Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb, TGF‐β, and RA treatment

Abstract

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long-term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti-CD4 antibody (aCD4). Here, we investigated whether adding TGF-β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg-cell generation and function. Murine CD4 + T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF-β+RA or aCD4+Rapa. Addition of TGF-β+RA or Rapa resulted in an increase of CD25 + Foxp3 + -expressing T cells. Expression of CD40L and production of IFN-γ and IL-17 was abolished in aCD4+TGF-β+RA aTreg cells. Additionally, aCD4+TGF-β +RA aTreg cells showed the highest level of Helios and Neuropilin-1 co-expression. Although CD25 + Foxp3 + cells from all culture conditions displayed complete demethylation of the Treg-specific demethylated region, aCD4+TGF-β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF-β+RA aTreg cells suppressed effector T-cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF-β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells. Keywords: Alloreactive regulatory T cells · Effector T cells · Nondepleting anti-CD4 monoclonal antibody · Retinoic acid · TGF-βAdditional supporting information may be found in the online version of this article at the publisher's web-site In recent years, adoptive transfer of Treg cells has gained major attention as an alternative or complementary therapy to conventional immunosuppressive treatments with the ultimate aim of reducing the side effects of conventional drugs [12,13]. Since only 5-10% of the circulating CD4 + cells in an organism are Foxp3 + Treg cells, their potential use for cell therapy seems to be limited and the peripheral population would require expansion [14]. Isolated CD4 + CD25 + cells frequently undergo expansion in the presence of aCD3/ aCD28 Ab and IL-2. Allo-specific expanded Treg cells seem to be more potent in suppressing chronic rejection, graft versus host disease (GvHD) and autoimmune diseases than polyclonal Treg cells. For example it was shown that antigen-specific expanded Treg (alloreactive Treg (aTreg)) cells could suppress experimental autoimmune diabetes more effectively than polyclonally expanded Treg cells [15].We have shown previously that in vitro culture of total murine CD4 + or CD25 − CD4 + cells in the presence of alloantigen and a nondepleting anti-CD4 antibody results in the enrichment of CD25 + CD62L + Foxp3 + T cells effective in controlling graft survival in vivo in an alloantigen-specific manner [16]. Although the in vitro enriched aTreg cells were effective in vivo, the protocol still has some limitations. To obtain almost pure Treg-c...

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection

“…A previous report by Sawitzki and colleagues showed that alloreactive Treg cells generated in vitro by combined anti-CD4 mAb, TGF-β, and RA treatment significantly prolonged graft survival due to the stabilization of Foxp3 expression [57]. In contrast to our setting, they generated alloreactive Treg cells by the addition of an anti-CD4 antibody, which has been described to perturb T-cell receptor signaling.…”

“…Moreover, we demonstrated that the RA-iTreg cells generated were de novo Treg cells and not a result of the expansion of already existing Treg cells because a highly purified population of naive T cells from the Foxp3-GFP knock-in mice was able to generate Treg cells in the presence of TILRA. A previous report by Sawitzki and colleagues showed that alloreactive Treg cells generated in vitro by combined anti-CD4 mAb, TGF-β, and RA treatment significantly prolonged graft survival due to the stabilization of Foxp3 expression [57]. In contrast to our setting, they generated alloreactive Treg cells by the addition of an anti-CD4 antibody, which has been described to perturb T-cell receptor signaling.…”

Relationship between CD4 Regulatory T Cells and Anergy In Vivo

“…However, this notion has since been challenged because activated T reg cells (which include both tT reg cells and pT reg cells) as well as inducible T reg cells generated in vitro from naive CD4 + T cells can express Nrp1 (73, 74). Similar to natural polyclonal T reg cells, anergy-derived polyclonal T reg cells demonstrated an ability to protect lymphopenic Tcra −/− mice from inflammatory bowel disease (49).…”

Neuropilin-1 in Transplantation Tolerance

“…Taken together, these results demonstrate the potential use of Nrp1+ T cells to abolish or diminish allograft effector responses, which can be complemented with current drugs to maximize the tolerogenic effect. In line with this report, Schliesser et al generated CD4+CD25+Foxp3+ Treg cells from total mouse CD4+ T-cell in vitro through treatments with anti-CD4, TGF-β, and Retinoic acid (RA) or anti-CD4 plus rapamycin, and showed that the majority of Foxp3-expressing Tregs generated under anti-CD4, TGF-β, and RA condition express Nrp1, mainly due to the expansion of nTregs instead of de novo iTregs (89). They also showed that anti-CD4, TGF-β, and RA expanded Tregs exhibited the highest stability and suppressive capacities both in vitro and in a skin transplantation setting, highlighting the role of Nrp1 in Treg cells.…”