CD4 ؉ CD25 ؉ T regulatory (T reg ) cells have been shown to critically regulate self and allograft tolerance in mice. Studies of human T reg cells have been hindered by low numbers present in peripheral blood and difficult purification. We found that cord blood was a superior source for T reg -cell isolation and cell line generation compared with adult blood. Cord blood CD4 ؉ CD25 ؉ cells were readily purified and generated cell lines that consistently exhibited potent suppressor activity, with more than 95% suppression of allogeneic mixed lymphocyte reactions (MLRs) ( IntroductionNaturally arising CD4 ϩ CD25 ϩ T regulatory (T reg ) cells can restrict or alter most types of immune responses. 1 Initially they were described to be critical for the control of autoimmunity 2-3 and were found on adoptive transfer to prevent experimental autoimmune diseases. More recently, T reg cells have been shown to suppress allogeneic immune responses and can prevent transplant rejection. [4][5] In addition, these cells can restrain antitumor [6][7] and antimicrobial immune responses. 8 Thus, CD4 ϩ CD25 ϩ T reg cells appear to be central control elements of immunoregulation, and understanding their biology will be important for efforts aimed at therapeutically manipulating immune responses.T reg cells are best characterized in mice, where they constitute 5% to 10% of lymph node and spleen CD4 ϩ T-cell populations. They are generated both through central thymic developmental mechanisms in pathogen-free mice and also arise by poorly defined peripheral generation or expansion mechanisms. 9-10 To date they have primarily been defined by coexpression of CD4 ϩ and CD25 ϩ antigens on fresh isolation. CD25 as well as other markers of murine T reg cells, cytotoxic T-lymphocyte antigen-4 (CTLA4 [CD152]) and glucocorticoid-induced tumor necrosis factor (TNF)-like receptor (GITR), are all activation antigens on conventional T cells and therefore are not specific. FoxP3, a nuclear protein thought to function as a transcriptional repressor, is a newer marker considered to be more specific for T reg cells. 11 It has been demonstrated that after activation (T-cell receptor-based, antigenspecific, or anti-CD3), T reg cells can nonspecifically suppress proliferation of both CD4 ϩ and CD8 ϩ T cells. The mechanism of suppression is unclear and in vitro appears to require cell-cell contact. A functional result of suppression is impaired production of interleukin-2 (IL-2). [12][13] In vivo, the suppression mechanism is more controversial, with some studies showing dependence on immunosuppressive cytokines, 14 which are not required for in vitro suppression.Studies in mouse models of bone marrow transplantation (BMT) have shown that fresh or culture-expanded CD4 ϩ CD25 ϩ cells can delay or prevent disease. [15][16][17] Previous studies have shown that ex vivo polyclonally expanded T reg cells, with anti-CD3 plus IL-2 (for 10 days), can be effective in preventing graft versus host disease (GVHD). 15 Ex vivo expansion of T reg cells with irradiated allog...

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