11. Regulatory T-cells and cord blood (2005)

A subpopulation of naïve CD4+ T cells that co-expresses CD25, the IL-2R alpha chain, has been shown to have potent suppressor activity. The frequencies of CD4+CD25bright cells are higher in rodents than in humans (8-12% vs 1-3%, respectively, of CD4+ T cells). CD4+CD25+ cells have been shown to play a vital role in the induction and maintenance of peripheral self-tolerance and prevention of systemic autoimmune disorders, including autoimmune gastritis, intestinal inflammation, and autoimmune diabetes in diabetes-prone (non-obese diabetes: NOD) mice. Sakaguchi found that nude mice rejected allogeneic skin grafts faster if transferred lymphocytes were first depleted of CD25+ cells indicating that CD25+ cells suppress alloresponses. We have shown that CD4+CD25+ regulate T cell alloresponses and tolerance induction. Depletion of CD4+25+ cells augments a mixed lymphocyte reaction (MLR) culture whereas adding a graded number of CD4+CD25+ T cells resulted in a dose-response reduction in alloproliferation. Costimulatory pathway blockade can induce tolerance to alloantigens in MLR cultures; depletion of CD4+25+ cells from the responder population precludes tolerance induction, while addback of cells restores tolerance induction revealing an important role for CD4+25+ cells in this form of tolerance induction. Several laboratories including ours have shown that CD4+25+ T cells present in the donor spleen graft downregulates graft-versus-host disease (GVHD) lethality while CD4+CD25+ cells depletion from the donor graft prior to in vivo infusion resulted in accelerated GVHD morbidity and increased mortality indicating that CD4+CD25+ cells play a role in regulation of GVHD. Supplementation of the donor graft with donor but not host CD4+25+ cells had a marked amelioration of GVHD lethality in multiple strain combinations. CD4+CD25+ cells became more potent suppressor cells after activation and expansion with anti-CD3 mAb and IL-2. Therefore, it is not surprising that ex vivo activation and expansion of CD4+25+ cells by exposure to reagents that induce TCR ligation and in the presence of a high concentration of IL-2 resulted in significant expansion of this cell population and increased their anti-GVHD potency. Expansion of CD4+25+ cells could be achieved by exposure to anti-CD3 mAb or alloantigen-bearing antigen-presenting cells in the presence of IL-2. Elucidation of other mechanisms by which activated CD25+ cells inhibit GVHD remains unclear. The literature suggests several possibilities that are not necessarily mutually exclusive. In vitro suppression is dependent upon cell-cell contact. Data from Nakamura et al suggested that cell surface-bound TGFβ was responsible for the cell contact-dependent suppression, in contrast to data from Shevach and colleagues. Thornton and Shevach showed that CD4+CD25+ T cells block IL-2 production by the CD4+CD25− cells at the RNA transcription level. The relative IL-2 paucity could inhibit the activation, proliferation and effector function of both CD4+CD25− and CD8+ T cells. Other data indicate that CD152, glucocorticoid-induced TNF receptor (GITR), or the transcription factor, FoxP3, in CD4+CD25+ cells or the activation of CD4+CD25+ cells by signaling of toll-like receptors each play an important role in their regulatory function. Of clinical significance, three groups have shown that CD4+25+ cells do not inhibit a GVL effect. Edinger et. al. demonstrated that cytolytic T cell mediated killing of two different B cell leukemia/lymphoma cell lines was preserved despite inhibition of GVHD lethality by CD4+25+ cell infusion. Jones et al showed that CD8+ T cell mediated GVHD was inhibited without precluding elimination of acute myeloid leukemia cells if CD4+25+ cells were administered 10 days post-BMT. Trenado et al demonstrated that ex vivo expanded CD4+25+ cells controlled GVHD but did not inhibit elimination of a B cell leukemia/lymphoma cell line in contrast to the graft-versus-tumor (GVT) response to a mastocytoma cell line which was impaired. Collectively, these preclinical data indicate that Tregs inhibit GVHD with the retention of GVL/GVT in many instances. CD4+25+ T cells are heterogeneous in their expression of cell surface antigens. For example, about 2/3 of CD4+25+ T cells express high levels of the lymph node (LN) homing receptor, L-selectin (CD62L). Although both L-Selhi and L-Sello CD4+25+ subsets have in vitro suppressor function, LSelhi Tregs could have superior therapeutic benefit due to inhibition of critical early GVHD priming events because of their ability to home to peripheral LNs via the binding of the L-Sel molecule to determinants on high endothelial venules in these LNs. Initial homing of L-Selhi Tregs to the LNs would optimize the likelihood of interfering with the priming and expansion of effector T cells thereby inhibiting an early phase of GVHD. Using whole body imaging, we have demonstrated that LSelhi CD4+25+ cells reduced numbers of green fluorescent protein (GFP)+ effector T cells in many types of tissues besides lymphoid organs. Despite the presumed ability of LSello CD4+25+ cells to migrate directly to sites of inflammation, imaging data indicate little demonstrable effect on the accumulation of GFP+ T cells in GVHD target organs in LSello-treated mice. In other studies, Ermann et al confirmed these findings and reported that only the L-Selhi subset of CD4+25+ T cells prevented severe tissue damage to the colon and protected recipients from lethal acute GVHD and concluded that the ability of Treg cells to efficiently enter the priming sites of pathogenic allo-reactive T cells appears to be a prerequisite for their protective function in acute GVHD. Thus, optimization of the homing and migration patterns of CD4+25+ cells may be critical in achieving the most potent anti-GVHD effects. Since CD4+CD25+ cells are capable of suppressing both CD4+ and CD8+ T cells, it is reasonable to presume that CD4+CD25+ cells may be beneficial in suppressing host anti-donor responses that can cause BM graft rejection. This has now been demonstrated by 3 separate groups using distinct model systems. Joffre et al showed that preactivated CD4+25+ cells prevented allogeneic BM graft rejection by host splenocytes given to lethally irradiated recipients. We have shown that L-selhi but not L-sello preactivated and expanded CD4+25+ cells prevent BM graft rejection in sublethally irradiated recipients via a TGFβ-independent mechanism. Hanash and Levy have shown that the addition of donor CD4+25+ cells resulted in greater lineage committed and multi-potential donor progenitors in recipient spleens one week post-transplant and significantly increased long-term multi-lineage donor chimerism in sublethally irradiated recipients. A major limitation for the clinical application of human CD4+CD25+ cells has been their paucity in peripheral blood. Only the CD4+25bright cells are potent suppressors and such cells typically are hypoproliferative. Although exogenous IL-2 can support human CD4+CD25+ cell proliferation the initial protocols published for human CD4+CD25+ T cells resulted in limited expansion. However, recent publications have solved this problem. Studies by Godfrey et al have used a non-cell sorter, bead-based approach to isolate CD4+25hi cells from adult peripheral blood mononuclear cells that were able to be expanded >100-fold in 2-3 weeks. These expanded CD4+25+ cells were far more potent suppressors of MLR cultures than fresh cells and retained the expression of L-selhi and other LN homing receptors. Hoffmann et al have shown that CD4+25bright cells can be isolated using cell sorting approaches and that such isolated cells can be expanded in great numbers while still retaining expression of L-selhi. Recent studies by Godfrey et al have shown that cord blood cell CD4+25+ cells are more readily isolated and more often lead to high levels of suppression as compared to adult peripheral blood mononuclear cells. These preliminary findings demonstrating a high degree of MLR suppression now have been extended in subsequent studies using >100 cord blood lines generated to date (B. Blazar and S. Porter, unpublished data). Thus, the stage is set for clinical trials of CD4+25+ cells for GVHD inhibition and therapy and engraftment promotion. A subpopulation of naïve CD4+ T cells that co-expresses CD25, the IL-2R alpha chain, has been shown to have potent suppressor activity. The frequencies of CD4+CD25bright cells are higher in rodents than in humans (8-12% vs 1-3%, respectively, of CD4+ T cells). CD4+CD25+ cells have been shown to play a vital role in the induction and maintenance of peripheral self-tolerance and prevention of systemic autoimmune disorders, including autoimmune gastritis, intestinal inflammation, and autoimmune diabetes in diabetes-prone (non-obese diabetes: NOD) mice. Sakaguchi found that nude mice rejected allogeneic skin grafts faster if transferred lymphocytes were first depleted of CD25+ cells indicating that CD25+ cells suppress alloresponses. We have shown that CD4+CD25+ regulate T cell alloresponses and tolerance induction. Depletion of CD4+25+ cells augments a mixed lymphocyte reaction (MLR) culture whereas adding a graded number of CD4+CD25+ T cells resulted in a dose-response reduction in alloproliferation. Costimulatory pathway blockade can induce tolerance to alloantigens in MLR cultures; depletion of CD4+25+ cells from the responder population precludes tolerance induction, while addback of cells restores tolerance induction revealing an important role for CD4+25+ cells in this form of tolerance induction. Several laboratories including ours have shown that CD4+25+ T cells present in the donor spleen graft downregulates graft-versus-host disease (GVHD) lethality while CD4+CD25+ cells depletion from the donor graft prior to in vivo infusion resulted in accelerated GVHD morbidity and increased mortality indicating that CD4+CD25+ cells play a role in regulation of GVHD. Supplementation of the donor graft with donor but not host CD4+25+ cells had a marked amelioration of GVHD lethality in multiple strain combinations. CD4+CD25+ cells became more potent suppressor cells after activation and expansion with anti-CD3 mAb and IL-2. Therefore, it is not surprising that ex vivo activation and expansion of CD4+25+ cells by exposure to reagents that induce TCR ligation and in the presence of a high concentration of IL-2 resulted in significant expansion of this cell population and increased their anti-GVHD potency. Expansion of CD4+25+ cells could be achieved by exposure to anti-CD3 mAb or alloantigen-bearing antigen-presenting cells in the presence of IL-2. Elucidation of other mechanisms by which activated CD25+ cells inhibit GVHD remains unclear. The literature suggests several possibilities that are not necessarily mutually exclusive. In vitro suppression is dependent upon cell-cell contact. Data from Nakamura et al suggested that cell surface-bound TGFβ was responsible for the cell contact-dependent suppression, in contrast to data from Shevach and colleagues. Thornton and Shevach showed that CD4+CD25+ T cells block IL-2 production by the CD4+CD25− cells at the RNA transcription level. The relative IL-2 paucity could inhibit the activation, proliferation and effector function of both CD4+CD25− and CD8+ T cells. Other data indicate that CD152, glucocorticoid-induced TNF receptor (GITR), or the transcription factor, FoxP3, in CD4+CD25+ cells or the activation of CD4+CD25+ cells by signaling of toll-like receptors each play an important role in their regulatory function. Of clinical significance, three groups have shown that CD4+25+ cells do not inhibit a GVL effect. Edinger et. al. demonstrated that cytolytic T cell mediated killing of two different B cell leukemia/lymphoma cell lines was preserved despite inhibition of GVHD lethality by CD4+25+ cell infusion. Jones et al showed that CD8+ T cell mediated GVHD was inhibited without precluding elimination of acute myeloid leukemia cells if CD4+25+ cells were administered 10 days post-BMT. Trenado et al demonstrated that ex vivo expanded CD4+25+ cells controlled GVHD but did not inhibit elimination of a B cell leukemia/lymphoma cell line in contrast to the graft-versus-tumor (GVT) response to a mastocytoma cell line which was impaired. Collectively, these preclinical data indicate that Tregs inhibit GVHD with the retention of GVL/GVT in many instances. CD4+25+ T cells are heterogeneous in their expression of cell surface antigens. For example, about 2/3 of CD4+25+ T cells express high levels of the lymph node (LN) homing receptor, L-selectin (CD62L). Although both L-Selhi and L-Sello CD4+25+ subsets have in vitro suppressor function, LSelhi Tregs could have superior therapeutic benefit due to inhibition of critical early GVHD priming events because of their ability to home to peripheral LNs via the binding of the L-Sel molecule to determinants on high endothelial venules in these LNs. Initial homing of L-Selhi Tregs to the LNs would optimize the likelihood of interfering with the priming and expansion of effector T cells thereby inhibiting an early phase of GVHD. Using whole body imaging, we have demonstrated that LSelhi CD4+25+ cells reduced numbers of green fluorescent protein (GFP)+ effector T cells in many types of tissues besides lymphoid organs. Despite the presumed ability of LSello CD4+25+ cells to migrate directly to sites of inflammation, imaging data indicate little demonstrable effect on the accumulation of GFP+ T cells in GVHD target organs in LSello-treated mice. In other studies, Ermann et al confirmed these findings and reported that only the L-Selhi subset of CD4+25+ T cells prevented severe tissue damage to the colon and protected recipients from lethal acute GVHD and concluded that the ability of Treg cells to efficiently enter the priming sites of pathogenic allo-reactive T cells appears to be a prerequisite for their protective function in acute GVHD. Thus, optimization of the homing and migration patterns of CD4+25+ cells may be critical in achieving the most potent anti-GVHD effects. Since CD4+CD25+ cells are capable of suppressing both CD4+ and CD8+ T cells, it is reasonable to presume that CD4+CD25+ cells may be beneficial in suppressing host anti-donor responses that can cause BM graft rejection. This has now been demonstrated by 3 separate groups using distinct model systems. Joffre et al showed that preactivated CD4+25+ cells prevented allogeneic BM graft rejection by host splenocytes given to lethally irradiated recipients. We have shown that L-selhi but not L-sello preactivated and expanded CD4+25+ cells prevent BM graft rejection in sublethally irradiated recipients via a TGFβ-independent mechanism. Hanash and Levy have shown that the addition of donor CD4+25+ cells resulted in greater lineage committed and multi-potential donor progenitors in recipient spleens one week post-transplant and significantly increased long-term multi-lineage donor chimerism in sublethally irradiated recipients. A major limitation for the clinical application of human CD4+CD25+ cells has been their paucity in peripheral blood. Only the CD4+25bright cells are potent suppressors and such cells typically are hypoproliferative. Although exogenous IL-2 can support human CD4+CD25+ cell proliferation the initial protocols published for human CD4+CD25+ T cells resulted in limited expansion. However, recent publications have solved this problem. Studies by Godfrey et al have used a non-cell sorter, bead-based approach to isolate CD4+25hi cells from adult peripheral blood mononuclear cells that were able to be expanded >100-fold in 2-3 weeks. These expanded CD4+25+ cells were far more potent suppressors of MLR cultures than fresh cells and retained the expression of L-selhi and other LN homing receptors. Hoffmann et al have shown that CD4+25bright cells can be isolated using cell sorting approaches and that such isolated cells can be expanded in great numbers while still retaining expression of L-selhi. Recent studies by Godfrey et al have shown that cord blood cell CD4+25+ cells are more readily isolated and more often lead to high levels of suppression as compared to adult peripheral blood mononuclear cells. These preliminary findings demonstrating a high degree of MLR suppression now have been extended in subsequent studies using >100 cord blood lines generated to date (B. Blazar and S. Porter, unpublished data). Thus, the stage is set for clinical trials of CD4+25+ cells for GVHD inhibition and therapy and engraftment promotion.