Large-scale in vitro expansion of polyclonal human CD4+CD25high regulatory T cells

Abstract

IntroductionImmunosuppression is an intrinsic capacity of the immune system and is partially mediated by T cells. The best-defined T-cell population with immunosuppressive activity is CD4 ϩ and constitutively expresses the interleukin 2 receptor (IL-2R) ␣-chain (CD25). 1 These thymus-derived suppressor cells 2,3 contribute to the maintenance of self-tolerance and thereby give protection from a variety of autoimmune diseases. 1,4 They control the size of the peripheral T-cell pool, 5 modulate immune responses after infection 6 and against tumors, 7,8 and contribute to tolerance induction after solid organ transplantation. 9,10 These findings confirm the central role of T reg cells in the modulation of immune responses under physiologic as well as pathologic conditions.We and others recently showed that donor-type CD4 ϩ CD25 ϩ T reg cells themselves do not induce graft-versus-host disease (GVHD) after major histocompatibility complex (MHC)-mismatched bone marrow transplantation but suppress GVHD induced by nonregulatory donor T cells. [11][12][13][14] Importantly, cotransfer of CD4 ϩ CD25 ϩ T reg cells neither interferes with stem cell engraftment [15][16][17] nor abrogates the beneficial antitumor activity of donor T-cell infusions. 15,18,19 These murine studies encouraged clinical transplanters to further investigate the role of donor CD4 ϩ CD25 ϩ T reg cells for the improvement of stem cell transplantation (SCT).CD4 ϩ T cells with high expression levels of CD25 have been isolated from human blood, peripheral lymphoid organs, umbilical cord blood, and thymus. [20][21][22][23][24][25] Phenotypically, they are comparable to their murine counterparts; that is, they constitutively express CD25, glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) and intracellular cytotoxic T lymphocyteassociated antigen-4 (CTLA-4). In addition, expression of the transcription factor forkhead box P3 (FoxP3), described recently as a key regulatory gene for the development of CD4 ϩ CD25 ϩ T cells in mice, 26 could now be demonstrated in human CD4 ϩ CD25 ϩ T reg cells. 27 Most importantly, however, functional characteristics of murine CD4 ϩ CD25 ϩ T reg cells could be confirmed for human CD4 ϩ CD25 ϩ/high T reg cells, such as hyporesponsiveness to T-cell receptor (TCR)-mediated stimulation and cytokine-independent, cell contact-dependent suppression of cocultured CD25 Ϫ T cells. 20,21,23,25 Thus, immunoregulation by CD4 ϩ CD25 ϩ T reg cells seems to be a conserved mechanism, and their adoptive transfer or therapeutic manipulation in vivo might therefore be an attractive strategy for the prevention or treatment of T cell-mediated diseases and, in particular, for the protection from GVHD after allogeneic SCT.The main obstacle for the clinical application of human CD4 ϩ CD25 ϩ T reg cells so far is their paucity in peripheral blood and, in consequence, the necessity to develop reliable expansion protocols. Although several studies demonstrated that exogenous IL-2 can abrogate the anergic state of human and muri...

Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation

“…CD25-enriched and -depleted cell populations were stained with anti-CD4 and respective additional antibodies to sort them into CD4 1 CD25 À and CD4 1 CD25 high T cells plus the indicated subpopulations (CD45RA 1/À and/or CD127 À ) on a FACS-ARIA s high-speed cell sorter (BD Biosciences). Sort gates for the isolation of CD4 1 CD25 high T cells excluded PI-positive dead cells and were otherwise set as described in detail before [11]. Sort gates for RA 1 Treg and CD 1 CD25 1 CD127 À subpopulations were set as shown in Fig.…”

“…Prerequisites for the initiation of such trials are (i) the availability of efficient in vitro expansion protocols for this rare cell population and (ii) the ability to unequivocally identify Treg to avoid contamination of Treg cultures with potentially harmful conventional effector T cells (Tconv). While efficient cell culture protocols have recently been established by us and others for the polyclonal as well as antigen-specific Treg cell expansion [11][12][13], the search for an exclusive surface marker for Treg is still ongoing.…”

“…1. Upon re-analysis, sorted cells routinely showed498% purity.T-cell expansion cultures were performed as described in detail before [11]. Briefly, T cells were stimulated with 100 ng/mL each of anti-CD3 (OKT3, Orthoclone s , a kind gift from Ortho Biotech (Neuss, Germany)), and anti-CD28 (CD28.2, BD Biosciences), presented by L929 cells bearing the human FcgRII (CD32) in the presence of 300 U/mL recombinant human IL-2 (Proleukin s , Chiron (Amsterdam, Netherlands)).…”

“…While efficient cell culture protocols have recently been established by us and others for the polyclonal as well as antigen-specific Treg cell expansion [11][12][13], the search for an exclusive surface marker for Treg is still ongoing.…”

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].…”