Identification and expansion of highly suppressive CD8+FoxP3+ regulatory T cells after experimental allogeneic bone marrow transplantation

Abstract

FoxP3 ؉ confers suppressive properties and is confined to regulatory T cells (T reg ) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4 ؉ T reg are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8 ؉ population of FoxP3 ؉ T reg that convert from CD8 ؉ conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8 ؉ T reg undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-␤. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4 ؉ FoxP3 ؉ population and is more potent in exerting class I-restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8 ؉ FoxP3 ؉ T reg are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8 ؉ FoxP3 ؉ T reg thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I-restricted T-cell responses after bone marrow transplantation. (Blood. 2012;119(24): 5898-5908) IntroductionThe development of GVHD remains a major limitation to the therapeutic potential of bone marrow transplantation (BMT). GVHD occurs as a result of immunologic damage to the host tissue by conventional T and NK cells in the transplanted donor graft. Natural regulatory T cells (T reg ) are defined by the expression of FoxP3 1 and are dedicated to suppression of immune responses. The importance of CD4 ϩ FoxP3 ϩ T reg in peripheral tolerance is well established, and the presence of naturally occurring CD4 ϩ T reg makes them an attractive target for adoptive transfer to control pathologic immune responses. Consequently, studies in GVHD using freshly isolated or ex vivo expanded donor CD4 ϩ T reg have demonstrated a delay or prevention of GVHD. [2][3][4][5] Conversely, depletion of CD4 ϩ T reg from the donor graft results in an increased severity of GVHD. 2 A limitation to using naive T reg in the adoptive transfer setting is the high ratio required compared with FoxP3 Ϫ cells in the ingoing graft (1:1); thus, the number of T reg required is restrictive. This has resulted in difficulties transferring adoptive cell therapy into the clinic with multiple groups now focusing on ex vivo expansion protocols in an endeavor to overcome this limitation. [6][7][8] In contrast to the extensive research being completed on naive CD4 ϩ T reg , studies investigating the role of T reg undergoing conversion in the periphery after BMT are lacking. The identification of FoxP3 as a T reg marker 1 and subsequent development of reagents that allow for identification (B6.FoxP3-GFP), 9 tracking (B6.FoxP3.LuciDTR) 10 and depletion (B6.DEREG) 11 of these cells has allowed for a more thorough investigation. We have used these reagents to characterize T reg populations after transplantation with the aim of identifying m...

Heterogeneity in the Differentiation and Function of CD8+ T Cells

“…This strong anti- Regulation of T-cell-mediated responses (Kim and Cantor 2011;Robb et al 2012;Tsai et al 2010) tumor activity was dependent on the marker cytokine of Tc9 cells, IL-9, but not on IFN-c. In this setting, adoptively transferred Tc9 cells converted after 3 weeks to IFN-c and IL-2 producers and displayed a phenotype similar to Tc1 memory cells characterized by KLRG1 lo and IL-7Ra expression (Lu et al 2014).…”

“…Treg cells with preferential tropism for the gastrointestinal tract can be detected. These cells prevent graft versus host disease (GvHD) by inhibition of MHC class I-restricted T-cell responses and the suppressive T-cell population can be expanded by co-administration of rapamycin and IL-2/anti-IL-2 antibody complexes (Robb et al 2012 (Cho et al 2012) and human Tc17 cells predominantly belong to memory subsets (Kondo et al 2009), suggesting that at least in humans both subpopulations are able to establish memory cells. In a mouse tumor model, transferred Tc9 cells acquired a Tc1 memory phenotype, indicating that memory formation was associated with the transition into Tc1 cells (Lu et al 2014).…”

Induced Regulatory T Cells Promote Tolerance When Stabilized by Rapamycin and IL-2 In Vivo

“…Foxp3-expressing regulatory T cells (Tregs) include two major groups: those generated at steady-state within the thymus ("so-called" natural Tregs [nTreg]) and those induced regulatory T cells (iTreg) that are generated in the periphery from Foxp3 2 T cells. Both play an important role in immune regulation, including that after BMT (7)(8)(9)(10). The adoptive transfer of freshly isolated or in vitro-expanded nTregs was shown to be an effective means to suppress GVHD and restore immune tolerance (11)(12)(13).…”

“…We (7) and other investigators (30,31) recently described the in vivo generation of highly suppressive CD8 + Foxp3 + iTregs in response to alloantigen. These iTregs appear to be more potent at suppressing MHC class I-restricted immune responses than are their CD4 counterparts (7). The in vitro generation and function of these CD8 iTregs has received only limited study.…”

Antigen-Encoding Bone Marrow Terminates Islet-Directed Memory CD8+ T-Cell Responses to Alleviate Islet Transplant Rejection

“…Ablation of Memory OVA-Specific CD8 + T-Cell Responses by OVA-Encoding BM Does Not Establish OVA-Specific "Regulation" Using irradiation to facilitate BM engraftment has been associated with generation or expansion of CD4 + or CD8 + Treg populations that modulate subsequent immune responses (23,24). To determine whether OVA-encoding BM transfer induced a regulatory response that inhibited subsequent OVA-specific T-cell activation, we adapted a widely used "regulation" assay and compared proliferation of a second adoptively transferred population of OVAspecific CD8 + T cells in mice where OVA-specific memory CD8 + T cells had been inactivated or not by BM transfer.…”