Homeostatic Expansion of Donor FoxP3+ CD25+CD4+ T Cells in Recipients of CD4+ Cells Depleted Allogeneic Hematopoietic Graft.

Background: In allogeneic BMT patients, the presence of allo-reactive donor CD4+ T cells in the graft were reported to be the primary cause of GvHD. Moreover, donor T-cells are required to promote the stem cell engraftment and to decrease the disease relapse. A number of studies also reported that a subset of CD4+CD25+ T cells usually generated de novo from the thymus that expressed FoxP3 regulate the T cells allo-reactivity in vivo. Thus, to establish a therapeutically useful adoptive T-cells immunotherapy, we depleted the CD4+ T cells from the graft and transplanted along with T cell depleted (TCD) BM cells in clinically relevant parent to F1 experimental allogeneic BMT model. Our hypothesis is that CD4-depleted graft will not cause GvHD, preserve the thymic function, homeostatically produce donor BM-derived CD4+ T cells along with FoxP3+CD4+CD25+ regulatory T cells with beneficial anti-opportunistic infection and anti-tumor effects.

Methods: We used a parent (C57BL/6) to (C57BL/6 X BALB/c)CB6F1 allogeneic BMT model with a combination of TCD BM and splenocytes as the hematopoietic graft. CD4+ or CD8+ cells were selectively depleted from the splenocytes of C57BL/6 donor mice using MACS column. 1×10⁶ CD4-depleted splenocytes or a mixture of 2×10⁶ CD8-depleted and 1×10⁶ CD4-depleted splenocytes and/or grafts containing 10×10⁶ unfractionated splenocytes along with 5×106 TCD BM cells harvested from the congeneic C57BL/6 donor mice, were adoptively transferred to lethally irradiated (11Gy) CB6F1 mice. GvHD was monitored twice weekly by weight loss and other clinical signs. After 50 days post transplant recipients mice were bled or sacrificed and lymphocytes isolated from blood and different organs were analyzed by multicolor FACS.

Results: Within 50 days of transplant the recipients of CD4-depleted splenocytes had 100% survival without GvHD whereas recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes suffered from severe GvHD (%weight loss below 20%) with 50% survival. Surprisingly, very significantly expansion of total CD4+ T cells (37% ± 7% of lymphocytes, CD4:CD8 ratio 6:1) occurred in the blood of recipients of CD4-depleted splenocytes. In contrast the recipients of mixture of CD4- and CD8-depleted splenocytes DLI or whole splenocytes had only few CD4+ T cells (~2% ± 2% of lymphocytes, CD4:CD8 ratio 1:2). Over 90% of the CD4+ T cells in the blood of recipients of CD4-depleted splenocytes were from the donor BM and included significantly higher number of CD25+CD4+ T cells compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes. Similarly, significantly increased numbers of FoxP3+CD25+CD4+ regularity T cells were also found in the spleen and thymus of recipients of CD4-depleted splenocytes compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes (p<0.005).

Conclusion: Adoptive immunotherapy with the CD4-depleted hematopoietic graft results better immune reconstitution, caused extensive homeostatic expansion of donor stem cell-derived CD4+ T cells including significantly increased levels of FoxP3+CD25+ CD4+ regulatory T cells derived from de novo thymopoiesis without GvHD. The presence of donor FoxP3+CD25+ CD4+ regulatory T cells in the hematopoietic graft are not necessary for post-transplant expansion of donor stem-cell-derived regulatory T-cells via thymopoiesis.