle at a dose level of 3 mg/kg. IGM-8444 will be dosed prior to administering FOLFIRI + bevacizumab in Treatment Arm 1. FOLFIRI + bevacizumab will be administered in accordance with prescribing guidelines and institutional standards. Patients will continue this regimen until disease progression or unacceptable toxicity. The primary endpoint will be progression-free survival as determined by study investigators. Secondary endpoints include frequency and severity of adverse events, overall response rate, duration of response, and overall survival as determined by study investigators. The randomized study is currently open with patients actively enrolling. The planned study size is 110 patients. Clinical Trial Registry Number NCT04553692 Citation Format: Susanna Ulahannan, Meredith Pelster, Patricia LoRusso, Gerald Falchook, Judy Wang, Minal Barve, Jaspreet Grewal, Warren Chow, Jason Henry, Michael Cecchini, Vivek Subbiah, Maya Leabman, Genevive Hernandez, Yao Li, Angus Sinclair, Beatrice Wang, Maya Kotturi, Eric Humke, Sean Ahern, Leslie Ennis, Melaine Caruano, Sapeck Agrawal. A randomized Phase 1b study of IGM-8444 in combination with FOLFIRI + bevacizumab compared to FOLFIRI + bevacizumab alone in second line metastatic colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C031.

Abstract Introduction: IGM-8444 is an engineered DR5 agonist IgM antibody with 10 binding sites that efficiently multimerizes DR5 to induce tumor cell apoptosis. The first-in-human Phase 1a/1b study of IGM-8444 (NCT04553692) is ongoing, and preliminary results show that IGM-8444 is well-tolerated as a single agent (SA) and in combination with chemotherapeutic and targeted agents. Encouraging efficacy is also observed with IGM-8444 combined with FOLFIRI +/- bevacizumab (bev) in patients with relapsed/refractory mCRC. Here we present pharmacokinetic (PK) and pharmacodynamic biomarker data from Phase 1a participants treated with IGM-8444 as SA or in combination at doses of 0.15 to 5 mg/kg given weekly (QW) or 0.3 to 10 mg/kg given every 2 weeks (Q2W). Methods: Blood samples for PK and biomarker analysis were collected at multiple timepoints during cycles 1 and 2. Sparse sampling was utilized at later cycles. Plasma biomarker evaluation included measurement of M30 by ELISA and total caspase-3 (casp-3) by MSD. Tumor biomarkers were assessed in baseline and on-treatment tissue through immunohistochemistry for DR5 and cleaved caspase-3 (CC3). Initial population PK modeling was conducted to assess the PK characteristics of IGM-8444 as SA and in combination, and to enable simulations of different doses and dosing regimen scenarios. Results: Treatment-related increases in plasma levels of apoptotic markers casp-3 (up to 25-fold) and M30 (up to 6-fold) were observed across dose levels. These changes occurred independently of any ALT or AST fluctuations, suggesting tumor-specific apoptosis induction. Dose-dependent increases in casp-3 and CC3 were observed in blood and tumor, respectively, indicating activation of DR5 pathway. At 3 mg/kg, on-treatment tumor CC3 levels were up to 10-fold higher in FOLFIRI combo, signifying enhanced apoptosis when IGM-8444 is combined with chemotherapy. Population PK analysis indicates that IGM-8444 PK is best described by a two-compartment model with parallel linear and nonlinear clearance. Terminal half-life at 10 mg/kg Q2W was >2 days. PK simulations suggest that IGM-8444 administered at 3 or 10 mg/kg Q2W sustains average concentrations within the range as those observed at efficacious doses of IGM-8444 in preclinical mouse tumor models, where SA IGM-8444 exhibits dose-dependent efficacy with no evidence of a bell-shaped response. To date, ADAs have been detected in ~25% of participants. ADA titers in confirmed ADA-positive participants remain relatively low and the available data do not indicate a significant effect of ADAs on the PK of IGM-8444. Conclusions: Pharmacodynamic changes in blood and tissue confirm the biologic activity of IGM-8444 and demonstrates that targeted therapy with an engineered IgM antibody can result in tumor penetration and target engagement within tumor tissue. Taken together, the biomarker and PK data support the use of IGM-8444 at a dose of 3 mg/kg or above for combination studies. The randomized Phase 1b is underway and evaluates the activity of FOLFIRI+bev +/- IGM-8444 in 2L mCRC. Citation Format: Genevive Hernandez, Sanela Bilic, Yinghui Guan, John So, Sabya Bhattacharya, Beatrice Wang, Maya Kotturi, Miho Oyasu, Melanie Desbois, Yuan Cao, Eric Humke, Sean Ahern, Angus Sinclair, Susanna Ulahannan, Vivek Subbiah, Merideth Pelster, Patricia Lorusso, Gerald Falchook, Judy Wang, Minal Barve, Jaspreet Grewal, Warren Chow, Jason Henry, Michael Cecchini, Chris Takimoto, Maya Leabman. Pharmacokinetics and pharmacodynamics of IGM-8444, a first-in-class engineered pentameric DR5-targeting agonist IgM monoclonal antibody, in patients with R/R and newly diagnosed cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A137.

S60pts will receive salvage therapy.If MRD-negative pts convert to MRD-positive status, they will be treated as MRD-positive pts.The primary endpoint is MRD-complete response rate after tx with tec + dara (intensification).Secondary endpoints include percentage of pts who convert to MRD negativity after ERI; percentage of pts with sustained MRD negativity at pre-defined time points in both tx arms; OS; progression-free survival; event-free survival; time to next tx; duration of response; and safety.Exploratory endpoints are immune profiling and genetic characterization.Results: Enrollment is expected to begin in May 2023 (target recruitment N=30).Conclusions: This study will provide needed data on the effect of MRD-guided individualized tx approaches in management of pts with HRMM, an underrepresented population with a high unmet need.

Background IGM-7354 is an engineered, humanized high affinity, high avidity anti-PD-L1 pentameric IgM antibody with an IL-15Rα chain and IL-15 fused to the joining (J) chain. IGM-7354 was designed to deliver IL-15-mediated stimulation of NK and CD8+ T cells to PD-L1 expressing tumors and antigen-presenting cells, to enhance anti-tumor immune responses. Methods IGM-7354 has 10 binding sites for human PD-L1 that cross-react with cynomolgus monkey (cyno) PD-L1, but not with rodent PD-L1. The IL-15 component of IGM-7354 binds to human and cyno β chain of the trimeric IL-15 receptor with similar affinities but has weaker affinity to rodent IL-15Rβ. In vitro activity of IGM-7354 on NK or CD8+ T cells was assessed using PBMCs from healthy donors. In vivo pharmacodynamic studies were conducted in cynos and humanized mice engrafted with human CD34+ cells from cord blood in the absence of tumor (BRGSF-HIS mice) or with human PBMC in MDA-MB-231 human tumor-bearing animals (MHC-/- NSG mice). Immune profiling was done by flow cytometry. Serum cytokines and chemokines were analyzed by ELISA or Luminex assays. Results Using in vitro assays with human and cyno PBMCs, IGM-7354 dose-dependently enhanced the proliferation of NK and CD8+ T cells. These immune subsets were further phenotypically characterized in vivo in the humanized BRGSF mouse model. Increases in NKp30, Granzyme B, and the proliferation marker Ki67 in NK cells were observed in animals treated with IGM-7354 as low as 1 mg/kg. IGM-7354 also enhanced the proliferation of CD8+ T cells with an increase of serum soluble CD25, suggestive of T cell activation. In MDA-MB-231 tumor-bearing mice engrafted with human PBMCs, the frequencies of proliferating CD8+ T cells, effector memory and CD39+ TCF-1- CD8+ T cells were increased following IGM-7354 treatment. In cynos, intravenous infusion of IGM-7354 was well tolerated at dose levels up to 10 mg/kg, with increased proliferating NK and effector memory CD8+ T cells in blood and lymphoid tissues. Soluble CD25 was also elevated in the serum of treated monkeys. Conclusions IGM-7354 administration in humanized mouse models and cynomolgus monkeys demonstrated potent activation and expansion of NK cells, effector memory CD8+ T cells, and increased levels of soluble CD25 in the serum. This approach may enhance targeted delivery of the immunostimulatory cytokine IL-15 through high affinity and high avidity binding to PD-L1 potentially improving anti-tumor responses and minimizing toxicity. Ethics Approval The animal studies were approved by the Testing Facilities' IACUC.

Anti-PD-1/PD-L1 therapies are efficacious in certain cancer indications, but often patients relapse following a primary response Therefore rational combinations are needed to enhance initial and durable responses of anti-PD-1/PD-L1 therapies. Immunostimulatory cytokine, IL-15 is an attractive combination partner to enhance anti-tumor NK and memory CD8+ T cell expansion and survival. We are developing IGM-7354, a high affinity, high avidity anti-PD-L1 pentameric IgM antibody with an IL-15Rα chain and IL-15 fused to the joining (J) chain, designed to deliver IL-15 to PD-L1 expressing tumors for enhancing anti-tumor immune responses. IGM-7354 was generated by grafting heavy chain variable regions of a high affinity humanized anti-PD-L1 IgG onto the IgM heavy chain framework, co-expressed with the light chain and the J chain which included a single IL-15Rα and IL-15 fusion. Binding ELISAs were performed using recombinant antigens. IGM-7354 bound human and cynomolgus monkey PD-L1 with similar affinities but did not bind to rat or mouse PD-L1. In addition, the IL-15 component of IGM-7354 bound to human and cynomolgus β chain of the trimeric IL-15 receptor with similar affinities, but with weaker binding affinity to rodent IL-15Rβ. Using in vitro assays with human and cynomolgus monkey PBMCs, IGM-7354 dose-dependently enhanced the proliferation of NK and CD8+ T cells. Furthermore, in an in vitro MLR setting, IGM-7354 was able to reverse T cell exhaustion beyond that of an IL-15/IL15Rα complex or anti-PD-L1 IgM or IgG alone, as demonstrated by an increase in activation and effector cytokine secretion. In vitro cytotoxicity and ADCC assays were performed with luciferase-tagged human cancer cell lines and PBMCs. IGM-7354 enhanced in vitro killing of PD-L1-expressing cancer cells by human PBMCs in monotherapy and in combination with other therapeutic agents. Efficacy and pharmacodynamic studies in an MDA-MB-231 xenograft mouse model showed dose-dependent increases in circulating NK and CD8+ T cells and tumor-infiltrating lymphocytes, which correlated with tumor regression. In cynomolgus monkeys, IGM-7354 markedly induced the proliferation of NK and CD8+ T cells in a dose-dependent manner with a preferential expansion of effector memory CD8+ T cells and γδ T cells in the periphery. IGM-7354 stimulates NK and CD8+ T cell expansion in vitro and in vivo plus induces tumor regressions in mouse tumor models in monotherapy or combination with various agents. This approach may enhance tumor localization of the immunostimulatory cytokine IL-15 through high affinity and high avidity binding to PD-L1 thereby improving anti-tumor responses and minimizing toxicity. Citation Format: Thierry Giffon, Melanie Desbois, Poonam Yakkundi, Keerthana Sekar, Marigold Manlusoc, Rodnie Rosete, Daniel Machado, Susan Calhoun, Tasnim Kothambawala, Dean Ng, Vu Tran, Avneesh Saini, Abhinav Jain, Beatrice Wang, Maya Kotturi, Bruce Keyt, Angus Sinclair. Enhanced NK and CD8+ T cell proliferation, tumor cytotoxicity and reversal of T cell exhaustion with IGM-7354, an anti-PD-L1 IgM antibody and IL-15 cytokine fusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3438.

BackgroundWhile approved PD-1/PD-L1 inhibitory antibodies have demonstrated clinical efficacy in certain cancer patients, relapse following a primary response is often observed. Enhancing anti-tumor immune responses with an immunostimulatory cytokine, IL-15 is an attractive combination strategy to enhance anti-tumor NK and memory CD8+ T cell expansion and survival. We have developed IGM-7354, a high affinity, high avidity anti-PD-L1 pentameric IgM antibody with an IL-15Rα chain and IL-15 fused to the joining (J) chain, designed to deliver IL-15 to PD-L1 expressing tumors for enhancing anti-tumor immune responses.MethodsIGM-7354 was generated by grafting heavy chain variable regions of a high affinity humanized anti-PD-L1 IgG onto the IgM heavy chain framework, co-expressed with the light chain and the J chain which included a single IL-15Rα and IL-15 fusion. Binding ELISAs were performed using recombinant antigens. Human and cynomolgus monkey PBMCs were used for potency testing. Reversal of T cell exhaustion was tested using in vitro MLR. In vitro cytotoxicity assays were performed with luciferase-tagged MDA-MB-231 cells and PBMCs. In vivo pharmacodynamic studies were conducted in mice and cynomolgus monkeys.ResultsIGM-7354 bound human and cynomolgus monkey PD-L1 with the same affinity but did not bind to rat or mouse PD-L1. In addition, the IL-15 component of IGM-7354 bound to human and cynomolgus β chain of the trimeric IL-15 receptor with similar affinities, but with weaker binding affinity to rodent IL-15Rβ. Using in vitro assays with PBMCs, IGM-7354 dose dependently enhanced the proliferation of human and cynomolgus monkey NK and CD8+ T cells. Furthermore, IGM-7354 was able to reverse T cell exhaustion in an in vitro MLR beyond that of an IL-15/IL15Rα complex or anti-PD-L1 IgM or IgG alone, as demonstrated by an increase in activation and effector cytokine secretion. IGM-7354 also enhanced in vitro killing of PD-L1-expressing MDA-MB-231 breast cancer cells by human PBMCs. Pharmacodynamic studies in an MDA-MB-231 xenograft mouse model showed dose-dependent increases in circulating NK and CD8+ T cells and tumor infiltrating lymphocytes, which correlated with tumor regression. In cynomolgus monkeys, intravenous administration of IGM-7354 was well tolerated and dose dependently induced the proliferation of NK and CD8+ T cells.ConclusionsIGM-7354 stimulates NK and CD8+ T cell expansion in vitro and in vivo plus induces tumor regressions in mouse tumor models. This approach may enhance tumor localization of the immunostimulatory cytokine IL-15 through high affinity and high avidity binding to PD-L1 thereby improving anti-tumor responses and minimizing toxicity.Ethics ApprovalAll animal studies were conducted according to approved Institutional Animal Care and Use Committee (IACUC) protocols of the testing facilities.

3074 Background: While inhibitors of CTLA4 and PD1 have emerged as effective cancer therapies, the majority of treated patients do not derive long term benefit. Employing our computational discovery platform, we discovered PVRIG as an immune suppressive molecule expressed on T and NK cells and identified COM701, an antibody (Ab) targeting human PVRIG that enhances T cell function and anti-tumor responses. Methods: Anti-human PVRIG Ab COM701 was identified as an antagonistic Ab that enhanced T cell function in multiple assays. Antagonistic anti-mouse PVRIG Abs and PVRIG deficient (PVRIG-/-) mice were generated and characterized using syngeneic tumor models. Results: PVRIG was induced upon T cell activation, with long term activation leading to the highest expression. PVRL2 was identified as the ligand for PVRIG, placing PVRIG in the DNAM/TIGIT immunoreceptor axis. Compared to normal adjacent tissues, PVRIG and PVRL2 were both induced in the tumor microenvironment of several human cancers. To target PVRIG for therapeutic intervention, we identified COM701, a high affinity Ab that disrupts the interaction of PVRIG with PVRL2. COM701 enhanced CD8 T cell proliferation and IFN-g production in vitro and had an additive or synergistic effect on T cell activation when further combined with an anti-PD1 or anti-TIGIT Ab. Consistent with a checkpoint function for human PVRIG, mouse PVRIG-/- T cells showed increased function compared to wild type T cells. A surrogate antagonistic anti-mPVRIG Ab reduced growth of CT26 and B16 tumors when combined with an anti-PDL1 Ab in vivo. MC38 tumors also grew slower in PVRIG-/- mice compared to wild type mice and ex vivo analysis pointed to functional differences in anti-cancer immunity. Conclusions: We demonstrated that targeting PVRIG with COM701, a high affinity antagonistic Ab, increased human T cell function. We further showed that PVRIG was induced in the tumor microenvironment and that disruption of PVRIG/PVRL2 interaction resulted in reduced tumor growth in preclinical models. These data demonstrate that PVRIG is a promising target for the treatment of cancer and provide the rationale for COM701 as a potential cancer immunotherapy.

Abstract Background: While blockade of the CTLA4 and PD1 pathways has emerged as an effective treatment of cancer, the majority of patients do not derive long term benefit. This provides a rationale for identifying and targeting additional checkpoints. Employing our unique computational algorithms, we identified PVRIG, a new member of the B7/CD28 family. We report here the expression pattern, functional characterization, and anti-tumor activity of blocking antibodies targeting PVRIG as well as characterization of PVRIG KO mice. Materials and Methods: PVRIG is expressed by T and NK cells within the tumor microenvironment. We identified PVRL2 as its counterpart and characterized the PVRIG-PVRL2 interaction. Antibody discovery was carried out with phage display and hybridoma platforms and antibodies against the human protein were screened for their ability to enhance T-cell activity in vitro, while surrogate antibodies targeting the mouse protein were assessed in syngeneic models for effects on tumor growth. PVRIG -/- KO mice were generated and characterized including phenotyping and anti-tumor immune response. Results: PVRIG is expressed on different T cell subsets and on NK, NKT and γδ T-cells. Within T cells, memory subsets possess the highest level of PVRIG and its expression is induced upon long term activation with different stimuli. Within tumor microenvironment, PVRIG was found to be expressed on NK and CD8+ T cells in multiple cancers. A high affinity lead Ab was selected, COM701, for further clinical development and demonstrated blockade of the interaction of PVRIG with PVRL2 as well as enhancement of activation of both primary and tumor-derived effector immune cells through a PVRL2-dependent mechanism. Moreover, COM-701 showed notable enhancement of T cell function in-vitro when combined with PD1 or TIGIT Ab blockade. The lead antibody, COM-701, is currently in preclinical development. A surrogate antibody, that blocks PVRIG-PVRL2 interaction, was shown to inhibit growth of colon carcinoma and melanoma in syngeneic models upon combined treatment with anti-PDL1 antibody. Comparative analysis of PVRIG KO versus WT derived T cells revealed enhanced reactivity of PVRIG null T cells upon polyclonal activation in presence of PVRL2-Ig. Accordingly, MC38 tumors grew slower in PVRIG KO than in WT mice and ex vivo analysis pointed to the quantitative and functional differences in anticancer immunity developed in these mice. Conclusion: We describe the identification of PVRIG as a novel T cell immune checkpoint. We further demonstrate that antibody blockade of the PVRIG-PVRL2 interaction has the potential to be efficiently combined with PD1 or TIGIT blockade for enhancing anti-tumor immunity. COM-701 is a high affinity antagonistic antibody that is currently in preclinical development. Taken together, these data demonstrate the utility of targeting PVRIG in addition to other B7 family checkpoints for the treatment of cancer. Citation Format: Ofer Levy, Chris Chan, Gady Cojocaru, Spencer Liang, Eran Ophir, Sudipto Ganguly, Maya Kotturi, Tal Friedman, Benjamin Murter, Liat Dassa, Ling Leung, Shirley Greenwald, Meir Azulay, Sandeep Kumar, Zoya Alteber, Xiaoyu Pan, Andy Drake, Ran Salomon, Arthur Machlenkin, John Hunter, Zurit Levine, Drew Pardoll, Mark White. Discovery and development of COM701, a therapeutic antibody targeting the novel immune checkpoint PVRIG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 581. doi:10.1158/1538-7445.AM2017-581

BACKGROUND:The transporter associated with antigen processing (TAP) supplies cytosolic peptides into the endoplasmic reticulum for binding to major histocompatibility complex (MHC) class I molecules. Its specificity therefore influences the repertoire of peptides presented by MHC molecules. Compared to human TAP, murine TAP's binding specificity has not been characterized as well, even though murine systems are widely used for basic studies of antigen processing and presentation.METHODOLOGY/PRINCIPAL FINDINGS:We performed a detailed experimental analysis of murine TAP binding specificity by measuring the binding affinities of 323 peptides. Based on this experimental data, a computational model of murine TAP specificity was constructed. The model was compared to previously generated data on human and murine TAP specificities. In addition, the murine TAP specificities for known epitopes and random peptides were predicted and compared to assess the impact of murine TAP selectivity on epitope selection.CONCLUSIONS/SIGNIFICANCE:Comparisons to a previously constructed model of human TAP specificity confirms the well-established differences for peptide substrates with positively charged C-termini. In addition these comparisons show that several residues at the N-terminus of peptides which strongly influence binding to human TAP showed little effect on binding to murine TAP, and that the overall influence of the aminoterminal residues on peptide affinity for murine TAP is much lower than for the human transporter. Murine TAP also partly prefers different hydrophobic amino acids than human TAP in the carboxyterminal position. These species-dependent differences in specificity determined in vitro are shown to correlate with the epitope repertoire recognized in vivo. The quantitative model of binding specificity of murine TAP developed herein should be useful for interpreting epitope mapping and immunogenicity data obtained in humanized mouse models.

The primary CD8+ T cell response against lymphocytic choriomeningitis virus (LCMV) infection of C57BL/6J mice is directed against 28 H‐2b‐restricted epitopes derived from the glycoprotein, nucleoprotein and RNA polymerase L protein. Within these 28 epitopes a clear immunodominance hierarchy is observed. Some major epitopes dominate the response, while some epitopes are intermediate and others are relatively minor contributors to the overall response. To probe the mechanisms regulating CD8+ T cell immunodominance, we manipulated the number of epitopes seen during acute LCMV infection. Deletion of epitope‐specific responses, either by using a LCMV viral variant lacking 4 major epitopes, or C57BL/6J mice altogether lacking expression of H‐2Kb, resulted in an overall decrease of the response, minor compensatory effects, and no detectable responses against new epitopes. Increasing the number of epitopes available for recognition by use of H‐2b F1 hybrid mice, or delivery of LCMV antigens by recombinant vaccinia virus also did not dramatically alter the immunodominance pattern. These data suggest that immunodominance hierarchy might be intrinsic to the epitope and its antigen of origin. We are currently investigating these issues by directly measuring the hierarchies in MHC binding affinities, the yield of epitope production as a result of cellular processing, and by quantifying the existing naïve T cell repertoire by the use of a sensitive tetramer‐based enrichment method.

CD8+ T cell responses control lymphocytic choriomeningitis virus (LCMV) infection in H-2b mice. Although antigen-specific responses against LCMV infection are well studied, we found that up to one third of the CD8+CD44hi response to LCMV in H-2b mice was not accounted for by known epitopes. We screened peptides predicted to bind MHC Class I, and overlapping 15-mer peptides spanning the complete LCMV proteome for their capacity to induce interferon-γ (IFNγ) production from CD8+ T cells derived from LCMV-infected H-2b mice. We identified thirteen novel epitopes. These together with the 7 previously known epitopes account for the total CD8+CD44hi response, diminishing the possible role for bystander T cell activation. Strikingly, 9 out of the 13 new epitopes were derived from the viral L polymerase, which, until now, was not recognized as a target of the cellular response induced by LCMV infection. The L epitopes induced significant levels of in vivo cytotoxicity and conferred protection against LCMV challenge. Interestingly, protection from viral challenge was best correlated with the cytolytic potential of CD8+ T cells, whereas IFNγ production and peptide avidity appear to play a lesser role. Taken together, these findings illustrate that the LCMV-specific CD8+T cell response is more complex than previously appreciated.

Activation of CD4(+) T cells helps establish and sustain CD8(+) T cell responses and is required for the effective clearance of acute infection. CD4-deficient mice are unable to control persistent infection and CD4(+) T cells are usually defective in chronic and persistent infections. We investigated the question of how persistent infection impacted pre-existing lymphocytic choriomeningitis virus (LCMV)-specific CD4(+) T cell responses. We identified class II-restricted epitopes from the entire set of open reading frames from LCMV Armstrong in BALB/c mice (H-2(d)) acutely infected with LCMV Armstrong. Of nine epitopes identified, six were restricted by I-A(d), one by I-E(d) and two were dually restricted by both I-A(d) and I-E(d) molecules. Additional experiments revealed that CD4(+) T cell responses specific for these epitopes were not generated following infection with the immunosuppressive clone 13 strain of LCMV. Most importantly, in peptide-immunized mice, established CD4(+) T cell responses to these LCMV CD4 epitopes as well as nonviral, OVA-specific responses were actively suppressed following infection with LCMV clone 13 and were undetectable within 12 days after infection, suggesting an active inhibition of established helper responses. To address this dysfunction, we performed transfer experiments using both the Smarta and OT-II systems. OT-II cells were not detected after clone 13 infection, indicating physical deletion, while Smarta cells proliferated but were unable to produce IFN-gamma, suggesting impairment of the production of this cytokine. Thus, multiple mechanisms may be involved in the impairment of helper responses in the setting of early persistent infection.