Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells

Abstract

IntroductionCD4 ϩ CD25 ϩ Foxp3 ϩ regulatory T cells (Treg) are negative regulators of immune responses to self-and foreign antigens and play a critical role in maintaining immune tolerance by suppressing pathologic immune responses in autoimmune diseases, transplant allograft rejection, and graft-versus-host disease (GVHD). [1][2][3] On adoptive transfer in rodents, Treg were found to control experimental autoimmune diseases, 4 inhibit GVHD, 5,6 and prevent transplant allograft rejection, 7,8 indicating that Treg-based therapy has a great therapeutic potential for these diseases in humans.An important obstacle to Treg-based therapy has been the limited numbers of these cells that are available, as only approximately 1% to 2% of circulating human CD4 ϩ T cells are Treg. Several groups have developed protocols to expand a large number of polyclonal CD4 ϩ CD25 ϩ Treg in vitro with repeated stimulation by either CD3 and CD28 mAbs or artificial antigenpresenting cells (APCs) for activation through CD3 and CD28, together with exogenous high-dose interleukin-2 (IL-2). [9][10][11] However, polyclonal Treg may cause global immune suppression. 4,7 In addition, because there are only few antigen-specific Treg in the population of the polyclonal Treg, very large numbers of nonspecifically expanded Treg are required to inhibit bone-marrow allograft rejection in animal models. 12 All of these characteristics of polyclonal Treg hamper their clinical applications.In contrast, adoptive transfer of antigen-specific Treg has been shown to prevent and treat T cell-mediated inflammatory diseases with high efficiency. In animal models, small numbers of antigen-specific Treg can suppress experimental autoimmune diseases 13 and prevent GVHD and allograft rejection in bone marrow and solid organ transplantation. 14,15 Importantly, the transfer of antigen-specific Treg prevented target antigenmediated T-cell responses, such as GVHD and allograft rejection, but did not compromise host general immunity, including the graft-versus-tumor activity and antiviral immunity. 5,[15][16][17] Based on these studies, antigen-specific Treg has substantial promise for human immunotherapy.The reliable induction and expansion of rare antigen-specific Treg are technically challenging. Currently, several protocols for murine antigen-specific Treg induction and expansion have been reported in which either purified CD4 ϩ CD25 Ϫ or CD4 ϩ CD25 ϩ cells were cocultured with autologous dendritic cells (DCs) pulsed with alloantigen in the presence of high-dose IL-2 or directly cocultured with allogeneic DCs. 14,18-20 Similar protocol has also been reported for generation of human antigen-specific Treg recently. 21 In this protocol, antigen-specific CD4 ϩ CD25 ϩ Treg can be generated using the coculture of CD4 ϩ CD25 Ϫ T cells with allogeneic monocyte-derived DCs. However, the large-scale in vitro expansion of alloantigen-specific Treg is difficult because of certain features of DCs. For example, DCs are relatively rare in peripheral blood and are usually derived f...

CD40-activated B cells are more potent than immature dendritic cells to induce and expand CD4+ regulatory T cells

“…B cells from peripheral blood mononuclear cells (PBMCs) were stimulated via CD40 using NIH3T3 cells transfected with the human CD40 ligand (t-CD40-L cells) as described previously. 20,21 Briefly, PBMCs were cocultured with lethally irradiated (96 Gy) t-CD40-L cells in the presence of IL-4 (2 ng/ml; R&D systems, Minneapolis, MN, USA) and cyclosporine A (5.5310 27 M, Sigma Chemical Co., St. Louis, MO, USA) in Iscove's modified Dulbecco's medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated human AB serum, 50 mg/ml transferrin (Boehringer Mannheim, Indianapolis, IN, USA), 5 mg/ml insulin (Sigma Chemical Co.), and 15 mg/ml gentamicin (Gibco BRL) at 37 uC in 5% CO 2 . After 14 days of coculture, more than 99% of the viable suspended cells were CD19 positive.…”

“…20 Cultured CD4 high CD25 1 and CD4 1 CD25 2 T cells were sorted by FACSAria after 6 days of coculture with allogeneic CD40-B or imDCs from the same donors. The purity of sorted cells was routinely more than 99%.…”

“…In a previous study, 20 we developed a simple and cost-effective method to rapidly induce and expand large numbers of functional human alloantigen-specific CD4 1 Tregs from antigenically naive precursors in vitro using allogeneic CD40-activated B cells (CD40-B) as stimulators. Using this approach, naive CD4 1 CD25 2 T cells could be expanded eight folds into alloantigen-specific CD4 high CD25 1 Tregs after 3 weeks of culture in the absence of exogenous cytokines.…”

Alloantigen‐specific de novo‐induced Foxp3+ Treg revert in vivo and do not protect from experimental GVHD

“…However, the availability of sufficient numbers of donor Treg for cell-based therapies remains limited [4]. Besides the in vitro expansion of nTreg [5], an obvious approach to solve this problem would be the de novo induction and expansion of Foxp3 1 Treg from abundant naïve CD4 1 T cells with recipient alloantigens [6,7]. Instead of mediating unspecific suppression, such alloantigen-induced Treg potentially could provide the advantage of antigen-specific regulation, thereby reducing the risk of disease relapse and infections [8].…”

Co-culture of the 55-6 B cell hybridoma with the EL-4 thymoma cell. Effect on cell growth and monoclonal antibody production

“…In this regard, several in vitro models have been used to reproduce CD40 activation on B-cell and B-cell hybridomas by using soluble agonists or cellular ligands (Valle et al 1989;Bergman et al 1996;Tu et al 2008;Wiesner et al 2008). The effects observed in these in vitro models after CD40 activation (enhanced cell proliferation, cell survival and antibody production), make CD40 an ideal target for increasing the monoclonal antibody production by B-cell hybridomas, which are supposed to have a component of genes from normal B cells used for cell hybridization.…”