Rapamycin and IL-2 Prevents Lethal Acute Graft-Versus Host Disease by Expansion of Donor Type CD4⁺CD25⁺Foxp3⁺ Regulatory T Cells.

Abstract 1334

Poster Board I-356

Previous work has demonstrated that both rapamycin (RAPA) and IL-2 enhance CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) proliferation and function. We investigated whether the combination of RAPA and IL-2 administered in vivo could reduce acute graft-versus-host disease (aGVHD) induction and increase survival after bone marrow transplantation (BMT) by induction of Treg. T cell depleted bone marrow (TCD-BM) cells from wild type C57BL/6 mice were injected after lethal irradiation with (800 cGy) into Balb/c recipients on day 0. To induce aGVHD and evaluate the proliferation of donor T cells by in vivo bioluminescence imaging, 1 × 10⁶ conventional CD4⁺ and CD8⁺ T cells (Tcon) from luciferase-expressing (luc⁺) transgenic C57BL/6 were injected intravenously on the same day. RAPA was administered intraperitoneally (dose of 0.5 mg/kg/day) for 14 days and IL-2 (dose of 5 × 10⁴ IU) for 3 days (twice a day). RAPA plus IL-2 significantly reduced the expansion of luc⁺ Tcon more than RAPA (P=0.04) or IL-2 alone (P=0.002) or no treatment (P = 0.01). Weight loss and aGVHD score were significantly reduced in the mice which were injected with the combination of RAPA and IL-2 compared with those animals injected with RAPA (P = 0.03) or IL-2 alone (P = 0.05). The percentage of donor type CD4⁺CD25⁺ cells was increased and CD4⁺CD25⁻ T cells were reduced from thymic and extrathymic tissues after treatment with RAPA plus IL-2 on day 7 after BMT. The combination of RAPA and IL-2 resulted in a 2.4 fold (mesenteric LN), 2.7 fold (peripheral LN), 4.2 fold (spleen) and 2.1 fold (thymus) increase in the percentage of donor type CD4⁺CD25⁺Foxp3⁺ Treg. In animals receiving the combination of RAPA and IL-2, the percentage of CD4⁺CD25⁺Foxp3⁺ T cells increased from 8.98% (RAPA alone) to 24.2% (RAPA plus IL-2) at 7 days after BMT, representing a 2.7 fold expansion. The cell numbers of CD4⁺ and CD8⁺ T cells and CD4⁺CD25⁻ T cells were reduced and CD4⁺CD25⁺Foxp3⁺ Treg were expanded in the thymus and extrathymic tissues after administration RAPA and IL-2. To study the origin of the expanded Treg in RAPA plus IL-2, BALB/c recipient mice were injected with purified donor CD4⁺CD25^highFoxP3⁺ Treg and CD4⁺CD25⁻ and CD8⁺CD25⁻ (both Foxp3⁻) Tcon after lethal irradiation followed by RAPA plus IL-2 for 7 days. The combination of RAPA and IL-2 increased the expansion of donor type CD4⁺CD25⁺Foxp3⁺ Treg, but did not result in an increase in the conversion of Foxp3⁺ Treg from donor CD25⁻ Tcon. We also evaluated whether Foxp3⁺ non-Treg were present in the expansion of Treg by RAPA plus IL-2 by evaluating interferon-γ- and IL-2-production which was minimally detected in the Foxp3⁺ cells. In conclusion, the combination of RAPA and IL-2 after BMT synergistically promoted the expansion of donor CD4⁺CD25⁺Foxp3⁺ Treg resulting in prevention of lethal aGVHD.