Adoptive transfer of xenoantigen‑stimulated T cell receptor Vβ‑restricted human regulatory T cells prevents porcine islet xenograft rejection in humanized mice

Abstract

Polyclonal expansion of human regulatory T cells (Tregs) prevents xenogeneic rejection by suppressing effector T cell responses in vitro and in vivo. However, a major limitation to using polyclonally expanded Tregs is that they may cause pan-immunosuppressive effects. The present study was conducted to compare the ability of ex vivo expanded human xenoantigen-stimulated Tregs (Xeno-Treg) and polyclonal Tregs (Poly-Treg) to protect islet xenografts from rejection in NOD-SCID interleukin (IL)-2 receptor (IL2r)γ−/− mice. Human cluster of differentiation (CD)4+CD25+CD127lo Tregs, expanded either by stimulating with porcine peripheral blood mononuclear cells (PBMCs) or anti-CD3/CD28 beads, were characterized by immune cell phenotyping, T cell receptor (TCR) Vβ CDR3 spectratyping and performing suppressive activity assays in vitro. The efficiency of adoptively transferred ex vivo human Tregs was evaluated in vivo using neonatal porcine islet cell clusters (NICC) transplanted into NOD-SCID IL-2rγ−/− mice, which received human PBMCs with or without Xeno-Treg or Poly-Treg. Xeno-Treg, which expressed increased levels of human leukocyte antigen-DR and secreted higher levels of IL-10, demonstrated enhanced suppressive capacity in a pig-human mixed lymphocyte reaction. Spectratypes of TCR Vβ4, Vβ10, Vβ18 and Vβ20 in Xeno-Treg showed restriction and expanded clones at sizes of 205, 441, 332 and 196 respectively, compared to those of Poly-Treg. Reconstitution of mice with human PBMCs and Poly-Treg resulted in NICC xenograft rejection at 63 days. Adoptive transfer with human PBMCs and Xeno-Treg prolonged islet xenograft survival beyond 84 days, with grafts containing intact insulin-secreting cells surrounded by a small number of human CD45+ cells. This study demonstrated that adoptive transfer of ex vivo expanded human Xeno-Treg may potently prevent islet xenograft rejection in humanized NOD-SCID IL2rγ−/− mice compared with Poly-Treg. These findings suggested that adoptive Treg therapy may be used for immunomodulation in islet xenotransplantation by minimizing systemic immunosuppression.

Neonatal Pig Sertoli Cells Survive Xenotransplantation by Creating an Immune Modulatory Environment Involving CD4 and CD8 Regulatory T Cells

“…Human CD4 Tregs exposed to pig xenoantigens have been shown to display xenospecific suppressive activity in vitro and in some studies these cells prolonged graft survival once they were transferred to an in vivo model 43 . Furthermore, baboon or human CD4 Tregs exposed to pig xenoantigens prior to adoptive transfer prolonged graft survival and suppressed T-cell responses 44,45 . Moreover, tolerance to nonsequestered meiotic germ cell antigens in the testis is CD4 Treg dependent 46 .…”

Progress towards xenogenic tolerance

“…In mice reconstituted with Xeno-or Poly-Treg and PBMC at a ratio of 1:10, it was found that over 75% of NICC xenografts survived over 84 days in the Xeno-Treg group vs 48 days in the Poly-Treg group, suggesting Xeno-Tregs may have greater ability to suppress xenogeneic cellular rejection. 38 In NHP, our group has reported that polyclonal Tregs administered at the time of GalTKO/hCD47Tg stem cells prolong the duration of donor chimerism. In these animals, we tested the potential for tolerance development by transplanting donor or donor-matched skin grafts without any maintained immunosuppression.…”

“…A delayed allogenic kidney transplant was tolerated for over 290 days without immunosuppression in one of these animals, compared to a maximum of 4 weeks in those receiving BMT alone, suggesting that Tregs play a role in promoting chimerism and immunomodulation of allogeneic responses. 37 Recently, Jin et al 38 studied ex-vivo human Tregs expanded with xenoantigen stimulation (Xeno-Treg) and their effect on porcine neonatal islet cell clusters (NICC) in NOD-SCID IL-2rγ−/− mice. After adoptive transfer of human PBMC and infusion of ex-vivo expanded Xeno-Tregs or polyclonal Tregs (Poly-Tregs), NICC engraftment and survival was determined.…”

Cellular Immune Responses in Islet Xenograft Rejection

“…This was supported by data showing enhanced in vitro suppression of human-anti-pig T cell responses by human CD4 + CD25 +high CD127 - Tregs that were expanded in vitro in the presence of pig peripheral blood mononuclear cells (PBMCs) combined with IL-2/Rapamycin and anti-CD3/CD28 magnetic bead stimulation ( 113 , 114 ). These enhanced human-anti-pig ( 112 , 115 ) or baboon-anti-pig Tregs proved effective in prevention of islet xenograft rejection in humanized or baboonized mouse models ( 114 ).…”