Neoantigen-specific T cell help outperforms non-specific help in multi-antigen DNA vaccination against cancer

Abstract

CD4⁺ T helper antigens are essential components of cancer vaccines, but the relevance of the source of these MHC class II-restricted antigens remains underexplored. To compare the effectiveness of tumor-specific versus tumor-unrelated helper antigens, we designed three DNA vaccines for the murine MC-38 colon carcinoma, encoding CD8⁺ T cell neoantigens alone (noHELP) or in combination with either "universal" helper antigens (uniHELP) or helper neoantigens (neoHELP). Both types of helped vaccines increased the frequency of vaccine-induced CD8⁺ T cells, and particularly uniHELP increased the fraction of KLRG1⁺ and PD-1^low effector cells. However, when mice were subsequently injected with MC-38 cells, only neoHELP vaccination resulted in significantly better tumor control than noHELP. In contrast to uniHELP, neoHELP-induced tumor control was dependent on the presence of CD4⁺ T cells, while both vaccines relied on CD8⁺ T cells. In line with this, neoHELP variants containing wild-type counterparts of the CD4⁺ or CD8⁺ T cell neoantigens displayed reduced tumor control. These data indicate that optimal personalized cancer vaccines should include MHC class II-restricted neoantigens to elicit tumor-specific CD4⁺ T cell help.

Keywords: DNA vaccine; MT: Regular Issue; cancer vaccine; cytolytic T lymphocytes; helper T lymphocytes; immunotherapy; neoantigens; personalized medicine; synthetic DNA.

Graphical abstract

Figure 1

Multi-antigen DNA vaccines induce functional T cell responses against MHC class I- and MHC class II-restricted (neo)antigens (A) Schematic representation of the coding sequences of multi-neoantigen vaccines noHELP, uniHELP, and neoHELP. (B–E) Mice were vaccinated with the indicated vaccines three times, at 3-week intervals. Ten days after the final vaccination, spleen cells were cultured with dendritic cells loaded with indicated peptides for 5 h, and analyzed by intracellular cytokine staining (ICS). IL-2-, TNF-, and/or IFN-γ-positive CD4⁺ or CD8⁺ T cells upon stimulation with the indicated individual long synthetic peptides: (B) OVA24, (C) Irgq, Adpgk, Cpne1, Rpl18,^, (D) Ddr2, Pcdh18, Zmiz1, (E) TTFCp30, PADRE, HIV-nef58-68 (nef). Colors indicate T cells secreting a single cytokine (pastel color), two cytokines (dark color), or three cytokines (black). Data are derived from a single experiment with 5 (mock, noHELP, uniHELP) or 10 (neoHELP) mice per group, representative of 2 independent experiments (shown in Figures S3D–S3F). Dots represent individual values, bars and whiskers represent means and standard errors (SEM), respectively. Data were analyzed by a two-way ANOVA test followed by Tukey’s multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 2

CD4 helper antigens increase the frequency of vaccine-induced CD8⁺ T cells in blood (A) Schematic overview of the experiment: mice were vaccinated intradermally three times, at 3-week intervals, with the indicated vaccines. Mice were bled at the indicated time points to quantify antigen-specific CD8⁺ T cells by flow cytometry (Figures S2A and S2B). (B) Kinetics of OVA-specific CD8⁺ T cell responses in blood. Data are derived from a single experiment with 10 mice per group, representative of 3 experiments. Bars and whiskers represent means and standard errors (SEM), respectively. Data were analyzed in mixed-effects analysis followed by Tukey’s multiple comparisons test comparing noHELP, neoHELP, and uniHELP. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (C) OVA-, (D) Adpgk-, (E) Irgq-specific CD8⁺ T cell percentages in blood at the peak of the tertiary response, 52 days post primary vaccination. Data in (C)–(E) are from 3 independent experiments with 50 mice per group in total. Dots represent individual values, while bars and whiskers represent means and standard errors (SEM). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test comparing noHELP, neoHELP, and uniHELP. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 3

CD4 helper antigens promote phenotypic changes in vaccine-induced CD8⁺ T cells associated with improved effector function Mice were vaccinated intradermally three times, at 3-week intervals as indicated in Figure 2. Mice were bled at the peak of the T cell response following the third vaccination (day 52), after which expression of the indicated markers on OVA-specific CD8⁺ T cells was measured by flow cytometry. (A) Histograms show normalized counts of cells expressing surface markers CD25, CX3CR1, KLRG1, PD-1, and LAG3 on total CD8⁺ T cells of mock-vaccinated mice or OVA-specific CD8⁺ T cells, identified by Kb/OVA-tetramer staining, in mice vaccinated with noHELP, neoHELP, or uniHELP (Figures S2C and S2D). Marker expression on (B) OVA-specific CD8⁺ T cells or (C) tetramer-negative CD8⁺ T cells of mock-, noHELP-, neoHELP-, or uniHELP-vaccinated mice. Expression was quantified as mean fluorescence intensity (MFI) or percentage of marker-positive cells, as indicated. Dots represent individual values, bars and whiskers represent mean and standard error (SEM). Data were derived from a single experiment, representative of three experiments, with 10 mice per group. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test comparing noHELP, neoHELP, and uniHELP. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (D) CX3CR1^hi, (E) KLRG1, or (F) PD-1 expression on OVA-specific CD8⁺ T cells monitored in blood over time on days 13, 31, and 52 post primary vaccination. Data are derived from a single experiment, representative of three, with 10 (noHELP) or 30 (neoHELP and uniHELP) mice per group. Data from tetramer-negative (TM⁻) CD8⁺ T cells from all experimental groups were pooled and plotted as CD8 as background reference. Dots and whiskers represent means and standard errors (SEM), respectively. Data were tested in mixed-effects analysis followed by Tukey’s multiple comparisons test comparing noHELP, neoHELP, and uniHELP. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. nd, no OVA-specific T cells detected.

Figure 4

Tumor-derived, but not tumor-unrelated, CD4 helper antigens improve tumor control Mice were vaccinated intradermally with the indicated vaccines three times, at 3-week intervals, followed by subcutaneous injection of MC-38 colon carcinoma cells after another 3 weeks. (A) Tumor volumes of individual mice were tracked over time. Mice were euthanized when volumes exceeded 1,000 mm³. Numbers of tumor-free mice at day 70 post challenge are indicated. (B) Survival of mice was plotted over time. Data are derived from 2 independent experiments with 20 mice per experimental group in total. Statistical significance was determined using the Gehan-Breslow-Wilcoxon test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 5

Neoantigen-specific CD4⁺ and CD8⁺ T cells both contribute to tumor control (A) Schematic representation of the coding sequences of the neoantigen vaccines. Wild-type counterparts of neoantigen sequences in neoHELP_CD4wt and neoHELP_CD8wt are indicated in white. Mice (10 per group) were injected three times, at 3-week intervals, with these vaccines. Three weeks after the final vaccination (day 63), mice were injected subcutaneously with MC-38 colon carcinoma cells. Additional neoHELP and uniHELP groups were depleted of CD4⁺ or CD8⁺ T cells by intraperitoneal injection of depleting antibodies around the time of tumor challenge (days 57, 61, and 64). Tumor size was tracked (Figure S4) and mice were euthanized if it exceeded 1,000 mm³. (B–E) For clarity, survival is shown in four separate panels, depicting mice vaccinated with (B) mock, uniHELP, neoHELP, (C) mock, neoHELP, neoHELP_CD4wt, neoHELP_CD8wt, (D) mock, uniHELP, uniHELP + CD4⁺ T cell depletion, uniHELP + CD8⁺ T cell depletion, or (E) mock, neoHELP, neoHELP + CD4⁺ T cell depletion, neoHELP + CD8⁺ T cell depletion. Statistical significance was determined using the Gehan-Breslow-Wilcoxon test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.