Abstract PD2-06: Implications of Heterogeneity in Breast Tumor Cell MHC-I Expression on Immunity and Therapeutic Resistance
Authors: Taylor Brandie C. , Sun Xiaopeng , Balko Justin , Gonzalez-Ericsson Paula , Sanders Melinda 
Published: March 2023

Abstract
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&lt; 0.0001) and EMT6 models (Mantel-Cox: &lt; 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.