iNOS expression in CD4⁺ T cells limits treg induction by repressing TGFβ1: Combined iNOS inhibition and treg depletion unmask endogenous antitumor immunity

Purpose: Expression of inducible nitric oxide synthase (iNOS) in different cellular compartments may have divergent effects on immune function. We used a syngeneic tumor model to functionally characterize the role of iNOS in regulation of CD4⁺FOXP3⁺ regulatory T cells (Treg), and optimize the beneficial effects of iNOS inhibition on antitumor immunity.

Experimental Design: Wild-type (WT) or iNOS knockout mice bearing established MT-RET-1 melanoma were treated with the small-molecule iNOS inhibitor L-NIL and/or cyclophosphamide alone or in combination. The effect of iNOS inhibition or knockout on induction of Treg from mouse and human CD4⁺ T cells in ex vivo culture was determined in parallel in the presence or absence of TGFb1-depleting antibodies, and TGFb1 levels were assessed by ELISA.

Results: Whereas intratumoral myeloid-derived suppressor cells (MDSC) were suppressed by iNOS inhibition or knockout, systemic and intratumoral FOXP3⁺ Treg levels increased in tumor-bearing mice. iNOS inhibition or knockout similarly enhanced induction of Treg from activated cultured mouse splenocytes or purified human or mouse CD4⁺ T cells in a TGFb1-dependent manner. Although either iNOS inhibition or Treg depletion with low-dose cyclophosphamide alone had little effect on growth of established MT-RET1 melanoma, combination treatment potently inhibited MDSC and Treg, boosted tumor-infiltrating CD8⁺ T-cell levels, and arrested tumor growth in an immune-dependent fashion.

Conclusions: iNOS expression in CD4⁺ T cells suppresses Treg induction by inhibiting TGFb1 production. Our data suggest that iNOS expression has divergent effects on induction of myeloid and lymphoid-derived regulatory populations, and strongly support development of combinatorial treatment approaches that target these populations simultaneously.