Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion

Abstract

Thymus-derived CD4 ؉ CD25 ؉ regulatory T cells suppress autoreactive CD4 ؉ and CD8 ؉ T cells and thereby protect from autoimmunity. In animal models, adoptive transfer of CD4 ؉ CD25 ؉ regulatory T cells has been shown to prevent and even cure autoimmune diseases as well as pathogenic alloresponses after solid organ and stem-cell transplantations. We recently described methods for the efficient in vitro expansion of human regulatory T cells for clinical applications. We now demonstrate that only CCR7-and L-selectin (CD62L) - IntroductionSelf-tolerance within the T-cell compartment is primarily ensured by positive and negative selection during T-cell development in the thymus. Autoreactive T cells that escape central deletion are frequently controlled by peripheral tolerance mechanisms, including cell-mediated suppression by CD4 ϩ CD25 ϩ regulatory T (Treg) cells. [1][2][3] Thymic maturation and suppressive function of natural Treg cells depend on expression of the transcriptional repressor forkhead box P3 (FOXP3), as nonsense mutations in this gene result in loss of Treg-cell function and severe autoimmunity in mice and humans. [4][5][6][7][8] Apart from suppression of autoreactive T cells, FOXP3 ϩ CD4 ϩ CD25 ϩ Treg cells also dampen immune responses against infectious pathogens, 9 cancer, 10 and allogeneic organ 11 and stem-cell grafts. 12 Thus, depletion of Treg cells seems a promising strategy to augment immune responsiveness to tumors, chronic infections, and vaccination, while an enhancement of Treg-cell activity is envisaged for the prevention and treatment of T-cellinduced diseases. 13 In animal models, the adoptive transfer of CD4 ϩ CD25 ϩ Treg cells has been shown to protect from type 1 diabetes 14,15 or experimental autoimmune encephalomyelitis 16 and even revert ongoing disease in colitis 17,18 and arthritis. 19 Similarly, adoptively transferred Treg cells protected against rejection and graft-versus-host disease (GVHD) after allogeneic organ transplantation and bone marrow transplantation (BMT), respectively. 11,[20][21][22][23][24][25] In human peripheral blood, natural Treg cells mainly reside within the subpopulation of CD4 ϩ T cells with high CD25 expression levels (CD25 high ), 26 while cells with intermediate CD25 expression (CD25 int ) consist mainly of recently activated and memory T cells, with only 5% to 15% FOXP3 ϩ Treg cells (P.H. and M.E., unpublished results, June 2006). Due to lack of Treg cell-specific surface markers, isolation of CD4 ϩ CD25 high T cells is thus far considered the most promising strategy for the generation of pure Treg-cell products. 27 As they represent only 1% to 3% of peripheral-blood mononuclear cells (PBMCs), we and others recently described methods for the in vitro expansion of Treg cells for future clinical trials. [28][29][30] Cross-linking of stimulating CD3 and CD28 antibodies by beads or Fc receptor-bearing fibroblasts in combination with high-dose interleukin-2 (IL-2) resulted in a 3-to 4-log expansion within 3 to 4 weeks. Cultured cells maintained Treg-c...

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

“…This happened although reproducibly more than 90% of the sorted cells expressed FOXP3 on the day of isolation ( [16] and H. and M. E. unpublished data). Possible explanations for the observed heterogeneity could be either contamination of the starting population with CD25 1 , non-regulatory Tconv that preferentially expand in culture over time, or, alternatively, the presence of a substantial number of ''converted'' or ''induced'' Treg that only transiently express FOXP3 after recent in vivo activation [22,23] and that re-convert to effector T cells during in vitro culture.…”

“…We previously showed that the heterogeneity in FOXP3 expression observed after prolonged in vitro expansion of bulk CD4 1 CD25 high T cells was predominantly caused by the emergence of FOXP3 À cells in the CD45RA À subfraction of the initial cell population [16]. Because at that time we had not included CD127 in our sorting strategy, we could not formally rule out contamination of the starting population with activated Tconv cells (which would be expected to reside primarily among CD45RA À memory cells).…”

“…We previously demonstrated that these CD45RA 1 CD4 1 CD25 high T cells (RA 1 Treg) homogeneously maintain their Treg cell phenotype and function upon in vitro expansion, whereas CD4 1 T cells selected solely for CD25 high expression were highly variable with respect to FOXP3 expression (50.5 to 72.8% FOXP3 1 cells) after long-term culture [16]. Based on these findings, we proposed that the selection of CD45RA 1 naïve Treg is the best strategy for the generation of homogeneous Treg cell products for future clinical trials.…”

“…3). Thus, epigenetic DNA modifications in the FOXP3 gene locus indicate that CD127 À Treg partially lose their lineage-specific signature during in vitro expansion, whereas this is not the case for RA 1 Treg isolated from the same donors.Heterogeneity of CD127 À Treg cultures is caused by CD45RA À Treg in the starting populationWe previously showed that the heterogeneity in FOXP3 expression observed after prolonged in vitro expansion of bulk CD4 1 CD25 high T cells was predominantly caused by the emergence of FOXP3 À cells in the CD45RA À subfraction of the initial cell population [16]. Because at that time we had not included CD127 in our sorting strategy, we could not formally rule out contamination of the starting population with activated Tconv cells (which would be expected to reside primarily among CD45RA À memory cells).…”

“…For this purpose, RA 1 and CD127 À Treg were isolated from the same leukapheresis products by high-speed flow cytometric cell sorting (FACS). Both cell populations showed 498% purity with regard to the applied sort gates: RA 1 Treg were isolated according to their particular expression levels of CD25 (expression level above that of CD4 À PBMC) and CD45RA (only CD45RA high cells), as described previously [16] and detailed in Fig. 1A Fig.…”