Abstract A86: Comparative immunophenotypic analysis of immunogenically warm and cold syngeneic tumor models at baseline and after anti-mCTLA-4 treatment
Authors: Draper David , Lapinski Philip , Roys Stacey , Wise Scott , Franklin Maryland Rosenfeld 
Published: March 2020

To examine how baseline tumor immunophenotyes can influence therapeutic outcome, we used the CT26 colorectal and 4T1-Luc (4T1) breast cancer models, which are immunologically warm and cold tumors, respectively. We treated tumor-bearing mice with control or anti-mCTLA-4 antibodies (anti-CTLA-4), or left them untreated, and excised tumors to measure immune cell infiltrates, TIL cytokine/granzyme B production, and activation marker expression. Baseline analysis in untreated mice revealed that CD8+ T-cell infiltrates were similar between models (5-6% of total CD45+ cells). NKT cells were reduced in CT26 (0.5% vs. 1.2%). The myeloid compartment in CT26 was mostly M2 macrophages (43% of CD45+ cells), whereas G-MDSCs dominated in 4T1 (40% of CD45+ cells). CD69 and PD-1 activation markers were expressed on &gt;90% of CD8+ T cells from CT26 tumors compared to 4T1, which were &lt;40% positive for both markers. Following ex vivo stimulation, the IFNγ response in CD8+ T cells was higher in CT26-derived cells. Treatment with anti-CTLA4 triggered distinct responses in the two models. Delayed tumor growth was observed in CT26 (74% TGI), but no effect in 4T1 was shown. In CT26 tumors, CD8+ T-cell and NKT cell infiltration increased, with a corresponding decrease in M2 macrophages. In 4T1 tumors, only increased NKT cell infiltration occurred. Several pharmacodynamic changes were observed in immune cell activation in CT26 but absent in 4T1. IFNγ production increased in CT26-derived CD4+ and CD8+ T cells, as well as NKT cell subsets (70%, 25%, and 17%, respectively compared to control). This included an increase in polyfunctional T cells that produced both IFNγ and TNFα. Moreover, we found that tumors with the highest frequency of IFNγ/TNFα double positive T cells had slower growth, suggesting that polyfunctional T-cell differentiation correlates with tumor growth delay. Granzyme B was 31% reduced in NKT cells in CT26. Together with increased CD107a expression on these cells, the observed granzyme B reduction was likely due to treatment-induced degranulation. Notably, a correlation between granzyme B expression and delayed tumor growth was also observed, albeit indirect. In summary, untreated CT26 tumors contained TILs that expressed high levels of CD69/PD-1 and moderately expressed IFNγ, and subsequently responded to anti-CTLA-4 treatment with increased polyfunctional T-cell recruitment, NKT cell activity, and reduced M2 macrophage infiltration. In contrast, 4T1 tumors contained low levels of both CD69/PD-1 expression and TIL cytokine expression, which is characteristic of an immunosuppressive tumor microenvironment, and did not display any remarkable response to therapy. Collectively, these results suggest baseline tumor profiles can provide insight into responsiveness to therapy and assist in model selection for preclinical research. Future studies to more broadly profile baseline TIL functional states across different syngeneic models should prove valuable during study design for targeted I/O therapy development.

Citation Format: David Draper, Philip Lapinski, Stacey Roys, Scott Wise, Maryland Rosenfeld Franklin. Comparative immunophenotypic analysis of immunogenically warm and cold syngeneic tumor models at baseline and after anti-mCTLA-4 treatment [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 A86.