title: iNOS expression in CD4+ T cells limits Treg induction by repressing TGFβ1: combined iNOS inhibition and Treg depletion unmask endogenous antitumor immunity.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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 TGFβ1-depleting antibodies, and TGFβ1 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 TGFβ1-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 TGFβ1 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.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, The Icahn School of Medicine at Mount Sinai, New York, New York. Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, New York, New York. Department of Dermatology, The Icahn School of Medicine at Mount Sinai, New York, New York. Immunology Institute, The Icahn School of Medicine at Mount Sinai, New York, New York. Tisch Cancer Institute, The Icahn School of Medicine at Mount Sinai, New York, New York. Head and Neck Cancer Research Program, The Icahn School of Medicine at Mount Sinai, New York, New York.
authors.forename: Padmini
authors.initials: P
authors.lastname: Jayaraman
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, The Icahn School of Medicine at Mount Sinai, New York, New York.
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Alfarano
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, The Icahn School of Medicine at Mount Sinai, New York, New York.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Svider
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, The Icahn School of Medicine at Mount Sinai, New York, New York. Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, New York, New York. Department of Dermatology, The Icahn School of Medicine at Mount Sinai, New York, New York. Immunology Institute, The Icahn School of Medicine at Mount Sinai, New York, New York. Tisch Cancer Institute, The Icahn School of Medicine at Mount Sinai, New York, New York. Head and Neck Cancer Research Program, The Icahn School of Medicine at Mount Sinai, New York, New York.
authors.forename: Falguni
authors.initials: F
authors.lastname: Parikh
authors.affiliation: Immunology Institute, The Icahn School of Medicine at Mount Sinai, New York, New York.
authors.forename: Geming
authors.initials: G
authors.lastname: Lu
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, The Icahn School of Medicine at Mount Sinai, New York, New York.
authors.forename: Sarah
authors.initials: S
authors.lastname: Kidwai
authors.affiliation: Immunology Institute, The Icahn School of Medicine at Mount Sinai, New York, New York.
authors.forename: Huabao
authors.initials: H
authors.lastname: Xiong
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, The Icahn School of Medicine at Mount Sinai, New York, New York. Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, New York, New York. Department of Dermatology, The Icahn School of Medicine at Mount Sinai, New York, New York. Immunology Institute, The Icahn School of Medicine at Mount Sinai, New York, New York. Tisch Cancer Institute, The Icahn School of Medicine at Mount Sinai, New York, New York. Head and Neck Cancer Research Program, The Icahn School of Medicine at Mount Sinai, New York, New York. andrew.sikora@BCM.edu.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Sikora
mesh_terms: D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000008:administration & dosage / Q000494:pharmacology; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D045744:Cell Line, Tumor; D003520:Cyclophosphamide / Q000008:administration & dosage / Q000494:pharmacology; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D008213:Lymphocyte Activation; D008212:Lymphocyte Depletion; D016246:Lymphocytes, Tumor-Infiltrating / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008239:Lysine / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D022423:Myeloid Cells / Q000276:immunology / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D014158:Transcription, Genetic; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*; D047368:Tumor Burden / Q000187:drug effects / Q000276:immunology
chemical_list: D018906:Antineoplastic Agents, Alkylating; D004791:Enzyme Inhibitors; C090663:N(6)-(1-iminoethyl)lysine; D053773:Transforming Growth Factor beta1; D003520:Cyclophosphamide; D052247:Nitric Oxide Synthase Type II; D008239:Lysine