NKG2A Blockade Potentiates CD8 T Cell Immunity Induced by Cancer Vaccines

Abstract

Tumor-infiltrating CD8 T cells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High-dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine whether NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1^b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1^b axis even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 T cells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.

Keywords: CD8 T cells; HLA-E; IFN-γ; NKG2A; Qa-1; acquired resistance; cancer vaccines; immune checkpoints; mouse tumor models; natural killer cells.

Figure 1:. Expression of the inhibiting NKG2A receptor is associated with worse clinical outcome

(A) Flow cytometry plots displaying the expression of NKG2A and co-receptor CD94 on TIL. (B) Pairwise proportions of NKG2A⁺ cells in PBMCs and matched TIL in head and neck squamous cell carcinoma (HNSCC) biopsies (n=19), paired Student’s t-test). (C) Frequencies of NKG2A⁺ cells in TIL subsets from HPV16 negative HNSCC (HPV-, n=5), HPV16-positive tumours without ex vivo immune reactivity (IR-, n=5) and HPV16-positive tumours displaying ex vivo immune reactivity to HPV16 (IR+, n=8). Means and SEM, one-way ANOVA with Tukey’s multiple comparison test (n=18). (D) KaplanMeier survival curves of HPV16+ HNSCC from the TCGA database (n=75). Interaction plots for transcript levels of the NKG2A-coding gene KLRC1 or HLA-E in either CD8 high and low subgroups or NCR1 (coding NKp46) high and low subgroups. Log-rank test. See also Figure S1.

Figure 2:. CD8 T cells that express NKG2A belong to CD103⁺ early effector tissue-resident cells

(A) Co-expression flow cytometry analysis of inhibitory receptors on CD8 TIL in HNSCC biopsies. Paired Student’s t-test. (B) T cell differentiation stage of NKG2A-positive CD8 TIL was determined by combined expression of these markers: naive (CCR7⁺ CD127^-), central memory (CCR7⁺ CD127⁺), SLEC (‘short lived effector cells’: CCR7^- CD45RO⁺ KLRG1+), TEMRA (‘CD45RA⁺ effectors’: CCR7’CD45RO’), effector memory (CCR7^- CD45RO⁺ KLRG1^- CD127⁺) and effector (CCR7^- CD45RO⁺ KLRG1^- CD127^-). (C) Density viSNE plot from CyTOF data on pre-gated CD3⁺CD8⁺ TIL. (D) Ratios of marker expression (based on the Mean Signal Intensity) on NKG2A⁺ versus NKG2A^- CD8 T cells in cervical carcinomas (n=6). Each symbol represents an individual tumor sample. See also Figures S1 and S2.

Figure 3:. Therapeutic vaccination induces NKG2A on tumor-infiltrating CD8 T cells

(A) Experimental layout of the vaccination protocol in the TC-1 model. (B) Tumor growth curves of untreated and vaccinated mice. Each line represents an individual mouse. (C) NKG2A expression on CD4 T cells, CD8 T cells and NK cells of intratumoral lymphocytes in untreated (black lines) or vaccinated (red lines) at day 24. (D) Enumeration of means with SEM (n=3–5 per group) of NKG2A⁺ lymphocytes. One way ANOVA with Dunnett’s multiple comparisons test. (E) Venn diagrams of flow cytometry results showing co-expression on CD8 T cells in the tumor. Means of 3–5 mice per group are depicted from one of three experiments with similar results. See also Figure S3.

Figure 4:. NKG2A blockade, but not PD-1 blockade, delays tumor relapse after peptide vaccination

(A) Progression free survival (PFS) time of mice bearing TC-1 tumors treated with vaccination and PD-1 blocking antibody. Means and SEM during the relapse phase and (B) therapy response rates according to RECIST criteria are plotted (NR, no response; PR, partial response; CR, complete response). (C) Survival curves of this experiment (n=8 per group). (D) PFS time of mice bearing TC-1 tumors treated with vaccination and NKG2A blocking antibody. Means and SEM during the relapse phase, (E) therapy response rates and (F) survival curves (n=16 per group). One-way ANOVA test (A and D) and log-rank test (C and F) were used for statistical analyses. See also Figures S4 and S5.

Figure 5:. Inflammation induces Qa-1^b expression on tumor cells

(A) Vaccination with synthetic long peptide in Qa-1^b-knockout mice bearing TC-1 tumors. Survival curves from two pooled independently performed experiments are shown and analysed by log-rank test (n=16 per group). (B) Qa-1^b expression on B16, RMA and TC-1 tumor cells in vivo. Thin lines represent control staining of tumor cells. Index of geometric mean fluorescence intensity (gMFI) (Qa- 1^b staining divided by control staining) and SEM, analysed with Student’s t-test. (C) Expression of Qa- 1^b and PD-L1 on B16, RMA and TC-1 tumor cells in vitro after incubation with recombinant interferon-γ (IFNƔ). gMFI and SD of 2 pooled experiments. See also Figure S5 and S6.

Figure 6:. Genetic knockdown of Qa-1^b in tumors enhances peptide vaccination therapy

(A) Outgrowth of wild type TC-1 tumors (WT) and Qa-1^b-knockout TC-1 tumors (Qa-1^b-). Each line represents an individual mouse. Numbers indicate fraction of tumor-free animals at day 60. (B) Mean and SEM of tumor sizes. Student’s t-test. (C) Outgrowth of wild type RMA tumors (WT) and Qa-1^b-knockout RMA tumors (Qa-1^b-). (D) Survival plots of these data from two pooled independently performed experiments with similar outcome, long-rank test. (E) Outgrowth of wild type B16 tumors (WT) and Qa-1^b-knockout B16 tumors (Qa-1^b-). (F) Survival plots of these data, logrank analysis. See also Figure S7.

Figure 7:. NK cells are dispensable for the enhanced peptide vaccination effect

(A) Mice were inoculated with WT RMA tumors or Qa-1^b- tumors and vaccinated with synthetic long peptides or control long peptide. Vaccine-induced CD8 T cell responses were measured in blood using intracellular cytokine staining. Student’s t-test for statistical analysis. (B) Tumor outgrowth plots depicting RMA tumor outgrowth in individual mice in three different treatment groups. Numbers indicate fraction of tumor-free animals at day 60. (C) Survival curves of these mice (n=10 to 20 mice per group). No statistical difference was observed using log-rank analysis when NK cells were depleted.