Expression of ICOS on human melanoma-infiltrating CD4+CD25highFoxp3+ T regulatory cells:: Implications and impact on tumor-mediated immune suppression

Objective: Interaction of ICOS with its ligand (ICOSL, B7-H2) promotes T cell responses. As CD4(+)CD25(high)Foxp(3+) 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(+)CD25(high)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 ICOS(high) and ICOS(low) 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. ICOS(high) or ICOS(low) Treg were coincubated with RC +/- TCR and IL-2 stimulation. ICOS(high) 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 <= 0.001). ICOS(high) Treg up-regulated Treg markers (p <= 0.0016) and mediated stronger suppression (p:5 0.001) relative to ICOS(low) Treg. ICOS(high) Treg induced Tr1 cells in nonactivated RC and Th2 cells in preactivated RC. ICOS(high) Treg exposed to Tr1 cytokines expressed IL-10 and suppressed RC (92 +/- 12%) in contrast to ICOS(low) Treg, which mediated low suppression (21 +/- 15%; p <= 0.0028). Conclusion: ICOS(high) Treg can induce diverse immune responses in RC, depending on activation signals and cytokines present. ICOS(high) Treg induce Tr1 or Th2 cells depending on the activation state of RC. In a Tr1 cytokine milieu, ICOS(high) Treg transit to Tr1.