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

Abstract

The adoptive transfer of CD4 1 CD25 1 natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high-grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell-specific surface markers. Depletion of CD127 1 cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3 1 cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4 1 CD25 1 CD127 low Treg, correlating with loss of FOXP3 expression and emergence of pro-inflammatory cytokines. Further analysis identified CD45RA À FOXP3 1 memory-type Treg as the main source of converting cells, whereas CD45RA 1 FOXP3 1 Treg from the same donors showed no conversion within 3 wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell-type-specific characteristics after repetitive TCR stimulation.Key words: Cellular therapy . Immune regulation . Treg Supporting Information available online Introduction CD4 1 CD25 1 Treg are pivotal for the maintenance of peripheral self-tolerance and imbalances in this T-cell compartment have been shown to contribute to various autoimmune diseases [1]. In murine disease models, adoptively transferred Treg prevent, and in some cases, even cure autoimmunity [2,3]. In addition, they protect from graft rejection after allogeneic organ transplantation [4] as well as from graft-versus-host disease after MHCmismatched stem cell transplantation [5][6][7][8]. Recently, a limited number of Phase I clinical trials exploring the adoptive transfer of Treg have been initiated and several additional trials in various clinical settings are in preparation [9,10]. 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 1088Treg 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.Contrary to earlier reports, human CD4 1 CD25 high Treg in adult peripheral blood have recently been shown to comprise not only (self-)antigen-experienced, CD45RA À central and effector memory cells, but also a subpopulation of CD45RA 1 naïve recent thymic emigrants [14,15]. We previously demonstrated that these CD45RA 1 CD4 1 CD25 high T cells (RA 1 Treg) homogen...

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

“…However, this might not intrinsically be limited to iTreg since it has recently been shown that nTreg can also turn into Th17 effector cells after transfer into lymphopenic hosts [24]. Along the same lines, human nTreg have been reported to lose Foxp3 expression after repetitive TCR stimulation [25]. Intriguingly, we find that approximately $80% of the transferred nTreg stably remain Foxp3 1 even in the lymphopenic and inflammatory environment of acute GVHD.…”

Allogeneic Mature Human Dendritic Cells Generate Superior Alloreactive Regulatory T Cells in the Presence of IL-15

“…Mature moDC-expanded nTregs express high levels of FOXP3 and maintain the TSDR-demethylation status within the FOXP3 gene Long-term cultures of nTregs may result in loss of the nTregphenotype identified by diminished FOXP3 expression, which is associated with loss of suppressive capacity (47)(48)(49). In accordance with a previous publication (11), .90% of the freshly isolate nTregs expressed FOXP3, in contrast with a very low expression in T cells depleted for nTregs (effector T cells) (11).…”

Human Tumor Antigen-Specific Helper and Regulatory T Cells Share Common Epitope Specificity but Exhibit Distinct T Cell Repertoire

“…First, adaptive Foxp3 + Tregs in vitro appear to lose Foxp3 expression on restimulation without TGF-b and their TSDR demethylation levels, although higher than those of Th cells, were much lower than those of CD4 + CD25 high Tregs (26). Second, progressive loss of Foxp3 expression on repetitive in vitro stimulation has been reported in sorted CD4 + CD25 high CD127 2 Tregs as well as in CD4 + CD25 high CD45RA + Treg clones (40). In this study, the loss of Foxp3 expression was associated with the very heterogeneous profiles of Foxp3 expression between Treg clones as is the case for the TA-specific clones in our study.…”