CD4 T cell-activating neoantigens enhance personalized cancer vaccine efficacy

Abstract

Personalized cancer vaccines aim to activate and expand cytotoxic antitumor CD8+ T cells to recognize and kill tumor cells. However, the role of CD4+ T cell activation in the clinical benefit of these vaccines is not well defined. We previously established a personalized neoantigen vaccine (PancVAX) for the pancreatic cancer cell line Panc02, which activates tumor-specific CD8+ T cells but required combinatorial checkpoint modulators to achieve therapeutic efficacy. To determine the effects of neoantigen-specific CD4+ T cell activation, we generated a vaccine (PancVAX2) targeting both major histocompatibility complex class I- (MHCI-) and MHCII-specific neoantigens. Tumor-bearing mice vaccinated with PancVAX2 had significantly improved control of tumor growth and long-term survival benefit without concurrent administration of checkpoint inhibitors. PancVAX2 significantly enhanced priming and recruitment of neoantigen-specific CD8+ T cells into the tumor with lower PD-1 expression after reactivation compared with the CD8+ vaccine alone. Vaccine-induced neoantigen-specific Th1 CD4+ T cells in the tumor were associated with decreased Tregs. Consistent with this, PancVAX2 was associated with more proimmune myeloid-derived suppressor cells and M1-like macrophages in the tumor, demonstrating a less immunosuppressive tumor microenvironment. This study demonstrates the biological importance of prioritizing and including CD4+ T cell-specific neoantigens for personalized cancer vaccine modalities.

Keywords: Cancer immunotherapy; Immunology; MHC class 2; Oncology; T cells.

Figure 1. PancVAX plus ICIs elicit antitumoral CD8⁺ T cell immunity with minimal effect on CD4⁺ T cells.

C57BL/6 mice were implanted with 1 × 10⁶ Panc02s s.c. and vaccinated with PancVAX, s.c. tail base on days 12 and 19. At day 12, mice received 100 µg anti–PD-1, i.p., or isotype twice weekly with 2 doses of 100 µg anti–CTLA-4 or isotype, i.p., on days 15 and 19. (A) Tumor volumes were measured in mice treated with isotype (gray), anti–CTLA-4, and–PD-1 (blue), PancVAX with isotype (yellow), and PancVAX with anti–CTLA-4 and PD-1 (magenta). Shaded regions represent data as mean ± SEM. P values derived from 2-way ANOVA with Tukey’s multiple-comparison test (****P ≤ 0.0001). (B) Survival analysis. P values derived from Log-rank (Mantel-Cox) (***P ≤ 0.001, ****P ≤ 0.0001). (C) Uniform manifold approximation plot (UMAP) of 6,423 T cells from Panc02 tumors (day 35). Cells annotated as cycling T cells (pink), cytotoxic CD8⁺ T cells (teal), effector CD4⁺ T cells (gold), exhausted CD8⁺ T cells (orange), naive CD8⁺ T cells (blue), and regulatory T cells (olive). UMAP of distribution of T cell populations across groups: Isotype control (gray), anti–CTLA-4, and anti–PD-1 (blue), PancVAX with isotype (yellow), and PancVAX with anti–CTLA-4 and PD-1 (magenta). (D) Stacked bar plot of the proportions of T cell phenotype across groups. (E) Volcano plots of MAST tests for differential expression between Isotype control- and PancVAX-treated, PancVAX + Isotype–treated, and PancVAX + anti–PD-1 + anti–CTLA-4–treated CD8⁺ T cells in total (top), exhausted CD8⁺ T cells (middle), and CD4⁺ T cells (bottom). Genes with significant FDR-adjusted P values (adjusted P < 0.05) and average log₂-fold changes (|log₂FC| > 0.5) are colored red.

Figure 2. Murine pancreatic cancer MHCII predicted neoantigens are immunogenic in non–tumor-bearing mice.

(A) Pipeline to identify predicted MHCII restricted neoantigens that stimulate a CD4⁺ T cell response in vivo. (B) Non–tumor bearing C57BL/6 mice were vaccinated with 20 mer SLPs predicted to bind to H2-IA^b on days 0 and 7 (n = 3). Splenocytes from vaccinated mice were pooled on day 14, and CD4⁺ T cells were isolated using magnetic bead negative isolation kits. Isolated CD4⁺ T cells were cocultured overnight at a 1:1 ratio with I-A^b–expressing T-2 APCs pulsed with MHCII-specific peptides in an IFN-γ capture plate and assessed for reactivity by ELISpot the following day. The 20 mer neoepitopes that produced a significant immune response in the CD4 vaccinated group over the untreated group are displayed. Symbols represent technical replicates. To confirm CD4 and CD8 T cell specificity to each peptide, non–tumor-bearing C57BL/6 mice were vaccinated with PancVAX2 on days 0 and 7 (n = 4). (C and D) Splenocytes from vaccinated mice were isolated and pooled on day 14, and CD4⁺ and CD8⁺ T cells were separately sorted using magnetic bead negative isolation kits and cocultured overnight at a 1:1 ratio with matured murine BMDCs that had been pulsed with 5 μg/mL MHCI-specific (C) or MHCII-specific (D) peptides in an IFN-γ capture plate and assessed for reactivity by ELISpot the following day. Data are shown as mean ± SD. Significance was calculated by 2-way ANOVA followed by Sidak’s multiple-comparison test. *P ≤ 0.05, **P ≤ 0.01,****P ≤ 0.0001. Significance values for CD8 T cell cultures (black asterisks) and CD4 T cell cultures (blue asterisks) are shown.

Figure 3. PancVAX2 improves control of tumor growth and long-term survival associated with an increase in tumor infiltrating T cells.

(A) C57BL/6 mice were implanted with 3 × 10⁶ Panc02s, s.c., on day 0. Mice were vaccinated s.c. at base of tail on days 3 and 10 with PBS (n = 14) or CD4 Vaccine, CD8 Vaccine, or PancVAX2 (n = 15). (B) Average tumor volumes were plotted until day 33. Data shown as mean ± SEM. Significance on day 33 calculated by 2-way ANOVA followed by Sidak’s multiple-comparison test. (C) Spider plots of individual growth curves. Number of tumor free mice at day 70: PBS (n = 0), CD4 vaccine (n = 3), CD8 vaccine (n = 3), PancVAX2 (n = 8). (D) Kaplan-Meier survival curves. Vaccination time points shown as gray dashed line. Significance calculated by Log-rank (Mantel-Cox) test. (E) C57BL/6 mice implanted with 3 × 10⁶ Panc02s, s.c., on day 0. Mice were vaccinated s.c. on day 14 and 21 with PBS, CD4 Vaccine, CD8 Vaccine, or PancVAX2 (n = 5). On day 28, tumors were harvested, dissociated into a single-cell suspension, and analyzed by flow cytometry. Total CD4 and CD8 or activated (CD44⁺) CD4 and CD8 populations per mg tumor were quantified from the CD45⁺CD3⁺CD19^-NK1.1^– population. Symbols represent technical duplicate of individual mice. (F) C57BL/6 mice were implanted with 3 × 10⁶ Panc02s, s.c., on day 0. Mice were vaccinated s.c. on day 28 with PBS or CD4 Vaccine, CD8 Vaccine, or PancVAX2 (n = 4). On day 35, tumors were harvested, and CD8 T cells were isolated and cocultured in an IFN-γ capture plate at a 1:1 ratio with T2-H2-Kb or T2-H2-Db APCs pulsed with 5 µg/mL MHCI peptides for 24 hours. Symbols represent individual mice. Data are shown as mean ± SD. Significance was calculated by 2-way ANOVA followed by Sidak’s multiple-comparison test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 4. PancVAX2 enhances infiltration of neoantigen-specific cytotoxic CD8⁺ T cells into the tumor with a less exhausted phenotype.

C57BL/6 mice were implanted on the right flank, s.c., with 3 × 10⁶ Panc02 cells on day 0. Mice were vaccinated s.c. at the base of the tail on day 14 and 21 with CD8 vaccine or PancVAX2 (n = 5). On day 28, tumors were harvested and pooled within treatment groups and dissociated into a single-cell suspension, and CD8⁺ T cells were isolated by magnetic positive selection kit. Isolated CD8⁺ T cells were cocultured overnight at a 1:1 ratio with T2-H2-K^b APCs pulsed with 5 μg/mL MHCI-specific peptides. Cocultures were then stained for activation marker and effector cytokine expression by flow cytometry analysis. (A) Representative gating of CD8 and CD137 marker expression on tumor infiltrating T cells cocultured with T-2 APCs pulsed with OVA peptide (control) or peptide 44. (B) Quantification of CD137 upregulation on tumor infiltrating CD8⁺ T cells cocultured with immunogenic CD8 epitopes. (C) Cytokine expression (GzmB, IFN-γ, and IL-2) from activated (CD137⁺) CD8⁺ T cells that stimulated a greater response in PancVAX2-treated tumors than in CD8 vaccinated tumors. (D) PD-1 expression measured by median fluorescence intensity (MFI) on activated (CD137⁺) CD8⁺ T cells after peptide restimulation. Symbols represent technical triplicates of pooled CD8⁺ T cells. Data are shown as mean ± SD. Significance was calculated by 2-way ANOVA followed by Sidak’s multiple-comparison test. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 5. PancVAX2 recruits neoantigen-specific Th1 CD4⁺ T cells into the tumor and reduces infiltration of Tregs into the tumor.

C57BL/6 mice were implanted with 3 × 10⁶ Panc02 cells, s.c., on day 0. Mice were vaccinated s.c. at the base of the tail on day 14 and 21 with PancVAX2 (n = 5). On day 28, tumors were harvested and pooled and dissociated into a single-cell suspension, and isolated CD4 T cells were cocultured overnight at a 1:1 ratio with T2-H2-IA^b APCs pulsed with 5 µg/mL MHCII peptides. (A) Representative gating of CD4 and CD137 expression from intratumoral CD3⁺ cells after cocultured with OVAII peptide (control) or peptide 43. (B) Quantification of CD4⁺CD137⁺ T cells following restimulation with each MHCII-specific peptide. (C–G) Activated (CD137⁺) CD4 T cells were then assessed for Th1-like cytokine expression (IFN-γ, IL-2, and TNF-α) (C), cytotoxic effector cytokine expression (GzB) (D), Th2-like cytokine expression (IL-4) (E), Th17-like cytokine expression (IL-17) (F), and Treg activity (IL-10) (G). Symbols represent technical triplicates from pooled CD4 T cells. Data are shown as mean ± SD. Significance was calculated by 2-way ANOVA followed by Sidak’s multiple-comparison test. (H) C57BL/6 mice were implanted on the right flank, s.c, with 3 × 10⁶ Panc02 cells on day 0. Mice were vaccinated s.c. at the base of the tail on day 14 and 21 with CD4 Vaccine or CD8 Vaccine (n = 4). On day 28, tumors were harvested, dissociated into a single-cell suspension, and stained for flow cytometry analysis. CD45⁺CD3⁺CD4⁺ populations were assessed for the proportion of cells expressing transcription factors T-bet (Th1, left) or GATA3 (Th2, right). (I) Proportion of FOXP3⁺CD4⁺ T cells (left) and the ratio of CD45⁺CD3⁺CD8⁺ T cells to FOXP3⁺CD4⁺ T cells (right). Symbols represent technical duplicate of individual mice. Data are shown as mean ± SD. Significance was calculated by a 2-tailed unpaired t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 6. PancVAX2 induces proinflammatory TME changes in the myeloid population.

C57BL/6 mice were implanted on the right flank, s.c., with 3 × 10⁶ Panc02 cells on day 0. Mice were vaccinated s.c. at the base of the tail on day 14 and 21 with PBS, CD4 vaccine, CD8 vaccine, or PancVAX2 (n = 5). On day 28, tumors were harvested, dissociated into a single-cell suspension, and stained for flow cytometry analysis. (A) Representative gating strategy for intratumoral myeloid cell populations. From the live CD45⁺ population, CD3⁺ or CD19⁺ cells were excluded. To identify DCs, MHCII⁺CD11c⁺ cells were gated on from the F4/80^– population of the CD3^–CD19^– gate. Macrophages were gated as CD11b⁺F4/80⁺ cells from the CD3^–CD19^– gate. Of the F4/80^– population Ly6C^hi M-MDSCs and Ly6G⁺Ly6C^lo G-MDSCs were identified. (B) Quantification of each myeloid subpopulation per mg tumor for each vaccine group. (C–F) To evaluate the proinflammatory or tumor suppressive nature of each myeloid subpopulation, the ratio of CD86/PD-L1 expression by MFI was determined on DCs (C), G-MDSCs (D), M-MDSCs (E), and macrophages (left) or the M1/M2 ratio of tumor infiltrating macrophages (right) (F) was determined by evaluating the proportion of CD86⁺ to CD206⁺ macrophages. Symbols represent technical duplicate of individual mice (n = 5). Data are shown as mean ± SD. Significance was calculated by 2-way ANOVA followed by Sidak’s multiple-comparison test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.