Functional and phenotypic characteristics of CD4+CD25highFoxp3+ Treg clones obtained from peripheral blood of patients with cancer

Circulating human CD4(+)CD25(high)Foxp3(+) T cell populations (Treg) may contain activated CD4(+)CD25(+) T cells interfering with Treg evaluation. To gain insights into the phenotypic and functional characteristics of Treg in patients with cancer, we have analyzed CD4(+)CD25(high) populations at the clonal level. Single-cell sorted (SCS) CD4(+)CD25(high) T cells obtained from PBMC of normal controls (NC) or patients with squamous cell carcinoma of the head and neck (HNSCC) were plated at 1 cell/well in 96 well plates and expanded with anti-CD3/anti-CD28 Abs and 1,000 IU IL-2/mL in the presence or absence of rapamycin (1 nM). All generated clones were evaluated for the phenotype by flow cyometry and suppressor function in CFSE-based proliferation assays. Clones had heterogeneous CD25 expression levels. Cloning efficiency of CD4(+)CD25(high) T cells was low. CD25(high) clones expressed CTLA-4, Foxp3, CD62L, but little GITR and suppressed proliferation of autologous CD4(+)CD25(-) responder cells. Clones of activated CD4(+)CD25(interm./low) cells expressed intermediate to high levels of GITR and HLA-DR and did not suppress proliferation of responder cells. The number, suppressor phenotype and function of CD25(high) Treg clones were significantly enhanced in HNSCC patients relative to NC (p </= 0.001). CD4(+)CD25(+) populations comprise phenotypically and functionally distinct subsets of CD25(+) cells. Only a small fraction of these activated CD4(+) T cells are potent suppressor cells characterized by high expression levels of CD25, Foxp3, CTLA-4 and CD62L. The number of expandable Treg is increased in HNSCC patients.