Our studies have addressed several major questions: 1) We have demonstrated that human Foxp3+ T cells, activated with plate-bound anti-hCD3, are almost as potent inhibitors of the proliferation of mouse CD4+Foxp3- responders (stimulated with soluble anti-mouse CD3 and mouse DC), as mouse CD4+Foxp3+ Treg. This result strongly suggests that at least in vitro a major component of the suppressive function of Foxp3+ Treg is preserved across the species. One advantage of this model is that it allows us to attempt to block the suppressive capacity of the human T cells with mAbs to human cell surface antigens that will not interfere with the activation of the mouse responder cells. We have tested a panel of several mAbs and noted that suppression by the human Tregs is completely reversed by anti-hCD11a (LFA-1), but not by anti-human CD54 (ICAM-1). We conclude from this that hCD11a on the human Tregs interacts with mouse CD54 on either the mouse responder T cell or DC, rather than with human CD54 on the human Treg. Human Tregs are fully competent suppressors of mouse CD4+Foxp3- T cells from CD54-/- mice in the presence of wild-type mouse DC indicating that one target of the human Tregs is the mouse DC rather that the mouse responder T cell. Thus, the suppressive effects of human Treg on mouse responder T cells closely mimic the potential function of Tregs cells in vivo where our recent studies have focused on the DC as the target. 2) The major obstacle for the use of human Tregs in cell-based therapy is the difficulty of obtaining a highly purified population after ex vivo expansion. A CD4+FOXP3+ population of >90% purity can be isolated by FACS of the top 2-4% of CD4+ T cells with high CD25 expression (CD25hi) from peripheral blood, but frequently the percentage of FOXP3+ T cells decreases to 75% after one week and to 50% after two weeks of expansion by stimulation with anti-CD3/CD28 and IL-2. A major issue which has not been resolved is the validity of FOXP3 as a bona fide marker of human Tregs. As TGFbeta is present in the serum used for cultures, an induction of FOXP3 expression in contaminating FOXP3 T cells may occur during expansion cultures of partially purified Tregs. While the expanded population might appear to be highly enriched in FOXP3+ cells, many of these cells may be induced FOXP3+ cells that lack Treg functions. We have identified three cell surface markers, latency associated peptide (LAP), IL-1 receptor type II (CD121b), and IL-1 receptor type I (CD121a) that are selectively expressed on activated Tregs, but not on activated CD4+FOXP3- or induced FOXP3+ cells. We have used these cell surface markers to design a protocol that allows for purification of FOXP3+ Tregs from ex vivo expansion cultures starting with leukapheresis preparations and using only magnetic bead targeting reagents. The final Treg product is composed of >90% FOXP3+ cells that is highly anergic and suppressive in vitro. The identification and characterization of FOXP3+ Tregs represent one of the major advances in the field of immunology over the past decade. The major issue for the future is how to translate the large body of data obtained with mouse Tregs to man and to use Tregs as immunomodulatory agents in autoimmune diseases and transplantation. The identification of LAP, CD121a and CD121b on activated Tregs as highly specific markers and their use in protocols for the isolation of large numbers of highly purified Tregs should facilitate the rapid advancement of the therapeutic application and functional analysis of Tregs in human disease. This method provides an important advance for the preparation of Tregs for cell-based immunotherapy to treat or prevent autoimmunity and transplant-related complications. 3.TGF-beta is a highly pleiotropic cytokines that has critical functions in cell differentiation, tissue morphogenesis and modulation of cell growth, inflammation, matrix synthesis and apoptosis. TGF-beta is cleaved in the Golgi apparatus by a furin-like convertase to produce the dimeric propeptides called latency-associated peptide (LAP) that noncovalently associates with the dimeric mature TGF-beta to prevent its activity. TGF-beta can be secreted in a small latent form associated with LAP or this complex can further associate with latent-TGF-beta-binding protein (LTBP) to produce a large latent form for deposition onto the extracellular matrix. Small latent TGF-beta can be expressed on the membrane of many cell types, including megakaryocytes, platelets, immature dendritic cells and activated FOXP3+ regulatory T cells (Tregs) and has important functions in tissue healing and immune regulation. However, it is unknown how this membrane small latent TGF-beta is anchored to the cell surface. It has been recently shown that megakaryocytes and activated Tregs expressed high levels of mRNA for a member of the leucine-rich repeat family of proteins which has been termed GARP or LRRC32 and that platelets express this molecule on their membrane. We have demonstrate that GARP or LRRC32 functions as a carrier and cell surface receptor for latent TGF-beta. Using siRNA technology, we have shown in vitro that GARP+LAP- cells can bind latent TGF-beta, but we have not been able to determine whether in vivo the binding of latent TGF-beta to GARP occurs exclusively intracellularly or whether GARP can also bind secreted latent TGF-beta or latent TGF-beta associated with LTBP. Although the GARP/LAP complex is expressed by platelets, within the immune system GARP/LAP expression is mostly observed on activated functional FOXP3+ Tregs. We did not observe significant surface expression of GARP or LAP in CD19+/CD20+ B cells, CD14+ monocytes, CD8+ T cells, natural killer (NK) cells, NK T cells and immature/mature monocyte-derived DCs. Our functional studies indicate that the GARP/LAP complex does contribute to Treg mediated suppression in vitro. We have recently proposed that a major role of latent TGF-beta on the surface of murine Tregs is to convert responder T cells into FOXP3+ Tregs through a mechanism of infectious tolerance when both populations are activated in concert via their TCRs. This TGF-beta-mediated infectious tolerance mechanism appears to also be involved in human Tregs. As GARP and LAP are only expressed on TCR activated FOXP3+ Tregs, one major function of this complex is to target delivery of TGF-beta to sites of ongoing immune responses where Tregs can be activated by recognition of their cognate antigens on antigen presenting cells. Following release of active TGF-beta, it might act locally on FOXP3 T cells to convert them to FOXP3+ Tregs, to generate Th17 effectors if an inflammatory milieu is present, to act directly on DCs to modulate their functions, or to signal in an autocrine manner to maintain their functions.
Showing the most recent 10 out of 17 publications
