B7-H1-dependent sex-related differences in tumor immunity and immunotherapy responses

Abstract

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are immunopathogenic in cancers by impeding tumor-specific immunity. B7-homologue 1 (B7-H1) (CD274) is a cosignaling molecule with pleiotropic effects, including hindering antitumor immunity. In this study, we demonstrate sex-dependent, B7-H1-dependent differences in tumor immunity and response to immunotherapy in a hormone-independent cancer, murine B16 melanoma. Antitumor immunity was better in B7-H1(-/-) females versus males as a result of reduced regulatory T cell function in the B7-H1(-/-) females, and clinical response following B7-H1 blockade as tumor immunotherapy was significantly better in wild-type females than in males, owing to greater B7-H1 blockade-mediated reduction of Treg function in females. Wild-type female Tregs expressed significantly lower B7-H1 versus males but were insensitive to estrogen in vitro. Female B7-H1(-/-) Tregs were exquisitely sensitive to estrogen-mediated functional reduction in vitro, suggesting that B7-H1 effects occur before terminal Treg differentiation. Immune differences were independent of known B7-H1 ligands. Sex-dependent immune differences are seldom considered in designing immune therapy or interpreting immunotherapy treatment results. Our data demonstrate that sex is an important variable in tumor immunopathogenesis and immunotherapy responses through differential Treg function and B7-H1 signaling.

FIGURE 1

B7-H1^−/− females resist B16 better than males through superior antitumor immunity independent of PD-1 or CD80. A, Groups of five mice were challenged with 125,000 B16 cells/flank. Tumor growth was measured with Vernier calipers. B, Tumor-specific immunity was assessed as proliferation of CFSE-labeled, adoptively transferred OT-I cells using flow cytometry 4 d after transfer. C, Groups of five mice each were challenged with B16, as above. D, Treg (CD4⁺CD25^hi T cell) function was tested in groups of five naive mice each in vitro. DLN, draining lymph node; SPL, spleen.

FIGURE 2

Defective Treg function in B7-H1^−/− females contributes to increased tumor resistance and superior antitumor immunity. Groups of five mice were challenged with 125,000 B16 cells/flank, treated with DT 5 µg or PBS twice a week beginning 4 d later, and sacrificed 17 d after tumor challenge. Effects of DT on phenotypic Treg depletion in SPL and DLN (A), Treg (CD4⁺CD25^hi T cell) function (B), and tumor growth (C) were determined. D, Effects of DT on tumor-specific immunity was assessed by OT-I cell proliferation using flow cytometry as for Fig. 1B. E, Tregs were obtained from spleens of B16-bearing B7-H1^−/− females and tested for regulation of B7-H1^−/−CD4⁺CD25⁻ Eff from naive or tumor-bearing females. F, Granzyme B, IL-10, and TGF-β expression gated on CD3⁺CD4⁺CD25⁺Foxp3⁺ cells in B16-bearing mice. n = 6/group. G, OT-I cell proliferation and IFN-γ expression induced by CD11c⁺ DCs obtained from spleens and DLNs of B16-bearing mice 7 d after tumor challenge. n = 3/group. Control mice received DCs with no peptide. DLN, draining lymph node; Eff, effector T cell; OVAp, DC loaded with SIINFEKL peptide; SPL, spleen.

FIGURE 3

Adoptively transferred, functional female WT Tregs reverse immune and clinical differences in B7-H1^−/− females. RFP⁺ or RFP⁻CD4⁺ T cells were sorted from female FIR mice and transferred into B7-H1^−/− females. A, Mice were challenged with B16 the day after transfer. At sacrifice 14 d after tumor challenge, flow cytometry was used to detect CD8⁺ T cell IFN-γ production (B) and tumor-specific CD8⁺ T cells (OVA pentamer stain) or (C) Foxp3 expression in CD4⁺CD25^hi spleen cells. The percentage positive gated events is shown. D, Functional status of endogenous, transferred, or converted Tregs in B16-bearing mice was tested 15 d after adoptive transfer. E, Endogenous Treg (CD4⁺CD25^hi T cell) function was tested 14 d after B16 tumor challenge in mice not receiving adoptive cell transfers. F, Treg function 28 d after i.p. ID8 tumor challenge with 10 × 10⁶ ID8 cells. G, Ascites development (>30% weight increase) in mice challenged with ID8 tumor.

FIGURE 4

Differential sex-dependent B7-H1 responses contribute to sex-dependent differences in tumor immunity. Groups of five mice each were challenged with 125,000 B16 cells/flank, treated with anti–B7-H1 or isotype control Abs (200 µg/mouse) starting 1 d before tumor challenge and every 2 d thereafter, and sacrificed on day 16 after tumor challenge. A, Tumor growth was measured by Vernier calipers. B, Tregs (CD4⁺CD25^hi T cells) were sorted from spleen and tested for function. C, Splenocytes were analyzed for tumor-specific CD8⁺ T cells by flow cytometry using OVA-specific pentamers. αB7-H1 or isotype refer to in vivo treatments in panels B and C. D, B7-H1 expression in naive WT mice gated on CD3⁺CD4⁺CD25^hi Tregs cultured without or with 10⁻⁸ M E2 for 48 h (n = 6/group). E, B7-H1 expression in B16-bearing WT mice gated on CD3⁺CD4⁺CD25^hi Tregs (n = 6/group). F, Female B7-H1^−/− Tregs from naive mice were tested for in vitro Treg without or with 10⁻⁸ M E2 and without or with 10⁻⁶ M ICI182,780 (an estrogen receptor antagonist) (n = 6/group).