CTLA-4 blockade enhances polyfunctional NY-ESO-1 specific T cell responses in metastatic melanoma patients with clinical benefit

Abstract

Blockade of inhibitory signals mediated by cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to enhance T cell responses and induce durable clinical responses in patients with metastatic melanoma. The functional impact of anti-CTLA-4 therapy on human immune responses is still unclear. To explore this, we analyzed immune-related adverse events and immune responses in metastatic melanoma patients treated with ipilimumab, a fully human anti-CTLA-4 monoclonal antibody. Fifteen patients were selected on the basis of availability of suitable specimens for immunologic monitoring, and eight of these showed evidence of clinical benefit. Five of the eight patients with evidence of clinical benefit had NY-ESO-1 antibody, whereas none of seven clinical non-responders was seropositive for NY-ESO-1. All five NY-ESO-1 seropositive patients had clearly detectable CD4(+) and CD8(+) T cells against NY-ESO-1 following treatment with ipilimumab. One NY-ESO-1 seronegative clinical responder also had a NY-ESO-1 CD4(+) and CD8(+) T cell response, possibly related to prior vaccination with NY-ESO-1. Among five clinical non-responders analyzed, only one had a NY-ESO-1 CD4(+) T cell response and this patient did not have detectable anti-NY-ESO-1 antibody. Overall, NY-ESO-1-specific T cell responses increased in frequency and functionality during anti-CTLA-4 treatment, revealing a polyfunctional response pattern of IFN-gamma, MIP-1beta and TNF-alpha. We therefore suggest that CTLA-4 blockade enhanced NY-ESO-1 antigen-specific B cell and T cell immune responses in patients with durable objective clinical responses and stable disease. These data provide an immunologic rationale for the efficacy of anti-CTLA-4 therapy and call for immunotherapeutic designs that combine NY-ESO-1 vaccination with CTLA-4 blockade.

Fig. 1.

Antibody responses to NY-ESO-1 before and after anti-CTLA-4 antibody therapy. Reciprocal antibody titers against Escherichia coli-derived NY-ESO-1 protein were measured by ELISA and are shown for each available time point as indicated. Antibody titers below 100 were defined as negative. Five NY-ESO-1 seropositive patients are shown, while the other 10 patients are NY-ESO-1 seronegative.

Fig. 2.

NY-ESO-1 specific CD4⁺ and CD8+ T cell responses were induced after CTLA-4 blockade. Representative intracellular cytokine and chemokine staining of both CD4⁺ and CD8+ T cells responding to NY-ESO-1 pooled peptides from NY-ESO-1 seropositive patient IMF-8 at weeks 1 and 12. Single cytokine gates were set based on negative control (unstimulated) samples and were placed consistently across samples (Bottom).

Fig. 3.

CTLA-4 blockade induced T cell responses are polyfunctional. NY-ESO-1 specific T cells secrete MIP-1β and IFN-γ (A), or TNF-α and IFN-γ (B). Samples scored as positive are indicated with * over week1 baseline. (C) Functional composition of the CD4⁺ T cell response. All possible combinations of three function responses are shown on the x axis. Responses are grouped and color-coded. (D) Each slice on the pie charts represents the fraction of the total response that is CD4⁺ T cell positive for a given number of functions; (Top) calculated from five clinical non-responders, (Bottom) from eight patients with evidence of clinical benefit.

Fig. 4.

Polyfunctional NY-ESO-1 antigen-specific T cells secreted higher levels of IFN-γ after anti-CTLA-4 antibody treatment. (A) IFN-γ fluorescence of CD4⁺ (Top) and CD8⁺ (Bottom) T cells with different functional properties before and after anti-CTLA-4 antibody treatment. Representative example from patient IMF-8. (B) Compiled IFN-γ MFI of CD4⁺ T cells of different cytokine combinations.