Identification of a neo-epitope dominating endogenous CD8 T cell responses to MC-38 colorectal cancer

Abstract

The murine MC-38 colorectal cancer model is a commonly used model for cancer with high mutational burden, which is sensitive for immune checkpoint immunotherapy. We set out to analyze endogenous CD8⁺ T cell responses to MC-38 neo-antigens in tumor-bearing mice and after anti-PD-L1 checkpoint therapy. Through combination of whole-exome sequencing analysis with mass spectrometry of MHC class I eluted peptides we could identify eight candidate epitopes. Of these, a neo-epitope encoded by a point-mutation in the sequence of the ribosomal protein L18 (Rpl18) strongly dominated the CD8⁺ T cell response to our MC-38 cell-line in comparison to a previously described neo-epitope in the Adpgk protein. Therapeutic vaccination with synthetic peptides induced CD8⁺ T cell responses against the mutated Rpl18 epitope, which controlled tumor growth in vivo. This immunologically dominant response to mutated Rpl18 is of great importance in the development and optimization of immunotherapeutic strategies with the MC-38 tumor model.

Keywords: Neoantigen; PD-L1; checkpoint; immunotherapy; vaccination; whole exome sequencing.

Figure 1.

Strategy to identify MC-38 colorectal tumor specific neo-peptides. MC-38 DNA was isolated and its sequenced exome was compared to DNA from C57BL/6 wild-type mice for genetic variants. Corresponding amino acid sequences were used to predict potential binders to MHC class I K^b and D^b by NetMHC4.0 prediction algorithm. A combination of immunoprecipitation of MHC class I from MC-38 lysate and subsequent mass-spectrometry of eluted peptides was performed to establish MHC class I presentation.

Figure 2.

Irradiated MC-38 immunization induces CD8⁺ T cells specific for candidate neo-epitopes. (a) Mice were prime-boost vaccinated with subcutaneous injection of 5 × 10⁶ irradiated MC-38 tumor cells. (b) Cytokine production of CD8α⁺ T lymphocytes through stimulation with synthetic “long” peptide-loaded dendritic cells ex vivo and (c) after restimulation with irradiated MC-38 cells. (b) and (c) are from two independent experiments, n = 3 for each. Shown are representative cytokine (IFNγ and TNFα) staining plots from CD8α⁺ populations (left) and plotted percentages of double-positive IFNγ and TNFα cytokine producing populations of individual mice (right).

Figure 3.

Tumor-bearing mice have circulating and tumor-infiltrating CD8⁺ lymphocytes specific for Rpl18 neo-peptide. (a) Mice were inoculated with 3 × 10⁵ live MC-38 tumor cells. Lymphocytes isolated from spleen and tumor-infiltrating lymphocytes from the established tumors were stimulated with neo-peptides eight days post inoculation (n = 4). (b) IFNγ and TNFα cytokine production of CD8α⁺ T lymphocytes through stimulation with synthetic “long” peptides-loaded dendritic cells; from a single mouse (left) and all double-cytokine producing summarized (right). Where indicated, statistical significance was determined with student’s t test.

Figure 4.

Dominant Rpl18-specific CD8⁺ populations in immunologically protected mice through PD-L1 checkpoint blockade therapy. (a) Mice bearing palpable tumors 6 days after inoculation with MC-38 tumor cells were injected thrice with anti-PD-L1 in a three-day interval to induce complete tumor control. At eighty days post inoculation, mice were re-challenged with MC-38 to confirm immunological protection. Tumor-specific T cells were expanded through co-culture with irradiated MC-38 tumor cells for one week and analyzed by peptide-loaded dendritic cell stimulation o/n (n = 6). (b) A shortened treatment schedule was used to analyze Rpl18-specific T cells in mice during tumor regression (left panel). At twelve days post challenge, lymphocytes specific for Rpl18 neo-epitope were observed in spleens and LN’s through tetramer staining (middle panel) and ICS (right panel). Anti-PD-L1 treatment increased Rpl18-specific T cell frequencies in both lymphoid organs (n = 7, statistical significance was determined with student’s t test, representative results of two independent experiments).

Figure 5.

Specificity to mutated peptide and direct recognition of tumor cells. (a) Minimal synthetic neo-peptide was compared to N- and C-terminally extended “long” peptides in intracellular cytokine staining of activated T cells upon coculture with peptide-loaded dendritic cells. (n = 6, error bars depict SEM) (b) Rpl18-specific T cell bulks were stimulated with either mutated or wild-type extended peptides to determine specificity to the single amino-acid mutation. (c) In vitro recognition of live MC-38 vs B16F10 melanoma control tumor cells by tetramer-stained T cells in Rpl18+ (5 out of 6 original bulks) and Adpgk+ (3 out of 6) cultures. A representative gating-plot of a single culture is shown (top). Tumor-specific single and double cytokine production for both Adpgk and Rpl18-specific T cells is shown below.

Figure 6.

Therapeutic vaccination with Rpl18 neo-peptide-based vaccinations improves tumor control. (a) Schematic representation of therapeutic vaccination protocol. 3 × 10⁵ tumor cells were subcutaneously injected in the right flank. Eight days post inoculation, tumors established and a single synthetic neo-peptide vaccination was given intradermally at the tail-base. (b) Therapeutic vaccination with 10 nmol of extended synthetic peptide harboring neo-epitopes of Adpgk or Rpl18 delayed outgrowth of MC-38 tumors. At day 20, average tumor size in Adpgk-vaccinated mice was two-fold reduced (279 vs. 563 mm³, P = 0,1894), while Rpl18 vaccination reduced tumor size nearly 10-fold (60 mm³, P = 0,0163). This trend was maintained 22 days post inoculation (Adpgk: 574 vs. 817 mm³, P = 0,4098; Rpl18: 102 vs. 817 mm³, P = 0,0083). Subsequent median survival was extended in Rpl18 vaccinated mice (37 vs 23,5 days, P = 0,0445), while no significant extension was observed for Adpgk-vaccinated mice (26,5 days, P = 0,4244). (Eight mice per group; error bars indicate SEM; multiple t tests and Gehan-Breslow-Wilcoxon test were used for statistical analysis of tumor size and survival, respectively) (representative results of two independent experiments). (c) To improve vaccination efficacy we conjugated the Rpl18 neo-peptide to Upam TLR2-ligand (also known as Amplivant®). Therapeutic vaccination with 10 nmol of Rpl18-conjugate against established tumor significantly improved tumor control and survival compared to Upam-control (P = 0,0174; n = 8 vs n = 7 in control group; log rank tested) (depicted results are from a single experiment).