title: Expression of ICOS on human melanoma-infiltrating CD4+CD25highFoxp3+ T regulatory cells: implications and impact on tumor-mediated immune suppression.
abstract: OBJECTIVE
  Interaction of ICOS with its ligand (ICOSL, B7-H2) promotes T cell responses. As CD4+CD25highFoxp3+ naturally occurring T regulatory cells in melanoma patients express ICOS, we investigated the impact of ICOS on naturally occurring T regulatory cell function.
  
  
  METHODS
  Expression of ICOS and T regulatory (Treg) cell markers was determined on CD4+CD25high T cells in PBMC and tumor-infiltrating lymphocytes from melanoma patients (n=10) and PBMC of normal controls (n=10) by multicolor flow cytometry. Suppression mediated by sorted ICOShigh and ICOSlow Treg was assessed in CFSE-based suppression assays with autologous CD4+CD25- responder cells (RC). Transwell inserts separating Treg from RC were used to evaluate suppression mechanisms used by Treg. ICOShigh or ICOSlow Treg were coincubated with RC+/-TCR and IL-2 stimulation. ICOShigh and ICOS- Treg were also expanded under conditions previously shown to induce Tr1 from RC.
  
  
  RESULTS
  Treg in tumor-infiltrating lymphocytes expressed ICOS (mean fluorescence intensity=70+/-10), while Treg in PBMC had low ICOS expression (mean fluorescence intensity=3.5+/-2.5, p<or=0.001). ICOShigh Treg up-regulated Treg markers (p<or=0.0016) and mediated stronger suppression (p<or=0.001) relative to ICOSlow Treg. ICOShigh Treg induced Tr1 cells in nonactivated RC and Th2 cells in preactivated RC. ICOShigh Treg exposed to Tr1 cytokines expressed IL-10 and suppressed RC (92+/-12%) in contrast to ICOSlow Treg, which mediated low suppression (21+/-15%; p<or=0.0028).
  
  
  CONCLUSIONS
  ICOShigh Treg can induce diverse immune responses in RC, depending on activation signals and cytokines present. ICOShigh Treg induce Tr1 or Th2 cells depending on the activation state of RC. In a "Tr1" cytokine milieu, ICOShigh Treg transit to Tr1.
authors.affiliation: University of Pittsburgh Cancer Institute, Department of Medicine, School of Medicine, Pittsburgh, PA 15232, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Strauss
authors.forename: Christoph
authors.initials: C
authors.lastname: Bergmann
authors.forename: Miroslaw J
authors.initials: MJ
authors.lastname: Szczepanski
authors.forename: Stephan
authors.initials: S
authors.lastname: Lang
authors.forename: John M
authors.initials: JM
authors.lastname: Kirkwood
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Whiteside
mesh_terms: D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D014408:Biomarkers, Tumor / Q000096:biosynthesis; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000502:physiology; D051858:Forkhead Transcription Factors / Q000096:biosynthesis*; D006801:Humans; D007108:Immune Tolerance; D060889:Inducible T-Cell Co-Stimulator Protein; D053645:Interleukin-2 Receptor alpha Subunit / Q000096:biosynthesis*; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D008545:Melanoma / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000473:pathology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D014407:Tumor Cells, Cultured
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; D014408:Biomarkers, Tumor; D016207:Cytokines; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; C556692:ICOS protein, human; D060889:Inducible T-Cell Co-Stimulator Protein; D053645:Interleukin-2 Receptor alpha Subunit