Lack of immunoediting in murine pancreatic cancer reversed with neoantigen

Abstract

In carcinogen-driven cancers, a high mutational burden results in neoepitopes that can be recognized immunologically. Such carcinogen-induced tumors may evade this immune response through "immunoediting," whereby tumors adapt to immune pressure and escape T cell-mediated killing. Many tumors lack a high neoepitope burden, and it remains unclear whether immunoediting occurs in such cases. Here, we evaluated T cell immunity in an autochthonous mouse model of pancreatic cancer and found a low mutational burden, absence of predicted neoepitopes derived from tumor mutations, and resistance to checkpoint immunotherapy. Spontaneous tumor progression was identical in the presence or absence of T cells. Moreover, tumors arising in T cell-depleted mice grew unchecked in immune-competent hosts. However, introduction of the neoantigen ovalbumin (OVA) led to tumor rejection and T cell memory, but this did not occur in OVA immune-tolerant mice. Thus, immunoediting does not occur in this mouse model - a likely consequence, not a cause, of absent neoepitopes. Because many human tumors also have a low missense mutational load and minimal neoepitope burden, our findings have clinical implications for the design of immunotherapy for patients with such tumors.

Figure 1. T cell dependency of KPC pancreatic cancer.

(A) Experimental design for survival studies of syngeneic KPC mice treated with an isotype control antibody, depleted of CD4 and CD8 T cells (αCD4/αCD8), or depleted of CD8 T cells alone (αCD8) beginning at 3–5 weeks of age. n = 19–21 mice per cohort. Starting at 7–8 weeks of age, mice were monitored by ultrasound every other week for tumor development and examined daily for morbidity. (B) Representative image of a tumor at the time of diagnosis by abdominal ultrasound (volume = 19 mm³). IVC, inferior vena cava. (C) Tumor-free survival according to ultrasound monitoring (time to diagnosis) and overall survival according to daily monitoring for the 3 cohorts described in A. P values were determined by log-rank (Mantel-Cox) analysis. (D) H&E staining of a representative pancreatic tumor from each treatment cohort (original magnification, ×10). (E) Flow cytometric analysis of tumors at the time of euthanasia (4–6 mice per cohort) to assess infiltration by leukocytes (CD45⁺ cells as percentage of viable cells), macrophages (CD45⁺CD19^–F4/80⁺ as percentage of viable CD45⁺ cells), and immature myeloid cells (CD45⁺CD19^–Gr-1⁺CD11b⁺ as percentage of viable CD45⁺ cells). Data are shown as whisker plots (symbols represent individual experimental measurements; center line, mean; outer lines; 1 SD), with P values determined by 2-way ANOVA.

Figure 2. KPC-derived tumor cell lines grow progressively upon implantation, regardless of donor or host T cell status.

(A) Experimental design using syngeneic pancreatic ductal adenocarcinoma (PDA) cell lines generated from immune-competent KPC mice or KPC mice serially depleted of CD4/CD8 T cells beginning at 3–5 weeks of age, as shown in Figure 1A. Cell lines were implanted subcutaneously (S.C.) in syngeneic mice predepleted of CD4 and CD8 T cells or administered an isotype control antibody (n = 8–10 mice per cohort). Tumor growth was measured by caliper over time, and mice were monitored for overall survival. (B) Subcutaneous tumor growth of 4662 PDA cells in immune-competent syngeneic hosts (Isotype control) or immune-compromised mice (αCD4/αCD8), shown with high inoculum (5 × 10⁵ tumor cells) or low inoculum (10⁵ cells). For the lower dose, mice were also monitored for overall survival. Growth data are shown as spaghetti plots of individual mice, and P values indicate analysis by 2-way ANOVA. Survival data were analyzed by log-rank (Mantel-Cox) test. (C) Histology of 4662 implanted tumor after 3 weeks of growth (left panels) stained by H&E and Masson’s trichrome. Right panels show H&E and trichrome staining of an autochthonous KPC tumor. Original magnification, ×10. (D) Subcutaneous growth of CD4- and CD8-depleted KPC cell lines (1262, 1493, and 1638; generated as described in A) in immune-competent syngeneic isotype control or αCD4/αCD8 mice. n = 8–10 mice per cohort. P values shown were generated by 2-way ANOVA. (E) Numbers of predicted neoepitopes for the B16 murine melanoma cell line, the 4662 PDA cell line derived from an immune-competent KPC mouse, and 3 cell lines derived from T cell–depleted KPC mice (1262, 1493, and 1638). Predictions are shown for both the 50 nM binding threshold (black bars) and 100 nM threshold (gray bars).

Figure 3. Expression of a strong antigen in a KPC tumor induces CD8-dependent tumor elimination.

(A) Parental 4662 cells were retrovirally transduced with a Td-Tomato/ovalbumin-expressing (Tdt/OVA-expressing) construct and sorted to generate single-cell clones. (B) Flow cytometric analysis of the parental 4662 cell line compared to the V6.Ova clone. Cells were assessed for expression of Td-tomato, incubated with or without IFN-γ, and stained using a mAb against SIINFEKL-bound H2-K^b (MHC class I) and gated on viable (Live/Dead aqua-negative) cells. Data are representative of 3 independent experiments. (C) A high dose of the V6.Ova clone (10⁶ cells) was subcutaneously implanted in syngeneic mice treated with an isotype control antibody, CD8-depleting antibody (αCD8), CD4-depleting antibody (αCD4), or an NK cell–depleting antibody (αNK1.1) and monitored for growth over time by caliper. n = 7–8 mice per cohort (mean tumor volume was plotted with error bars representing + 1 SD; ****P < 0.0001 by 2-way ANOVA) (left). H&E staining of an implant at day 8 of an isotype-treated mouse showing presence of tumor cells with marked infiltration of lymphocytes (original magnification, ×10; inset, ×40) (right). (D) Tumor growth at a lower inoculum of 0.75 × 10⁶ V6.Ova cells was assessed in isotype-treated and CD8-depleted cohorts, which were also monitored for overall survival (n = 12–13 mice per cohort; P value by log-rank [Mantel-Cox] test). (E) Growth of V6.Ova tumor cells implanted orthotopically in mice treated with isotype control or αCD8 with an inoculum of 0.125 × 10⁶ cells. Mice were monitored for tumor growth by ultrasound and assessed for overall survival. n = 9–10 mice per cohort; data shown are pooled from 2 independent experiment experiments. P values were determined by 2-way ANOVA (tumor growth) and log-rank (Mantel-Cox) (overall survival). (F) A summary of subcutaneous and orthotopic growth of parental 4662 cells and isotype-treated or CD8-depleted V6.Ova-implanted cohorts. Numbers above bars indicate the number of mice growing tumors over the total number of mice tested. (G) C57BL/6 mice that rejected a subcutaneous V6.Ova implant after 6 weeks were either CD4/CD8 depleted or administered an isotype control and then rechallenged with parental 4662 on the opposite flank. A third naive cohort was simultaneously challenged with parental 4662 cells at the same dose of 10⁵ cells. n = 9–10 mice per cohort. Mice were followed by caliper for tumor growth and monitored for overall survival. P values represent analysis by 2-way ANOVA or log-rank (Mantel-Cox) tests, respectively; **P < 0.05; ***P < 0.001.

Figure 4. Selective outgrowth of Ova^– tumors in immune-competent mice.

(A) Experimental design of a competition assay between V6.Ova cells and negatively sorted (OvaNeg) cells subcutaneously implanted at the following ratios in immune-competent or CD8-depleted cohorts (n = 5 mice per cohort): 100% V6.Ova (7.5 × 10⁵ V6.Ova cells); 90% V6.Ova/10%OvaNeg (6.75 × 10⁵ V6.Ova cells and 0.75 × 10⁵ OvaNeg cells); 80% V6.Ova/20%OvaNeg (6.0 × 10⁵ V6.Ova cells and 1.5 × 10⁵ OvaNeg cells); and 100%OvaNeg (7.5 × 10⁵ OvaNeg cells). (B) Implants containing either a combination of Ova⁺ and Ova^– cells (90% V6.Ova and 80% V6.Ova) or a 100% population of V6.Ova or OvaNeg cells were assessed for tumor growth. Data are shown as the individual growth curves for each mouse per cohort (n = 5 mice per cohort). Three independent experiments were performed. P values represent 2-way ANOVA. Overall survival was assessed by log-rank (Mantel-Cox) for each cohort.

Figure 5. Immunity is dependent on cytotoxic Ova-specific CD8 T cells.

(A) From the experimental schema in Figure 4A, Ova-specific CD8⁺ T cells in 80% V6.OVA tumors at day 14 were compared with spleens and quantified in the tumor in all cohorts. n = 5–7 mice per cohort; data are representative of 3 independent experiments. ***P < 0.001 by unpaired 2-tailed Student’s t test. ****P < 0.0001 by 2-way ANOVA. Data are shown as whisker plots (symbols represent individual experimental measurements; center line, mean; outer lines; 1 SD). (B) Intratumoral CD8⁺ T cells in 80% V6.Ova-implanted mice were assessed by flow cytometry for intracellular levels of granzyme B and Tbet at day 21. Ki67 and IFN-γ expression was also assessed with or without stimulation with PMA/ionomycin. n = 6 mice per cohort. Data are representative of 2 independent experiments. ***P < 0.001 by unpaired 2-tailed Student’s t test. ****P < 0.0001 by 2-way ANOVA. (C) Tumor-enriched live cells (CD45^–CD31^–CD90^–) were assessed for Td-Tomato expression by flow cytometry; Td-tomato⁺ cells are shown as a percentage of this tumor-enriched population across cohorts. n = 5–7 mice per cohort. Data are representative of 3 independent experiments. ****P < 0.0001 calculated by 2-way ANOVA. Representative data are shown as a histogram of Td-tomato expression for each cohort. (D) H&E stain of an isotype-treated 80% V6.Ova tumor (original magnification, ×10).