Overcoming Rejection of Bone Marrow Allografts by Treg Cells in a Stringent Mouse Model for T Cell Mediated Rejection: Tolerance Induction by Third Party “Off-the-Shelf” Cells.

Recent reports have shown that donor or host CD4⁺CD25⁺ Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. More recent data suggests that in the context of T cell depleted BM allografting, engraftment was only mildly improved by Treg cells alone, or by Rapamycin (RAPA) alone, but it was markedly enhanced by using Treg cells in conjunction with RAPA. These studies were carried out in a mouse model specifically designed to measure T cell mediated graft rejection. In this model, lethally irradiated (11Gy) C3H mice were infused with 1x10⁴ purified host type T cells (HTC) and were transplanted one day later with 2x10⁶ BM cells from Balb-Nude donors, which are markedly depleted of T cells and do not induce GVHD. Rejection mediated by the HTC is manifested by severe aplasia and lethality within 21 days posttransplant. In 10 independent experiments none of the mice in the irradiation control survived (0/62), the majority of the mice receiving BM survived (58/63) while marked rejection, associated with poor survival (2/62) was found in the group receiving purified HTC prior to the BM transplant. In the present study we further tested in this model whether third party Treg cells could be used instead of donor or host Treg cells to overcome rejection of BM allografts. We initially tested freshly isolated lymph node CD4⁺CD25⁺ cells. C3H (H2^k) recipients received BM from Balb- Nude (H2^d) donors and the Treg cells were obtained from Balb/c or FVB (H2^q) donors. As in our previous study, while none of the recipients survived upon treatment with RAPA alone, using third party or donor type Treg cells in conjunction with RAPA led to survival of 9 of 13 and 7 of 10 mice respectively. Thus, the third party fresh Treg cells were as effective as the donor type cells in preventing graft rejection (P>0.05). Considering the low levels of CD4⁺CD25⁺ cells in peripheral blood or spleen, new strategies for growing these cells ex-vivo have been developed. Although, Treg cells exhibit low proliferative potential in-vitro upon TCR stimulation, the feasibility of growing mouse or human regulatory cells has been demonstrated mainly using the combination of TCR stimulation (either with an anti-TCR antibody or with allogeneic stimulator cells), costimulatory signals and high doses of IL-2. When tested in the same model, Treg cells ex-vivo expanded by stimulation against 4th party allogeneic cells, exhibited effective enhancement of engraftment of Balb-Nude BM. Thus, in four independent experiments, when assessing treatment with expanded Treg cells, of third party or donor type origin, the survival rate was 19 of 35 (54%) and 25 of 40 (62%) mice, respectively. Again, in both instances the marked potential of Treg cells to overcome T cell mediated rejection was exhibited only when co-administered with RAPA. In conclusion, our data strongly indicate that, at least in the bone marrow transplantation setting, third party Treg cells could afford a new viable ‘off-the-shelf’ source for tolerance induction. The use of third party Treg cells in contrast to donor type cells could allow advanced preparation of a large bank of Treg cells, with all the appropriate quality controls required for cell therapy. Further studies with human Treg cells in-vitro are required to ascertain the potential of third party cells as a valuable source for clinical transplantation.