Vaccination with High-Affinity Epitopes Impairs Antitumor Efficacy by Increasing PD-1 Expression on CD8+ T Cells

Abstract

Antitumor vaccines encoding self-antigens generally have low immunogenicity in clinical trials. Several approaches are aimed at improving vaccine immunogenicity, including efforts to alter encoded epitopes. Immunization with epitopes altered for increased affinity for the major histocompatibility complex (MHC) or T-cell receptor (TCR) elicits greater numbers of CD8 T cells but inferior antitumor responses. Our previous results suggested that programmed death 1 (PD-1) and its ligand (PD-L1) increased on antigen-specific CD8 T cells and tumor cells, respectively, after high-affinity vaccination. In this report, we use two murine models to investigate whether the dose, MHC affinity, or TCR affinity of an epitope affected the antitumor response via the PD-1/PD-L1 axis. T cells activated with high-affinity epitopes resulted in prolonged APC:T-cell contact time that led to elevated, persistent PD-1 expression, and expression of other checkpoint molecules, in vitro and in vivo Immunization with high-affinity epitopes also decreased antitumor efficacy in the absence of PD-1 blockade. Thus, APC:T-cell contact time can be altered by epitope affinity and lead to therapeutically relevant changes in vaccine efficacy mediated by changes in PD-1 expression. These findings have implications for the use of agents targeting PD-1 expression or function whenever high-affinity CD8 T cells are elicited or supplied by means of vaccination or adoptive transfer. Cancer Immunol Res; 5(8); 630-41. ©2017 AACR.

Figure 1. Increasing the MHC I affinity of SSX2-p103 resulted in increased activation, expansion, and PD-1 expression

A) T2 cells were incubated with the indicated peptides for six hours, stained for HLA-A2, and analyzed by flow cytometry. HLA-A2 MFI is displayed as the fold change over cells that were incubated with a non-specific peptide B) Mice were immunized with each peptide and after one week the spleens were collected. The percentage of CD8 T cells that were positive for SSX2-p103-tetramer staining was determined. C) Splenocytes from immunized mice were stimulated with the native SSX2-p103 peptide, and 4-1BB expression and intracellular cytokine staining for one or more T_h1 (IFNγ, TNFα, IL2) cytokines, was assessed by flow cytometry. D) PD-1 amounts on the tetramer⁺ cells from B. APL abbreviations are shown in Table 1. Letters along the X axis indicate AA changes in the SSX2-p103 sequence as indicated in Table 1. Nat = SSX2-p103 native sequence, Flu = HLA-A2 restricted flu epitope positive control. Results shown are representative of two independent experiments with N=6 replicates each, *= P < 0.05, two-way ANOVA with Bonferroni’s posttest. Representative flow cytometry gating strategy shown in Supplementary Fig. S1.

Figure 2. Amino acid substitutions in the SIINFEKL (OVA) peptide affect MHC-I or TCR affinity

A) TAP-deficient RMA-S cells were incubated with the indicated peptides (Table 1) for six hours, at which time the cells were stained for MHC-I and analyzed by flow cytometry. MHC-I MFI is displayed as the fold change over cells that were incubated with a non-specific peptide. B) OT-1 mouse splenocytes were incubated with the indicated peptides and cytokine expression was assessed by intracellular cytokine staining. The number of cells expressing IL2 and/or TNFα and/or IFNγ is shown as the percent of total CD8 T cells. Highlighted peptides were selected for further analysis. The letters along the x axis indicate AA changes in the SIINFEKL sequence as indicated in Table 1, ex. IP-FT = SIPNTEKL. No = vehicle control, NS = non-specific peptide control, SSX2 p103–111 (RLQGISPKI), PMA = PMA and ionomycin positive control. Results shown are representative of two independent experiments with N=6 replicates each, *= P < 0.05, two-way ANOVA followed by Bonferroni’s posttest correction. Representative flow cytometry gating strategy shown in Supplementary Fig. S2.

Figure 3. Varying the MHC-I affinity, TCR affinity or antigen dose alters the phenotype of CD8 T cells ex vivo

Mouse splenocytes (OT-1) were collected and immediately stimulated with SIINFEKL (OVA) or one of the variants that alters the MHC-I affinity (top), TCR affinity (middle) or different concentrations of SIINFEKL (bottom). The CD3 (TCR) levels, activation, and checkpoint receptor expression of CD8 T cells was monitored every 24 hours for 96 hours. Results are representative of two independent experiments with N=3 mice each. NS = non-specific peptide, SSX2 p103–111 (RLQGISPKI). Statistical analyses compare OVA to FT (top), OVA to NG (middle), and 2µg/mL to 0.1ng/mL OVA (bottom). For CD3 levels, OVA, FT, NG and 0.1ng/mL OVA were compared to NS. *= P < 0.05, two-way ANOVA followed by Bonferroni correction. Representative flow cytometry gating strategy shown in Supplementary Fig. S3.

Figure 4. Vaccination with high-affinity peptides led to persistent PD-1 and LAG3 expression in vivo

A) C57BL/6 mice received 2×10⁶ OT-1 cells by adoptive transfer and the next day were immunized with 100µg OVA, 100µg of one of the variants, or 0.1 µg (low dose) of OVA with Freund’s adjuvant (top, CFA) or alone in PBS (bottom, vehicle). Spleens were harvested from individual groups every two days for six days. The MFI of indicated receptors was assessed on SIINFEKL-tetramer⁺ CD45⁺ CD8⁺ T cells directly ex vivo. B) C57BL/6 mice were immunized as in panel A with 100µg FT, OVA, or NS control in CFA. Expression of 4-1BB (activation), PD-1, and LAG3 were assessed on splenocytes on days 2, 7, 14 and 28. Results shown are representative of two independent experiments with N=5 mice/group. Statistical comparisons are made between groups receiving OVA and FT. *= P < 0.05, two-way ANOVA with Bonferroni correction. (NS = non-specific peptide, SSX2 p103–111 (RLQGISPKI)). Representative flow cytometry gating strategy shown in Supplementary Fig. S4.

Figure 5. APC:T-cell contact time corresponds to antigen affinity or dose

Top) APCs were stained with CellTracker red (CMTPX), incubated with the indicated peptides or various concentrations of OVA (ng/mL), and allowed to incubate for one hour. Isolated OT-1 T cells stained with CellTracker green (CMFDA) were added to the peptide loaded APCs. Upon addition of the T cells, 20× time-lapse microscopy was conducted for one hour at one minute intervals to monitor APC:T-cell contact times. Images are representative of cells interacting for the mean time observed with the respective peptide. Color was removed from the surrounding cells for ease of tracking. Bottom) Contact time for each peptide was quantified. Prior to imaging, cells were removed, allowed to incubate together for 72 hours and analyzed by flow cytometry. Association between contact time and peptide affinity was determined via two-sided Kendall’s rank correlation. NS = non-specific peptide, SSX2 p103–111 (RLQGISPKI). * = P < 0.05, two-way ANOVA followed by Bonferroni correction. For contact time calculations, N=30 APC:T-cell interactions were evaluated for each experimental condition. For MFI of 4-1BB, PD-1 and LAG3 expression in the bottom panels, data shown is for 6-well replicates. Representative flow cytometry gating strategy shown in Supplementary Fig. S5.

Figure 6. Moderate affinity vaccine antigens elicited greatest antitumor response

Wild-type C57BL/6 (n=5) mice were injected with PD-L1 transduced E.G7-OVA tumor cells. When tumors were palpable (day 14), 2 × 10⁶ OT-1 T cells were adoptively transferred into the mice. The following day mice were immunized with the indicated peptides or 0.1 µg (low dose) of OVA in CFA; PD-1 blocking antibodies were given the day following vaccination. A) Tumor growth was monitored over time. Complete tumor responses were observed in 2/5 animals receiving FT, 1/5 animals receiving NG, and 2/5 animals receiving OVA+αPD1. On day 40, tumors were removed and analyzed by flow cytometry. B) The number CD8⁺ TIL was calculated as a percent of CD45⁺ cells. C) PD-L1 was assessed on the CD45⁻ population. D) Activation and immune checkpoint expression was assessed on CD45⁺CD3⁺CD4⁻CD8⁺ TIL. NS = non-specific peptide, SSX2 p103–111 (RLQGISPKI). Results are representative of four independent experiments, N=5 mice each. * = P < 0.05 two-way ANOVA with Bonferroni’s posttest correction. Representative flow cytometry gating strategy shown in Supplementary Fig. S6.