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1002 Intratumor heterogeneity in MHC-I expression drives immunotherapy resistance and exposes NKG2A as a therapeutic target in breast cancer
  1. Brandie C Taylor1,
  2. Xiaopeng Sun1,
  3. Paula Gonzalez Ericsson2,
  4. Violeta Sanchez2,
  5. Melinda Sanders2,
  6. Elizabeth Wescott1,
  7. Susan Opalenik2,
  8. Ann Hanna2,
  9. Brian Lehmann2,
  10. Vandana Abramson3,
  11. Jennifer Pietenpol3 and
  12. Justin M Balko2
  1. 1Vanderbilt University, Nashville, TN, USA
  2. 2Vanderbilt University Medical Center, Nashville, TN, USA
  3. 3Vanderbilt University School of Medicine, Nashville, TN, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Resistance to immune checkpoint inhibition (ICI) in patients with triple-negative breast cancer (TNBC) remains a common problem, with the underlying mechanisms not well understood. Tumor antigens bound to major histocompatibility complex-I (MHC-I) are required for CD8-mediated tumoricidal activity, and thus, anti-PD-1/L1 targeted ICI. However, many breast tumors downregulate, or heterogeneously express, MHC-I, potentially making them less susceptible to ICIs. This heterogeneity in MHC-I expression is not examined 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.

Methods We evaluated tumor specific MHC-I expression with clinical outcome via multiplexed immunofluorescence (mIF) staining on pre-treatment biopsies from metastatic TNBC patients (n=84) in a randomized Phase II clinical trial evaluating carboplatin ± atezolizumab (NCT03206203). Further, we evaluated MHC-I heterogenous expression across breast cancer subtypes at a single-cell level (n=314). To model patterns of MHC-I heterogeneity and how this impacts tumor immune infiltration and response to immunotherapy, we examined murine mammary cancer models of enforced MHC-I heterogeneity via scRNA sequencing, flow cytometry, and RNA gene expression profiling. Lastly, we applied spatial technologies on TNBC tumors (ROI=154) to characterize the immune cell infiltration patterns around regions of high, low, and heterogenous MHC-I expression.

Results The current study demonstrates that TNBC patients show remarkable intratumor heterogeneity in MHC-I expression patterns. In preclinical mouse models, complete loss of MHC-I negates anti-tumor immunity and ICI response, whereas intratumor MHC-I heterogeneity leads to increased local infiltration of natural killer (NK) cells. These findings are replicated in human breast cancers using spatial technologies where MHC-I heterogeneity is associated with clinical resistance to anti-PD-L1 therapy and increased NK:T cell ratios in breast tumors. MHC-I heterogeneous tumors require NKG2A to suppress NK cell function and to eliminate MHC-I negative tumor cells. Combining anti-NKG2A and anti-PD-L1 therapies restores complete response in heterogeneous MHC-I murine models, and it is dependent on both activated, tumor-infiltrating NK and CD8+ T cells.

Conclusions These data reinforce the growing interest on how tumor-specific antigen presentation via MHC-I plays a role in modifying anti-tumor immunity and ICI response. Together, they endorse the unmet translational and clinical need to address heterogeneity in MHC-I expression as a variable in understanding breast cancer anti-tumor immunity and response to immunotherapy. Moreover, these data showcase the potential to harness NK cell function in advancing cancer immunotherapy combinations.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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