Combination central tolerance and peripheral checkpoint blockade unleashes antimelanoma immunity

Abstract

Blockade of immune checkpoint proteins (e.g., CTLA-4, PD-1) improves overall survival in advanced melanoma; however, therapeutic benefit is limited to only a subset of patients. Because checkpoint blockade acts by "removing the brakes" on effector T cells, the efficacy of checkpoint blockade may be constrained by the limited pool of melanoma-reactive T cells in the periphery. In the thymus, autoimmune regulator (Aire) promotes deletion of T cells reactive against self-antigens that are also expressed by tumors. Thus, while protecting against autoimmunity, Aire also limits the generation of melanoma-reactive T cells. Here, we show that Aire deficiency in mice expands the pool of CD4+ T cells capable of melanoma cell eradication and has additive effects with anti-CTLA-4 antibody in slowing melanoma tumor growth and increasing survival. Moreover, pharmacologic blockade of central T cell tolerance and peripheral checkpoint blockade in combination enhanced antimelanoma immunity in a synergistic manner. In melanoma patients treated with anti-CTLA-4 antibody, clinical response to therapy was associated with a human Aire polymorphism. Together, these findings suggest that Aire-mediated central tolerance constrains the efficacy of peripheral checkpoint inhibition and point to simultaneous blockade of Aire and checkpoint inhibitors as a novel strategy to enhance antimelanoma immunity.

Keywords: Cancer immunotherapy; Cellular immune response; Immunology; Melanoma.

Figure 1. Association between Aire and antimelanoma effects of CTLA-4 blockade in mice and humans.

(A and B) WT and Aire^GW/+ mice were s.c. injected with 1 × 10⁵ B16 melanoma cells followed by anti–CTLA-4 (aCTLA-4) antibody or isotype control (iso) antibody treatment. Mice in each group were followed for B16 melanoma tumor growth and survival. n = 7–12 per group, cumulative of at least 2 independent experiments. Mann-Whitney U test. *P < 0.05. (C and D) Comparison of progression-free survival (PFS) and overall survival (OS) probability between patients with Aire SNP rs1055311 (TT versus CT/CC).

Figure 2. Aire deficiency increases CD4⁺ T cell cytolytic function to reduce melanoma growth.

CD4⁺ splenocytes were transferred from WT and Aire^GW/+ donors into RAG^–/– recipients, followed by s.c. B16 melanoma injection on day 7. Recipients were followed for tumor growth and survival. (A and B) B16 melanoma tumor growth and survival was measured after B16 inoculation in recipients; n = 15 per group. Mann-Whitney U test. *P < 0.05, **P < 0.01. (C–E) Tumor-infiltrating lymphocytes (TIL) were harvested on day 19 following B16 melanoma inoculation in recipients of either WT or Aire^GW/+ CD4⁺ splenocytes. Absolute numbers of CD4 tumor-infiltrating cells are shown in C. Two-tailed t test. Representative flow cytometry plots and average cumulative frequencies of Ki67⁺ (D) and KLRG1⁺ and granzyme B⁺ (E), among CD4⁺ T cells. Two-tailed t test. *P < 0.05, ****P < 0.0001. (F) Representative flow cytometry histograms and average absolute CellTrace Violet–labeled B16 cell numbers after coincubation with WT and Aire^GW/+ CD4⁺ T cells. Two-tailed t test. *P < 0.05. (G) Average relative B16 cell numbers (log₂) after coincubation with CD4⁺ T cells from WT and Aire^GW/+ mice, along with pan-granzyme inhibitor (3, 4 Dichloroisocoumarin). One-way ANOVA and two-tailed t tests, with P values adjusted using Hommel’s correction for multiple comparisons. *P < 0.05.

Figure 3. Additive effect of Aire deficiency and anti–CTLA-4 antibody on T cell responses.

(A and B) Tumor-infiltrating lymphocytes (TIL) were harvested on day 19 following B16 melanoma inoculation in WT and Aire^GW/+ mice treated with aCTLA-4 or isotype control antibody (iso). Representative flow cytometry plots and average cumulative frequencies of Ki67⁺ (A) and KLRG1⁺ and granzyme B⁺ (B) among CD4⁺ T cells. n = 9–12 in each group. One-way ANOVA and two-tailed t tests with P values adjusted using Hommel’s correction for multiple comparisons. *P < 0.05, **P < 0.01. (C) ELISPOT with splenocytes harvested on day 14 following B16 melanoma inoculation in WT and Aire^GW/+ mice treated with anti–CTLA-4 antibody (aCTLA-4) or iso. Cumulative spot forming cells (SFC)/5×10⁵ cells secreting IL-2 with OVA and TRP-1 was analyzed. Each data point represents an individual animal. One-way ANOVA and two-tailed t tests with P values adjusted using Hommel’s correction for multiple comparisons. *P < 0.05.

Figure 4. Additive antimelanoma effect of Aire deficiency and anti–CTLA-4 antibody in TRP-1 CD4⁺ TCR Tg mice.

(A and B) Aire sufficient (WT) and Aire^GW/+ TRP-1 TCR Tg mice were s.c. injected with 1 × 10⁵ B16 melanoma cells followed by anti–CTLA-4 antibody (aCTLA-4) or isotype control antibody (Iso) treatment, as outlined. Mice in each group were followed for B16 melanoma tumor growth (A) and survival (B). n = 7–12 per group, cumulative of at least 2 independent experiments. Mann-Whitney U test. *P < 0.05. (C) Examples of periporbital and tail vitiligo in Aire^GW/+ mice treated with aCTLA-4 antibody.

Figure 5. Aire deficiency does not exacerbate the development of experimental autoimmune colitis associated with anti–CTLA-4 antibody treatment.

(A) Percent initial body weight of RAG^–/– recipients after transfer of WT or Aire^GW/+CD4⁺CD25^–CD45RB^hi splenocytes. After transfer, recipients were treated with anti–CTLA-4 antibody (aCTLA-4), untreated, or treated with isotype control antibody (iso). Cumulative data from 2 independent experiments are shown. *P < 0.05 comparing aCTLA-4 antibody treatment versus untreated/iso treatment in recipients of WT cells. (B) Representative H&E-stained sections of descending colons and (C) average histopathological scores of colons from recipients of CD4⁺CD25^–CD45RB^hi splenocytes derived from WT and Aire^GW/+ mice and administered aCTLA-4 or untreated/iso (-). Gray arrowheads, immune cell infiltration in lamina propria; black arrowheads, immune cell infiltration in the submucosa. One-way ANOVA and two-tailed t tests with P values adjusted using Hommel’s correction for multiple comparisons. *P < 0.05.

Figure 6. Combination aRANKL and aCTLA-4 antibody administration enhances immune rejection of melanoma.

(A) Schematic of treatment regimen, in which anti-RANKL (aRANKL) or isotype control (iso) antibody was injected every other day for 11 days, followed by s.c. B16 melanoma injection (2 × 10⁴ cells) on day 18. GVAX with or without anti-CTLA-4 (aCTLA-4) antibody was given on days 3, 6, and 10 following melanoma injection. (B) Survival curves in GVAX-treated mice receiving indicated combinations of therapy. n = 10 for each group. Mann-Whitney U test. *P < 0.05. Tumor-infiltrating lymphocytes (TIL) were harvested on day 19 following 1 × 10⁵ B16 melanoma cell inoculation. Representative flow cytometry plots and average cumulative frequencies of Ki67⁺ (C) and KLRG1⁺ and granzyme B⁺ (D) among CD4⁺ T cells was measured. n = 9–12 in each group. One-way ANOVA and two-tailed t tests with P values adjusted using Hommel’s correction for multiple comparisons. *P < 0.05.