UV-induced somatic mutations elicit a functional T cell response in the YUMMER1.7 mouse melanoma model

Abstract

Human melanomas exhibit relatively high somatic mutation burden compared to other malignancies. These somatic mutations may produce neoantigens that are recognized by the immune system, leading to an antitumor response. By irradiating a parental mouse melanoma cell line carrying three driver mutations with UVB and expanding a single-cell clone, we generated a mutagenized model that exhibits high somatic mutation burden. When inoculated at low cell numbers in immunocompetent C57BL/6J mice, YUMMER1.7 (Yale University Mouse Melanoma Exposed to Radiation) regresses after a brief period of growth. This regression phenotype is dependent on T cells as YUMMER1.7 tumors grow significantly faster in immunodeficient Rag1^-/- mice and C57BL/6J mice depleted of CD4 and CD8 T cells. Interestingly, regression can be overcome by injecting higher cell numbers of YUMMER1.7, which results in tumors that grow without effective rejection. Mice that have previously rejected YUMMER1.7 tumors develop immunity against higher doses of YUMMER1.7 tumor challenge. In addition, escaping YUMMER1.7 tumors are sensitive to anti-CTLA-4 and anti-PD-1 therapy, establishing a new model for the evaluation of immune checkpoint inhibition and antitumor immune responses.

Keywords: Braf; UV radiation; immunotherapy; melanoma; mouse models.

Figure 1. Generation and Characterization of YUMMER1.7

(A) YUMMER1.7 was generated from YUMM1.7 using three rounds of UVB radiation and a single cell-derived clone was expanded. (B) Total point mutations in YUMMER1.7 compared to YUMM1.7 categorized based on the type of base substitution. (C) Growth of 100,000 cell YUMM1.7 and YUMMER1.7 tumors engrafted into wild-type C57BL/6J mice. (D) Growth of 100,000, 250,000, 500,000, and 1,000,000 cell YUMMER1.7 tumors engrafted into wild-type C57BL/6J mice. (E) Growth of 100,000 cell YUMM1.7 tumors and YUMMER1.7 tumors engrafted into Rag1^−/− C57BL/6J mice. (F) Growth of 100,000 cell YUMMER1.7 tumors engrafted into wild-type C57BL/6J mice that were treated with depleting antibodies against CD4, CD8 or both. Tumor growth curves are representative of two independent experiments (mean ± SEM, n = 5 each).

Figure 2. Immune infiltration, mitotic rate and apoptotic cell death within YUMM1.7-GFP and YUMMER1.7-GFP tumors

(A) GFP (melanoma), CD45, CD3, F4/80-positive cells as a percent of nucleated cells. Data are averages of counts from 3–4 independent tumors (mean ± SEM). (B) CD3, Foxp3 as a percent of nucleated cells and Foxp3 as a percentage of CD3 positive cells (orange). Data are averages of counts from 3–4 independent tumors (mean ± SEM). (C) Representative immunohistochemical images of YUMM1.7 and YUMMER1.7 tumors on day 20 with the indicated staining at 40X magnification. YR^LO = YUMMER1.7-GFP^LO, YR^HI = YUMMER1.7-GFP ^HI, YM = YUMM1.7-GFP. (D) Mitotic rate and cleaved caspase-3 staining for YUMM1.7-GFP and YUMMER1.7-GFP tumors over time (mean ± SEM, n = 3–4).

Figure 3. Immune infiltration of escaping YUMM1.7 and YUMMER1.7 tumors

(A) Density of CD4⁺ T cells per gram of tumor. (B) Density of CD8⁺ T cells per gram of tumor. (C) Percent of CD8⁺ T cells that are PD1⁺ TIGIT⁺. Data are from two independent experiments (mean ± SEM, n=2–3 each).

Figure 4. YUMMER1.7 but not YUMM1.7, is sensitive to checkpoint inhibitors anti-CTLA-4 and anti-PD-1

(A) Individual tumor growth curves for YUMMER1.7 tumors treated with anti-CTLA-4 (n = 5), anti-PD-1 (n = 5) or both (n = 4) compared to isotype control (n = 5). Data are representative of two independent experiments. (B) Kaplan-Meier survival curves for treated YUMMER1.7 and (C) YUMM1.7 tumors with an endpoint of tumor size > 1000 mm³ (n = 4–5).