Abstract
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. Singleβcell 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 antiβCD3/antiβCD28 Abs and 1,000 IU ILβ2/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 CFSEβbased proliferation assays. Clones had heterogeneous CD25 expression levels. Cloning efficiency of CD4^+^CD25^high^ T cells was low. CD25^high^ clones expressed CTLAβ4, Foxp3, CD62L, but little GITR and suppressed proliferation of autologous CD4^+^CD25^β^ responder cells. Clones of activated CD4^+^CD25^interm./low^ cells expressed intermediate to high levels of GITR and HLAβDR 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 β€ 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, CTLAβ4 and CD62L. The number of expandable Treg is increased in HNSCC patients. Β© 2007 WileyβLiss, Inc.