Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to immune checkpoint inhibitor (ICI) utility in anti-cancer therapy1. The pathogenesis of ICI-myocarditis is poorly understood. Pdcd1-/-Ctla4+/-mice recapitulate clinicopathologic features of ICI-myocarditis, including myocardial T cell in ltration2. Single cell RNA/T cell receptor (TCR) sequencing on the cardiac immune in ltrate of Pdcd1-/-Ctla4+/-mice identi ed activated, clonal CD8+ T cells as the dominant cell population. Treatment with anti-CD8, but not anti-CD4, depleting antibodies rescued survival of Pdcd1-/-Ctla4+/-mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients which required CD8+ T cells. Alpha-myosin, a cardiac speci c protein absent from the thymus3,4, was identi ed as the cognate antigen source for three MHC-I restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from two patients with ICI-myocarditis were expanded by alpha-myosin peptides, and these alpha-myosin expanded T cells shared TCR clonotypes with diseased heart and skeletal muscles, indicating that alpha-myosin may be a clinically important autoantigen in ICI-myocarditis. These studies underscore the critical role for cytotoxic CD8+ T cells, are the rst to identify a candidate autoantigen in ICI-myocarditis and yield new insights into ICI toxicity pathogenesis.Grant 5P30 CA68485-19 and the Shared Instrumentation Grant S10 OD023475-01A1 for the Leica Bond RX. The Vanderbilt VANTAGE Core, including A. Jones and L. Raju, provided technical assistance for this work. VANTAGE is supported in part by a CTSA Grant (5UL1 RR024975-03), the Vanderbilt Ingram Cancer Center (P30 CA68485), the Vanderbilt Vision Center (P30 EY08126) and the NIH/NCRR (G20 RR030956). Figures 1a and 4b were created with BioRender.com.Con ict Interest Disclosure M.L. Axelrod is listed as a coinventor on a provisional patent application for methods to predict therapeutic outcomes using blood-based gene expression patterns, that is owned by Vanderbilt University Medical Center, and is currently unlicensed. S.C. Wei is an employee of Spotlight Therapeutics, a consultant for BioEntre, and an inventor on a patent for a genetic mouse model of autoimmune adverse events and immune checkpoint blockade therapy (PCT/US2019/050551) pending to Board of Regents, The University of Texas System. J.C. Rathmell is a founder, scienti c advisory board member, and stockholder of Sitryx Therapeutics, a scienti c advisory board member and stockholder of Caribou Biosciences, a member of the scienti c advisory board of Nirogy Therapeutics, has consulted for Merck, P zer, and Mitobridge within the past three years, and has received research support from Incyte Corp., Calithera Biosciences, and Tempest Therapeutics. P.B. Ferrell receives research support from Incyte Corporation. D.B.Johnson has served on advisory boards or as a consultant for BMS, Catalyst

Immunotherapy has become a key therapeutic strategy in the treatment of many cancers. As a result, research efforts have been aimed at understanding mechanisms of resistance to immunotherapy and how anti-tumor immune response can be therapeutically enhanced. It has been shown that tumor cell recognition by the immune system plays a key role in effective response to T cell targeting therapies in patients. One mechanism by which tumor cells can avoid immunosurveillance is through the downregulation of Major Histocompatibility Complex I (MHC-I). Downregulation of MHC-I has been described as a mechanism of intrinsic and acquired resistance to immunotherapy in patients with cancer. Depending on the mechanism, the downregulation of MHC-I can sometimes be therapeutically restored to aid in anti-tumor immunity. In this article, we will review current research in MHC-I downregulation and its impact on immunotherapy response in patients, as well as possible strategies for therapeutic upregulation of MHC-I.

Background Immune checkpoint inhibitors (ICIs) targeting the PD-1/L1 axis are approved in early-stage treatment for triple-negative breast cancer (TNBC), but only a fraction of patients benefit. Tumor expressed antigens bound to major histocompatibility complex-I (MHC-I) are required for CD8-mediated tumoricidal activity, and thus, response to anti-PD-1/L1 targeted ICI. However, many breast tumors downregulate, or heterogeneously express, MHC-I, making them less susceptible to ICIs. Tumor cells downregulating MHC-I may be effectively targeted by natural killer (NK) cells due to ‘missing self’ signals. However, this heterogeneity in MHC-I expression is poorly modeled in most preclinical studies, limiting our understanding of how to overcome ICI resistance in the context of heterogeneous MHC-I expression, as is often observed clinically. Objective We aimed to 1) quantitatively delineate how intratumoral heterogeneity in MHC class I expression affects immune responses and immunotherapy outcomes in mouse models and 2) determine whether targeting inhibitory signals on NK cells can overcome ICI resistance in MHC-I heterogenous TNBC. Methods We performed quantitative immunofluorescence for MHC-I, CD8, CD56, and pan-cytokeratin on breast cancer tumors from diverse subtypes (n=314) to obtain single-cell-resolution MHC-I expression and spatial information of tumor and immune cells. Fluorescence intensity and spatial analysis were processed to output individual tumor/stromal cell MHC-I expression and the composition of the local tumor microenvironment. To determine the functional effect of MHC-I heterogeneity in vivo, we generated a CRISPR-guided B2m knockout (B2m-null) in a murine orthotopic model (EMT6). We then combined MHC-I-proficient and MHC-I-deficient isogenic lines at various ratios to model how populations of MHC-I loss affected the immune microenvironment. We also assessed a second, intrinsically heterogenous (MHC-I expression) TNBC model E0771. To evaluate changes in the microenvironment, we used flow cytometry and an immune NanoString panel to evaluate gene expression patterns in tumor cells and infiltrating immune cells. Results TNBC patients had the highest MHC-I expression level across tumor cells, but also the highest variability and probability of demonstrating bimodal MHC-I expression (consisting of high and low/absent cells within a single tumor). ER+ tumors were unimodally low. Using spatial analysis, we identified that heterogenous MHC-I tumors had significantly higher levels of infiltrating NK cells(Paired T-Test: p=0.03). In murine models, even 10% or less of MHC-I-null (B2m-null) EMT6 cells in the tumor injection resulted in acquisition of ICI resistance. Interestingly, heterogeneity in expression of MHC-I resulted in a substantial infiltration by NKG2A+ NK cells compared to MHC-I high or -low tumors (Student T-Test: p=.002). Activation of these infiltrating NK cells via anti-NKG2A and anti-PD-L1 combination treatment restored complete responses in heterogeneous EMT6 tumors, and significantly extended survival in both E0771 (Mantel-Cox: p< 0.0001) and EMT6 models (Mantel-Cox: < 0.0001). Additionally, anti-NKG2A and anti-PDL1 combination treatment improved complete response in the heterogenous MHC-I EMT6 model to 30% and in the parental EMT-6 tumors to 70%. Conclusion Combined therapy with anti-NKG2A (targeting NK cells) and anti-PD-L1 (targeting CD8+ T cells) can restore immunotherapy responses and overcome resistance due to lack of MHC-I expression in tumor cell subpopulations. Citation Format: Brandie C. Taylor, Xiaopeng Sun, Justin Balko, Paula Gonzalez-Ericsson, Melinda Sanders. Implications of Heterogeneity in Breast Tumor Cell MHC-I Expression on Immunity and Therapeutic Resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-06.

Immune checkpoint inhibitors (ICI) have significantly enhanced patient survival in many cancers but yield limited success in breast cancer. ICIs activate anti-tumor immunity by overriding the inhibition of tumor infiltrating lymphocytes (TILs). Clinical trials in triple negative breast cancer (TNBC) patients, who harbor TILs within tumor stroma, have demonstrated increased survival (IMpassion130) and pathologic complete response (KEYNOTE-522) to ICI leading to FDA-approval of ICI and chemotherapy combinations in metastatic TNBC. However, ICI benefit is heterogeneous among patients. We sought to model ICI response in vivo to evaluate therapeutic resistance and response heterogeneity and to ascertain predictive biomarkers for favorable ICI outcomes. An immunocompetent EMT6 orthotopic mammary tumor model was used to investigate the efficacy of anti-PD-L1. Matched longitudinal samples of the tumor microenvironment (TME) (collected by fine-needle aspiration) and peripheral blood (PBMC) from mice were profiled by bulk RNA and T-cell receptor sequencing. Anti-PD-L1 robustly suppressed primary tumor growth and extended survival beyond the control group. The addition of chemotherapy demonstrated moderate therapeutic efficacy but failed to enhance ICI benefit. Phenotypic profiling of the TME revealed increased T cells, DCs, and NK cells in anti-PD-L1 only and chemotherapy combination groups. Despite using a genetically identical tumor model and host, PD-L1 blockade induced heterogeneous responses, like clinical outcomes in TNBC patients, ranging from complete response (CR) to intrinsic resistance (IR). The primary TME showed upregulated signatures of cytotoxic T cell response and activation, specifically inflammatory interferon signaling (both prior to and post ICI administration) that corresponded to favorable outcomes to anti-PD-L1 in individual mice. Longitudinal analysis of the peripheral blood identified modest changes among mice at baseline that progressively deviated by response type (IR-vs-CR). Mice harbored enriched myeloid signatures and clonal T cell expansion during therapy corresponding to ICI resistance and response, respectively. Further investigations of matched peripheral blood and the primary TME signatures may identify systemic biomarkers and tumor antigen-specific T cell clones to accurately predict ICI response in patients and uncover mechanisms for sensitizing tumors refractory to ICI. Thus, we identify an in vivo model that emulates TNBC patient heterogeneous outcomes to ICI combinatorial approaches. We describe host-specific signatures, specifically from myeloid cells, that correlate with differential responses to ICI, which may serve as a basis for peripheral blood tracking of breast cancer patient responses. Citation Format: Ann Hanna, Xiaopeng Sun, Emily Tran, Quanhu Sheng, Brandie C. Taylor, Susan R. Opalenik, Justin M. Balko. Longitudinal local and peripheral immunologic changes associated with PD-L1 response in a murine breast cancer model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4152.