Selective Depletion of CD25+ Host-Reactive Donor Lymphocytes from Allografts Preserves a CD25− CD4+foxp3+ Fraction of T Cells and Thereby Provides a Source for Efficient Reconstitution of Regulatory T Cells and Additional GVHD Control.

Abstract

We previously showed that removal of host-reactive donor T cells from allografts by anti-CD25 immunotoxin (IT) is clinically feasible and reduces the frequency of severe graft-versus-host-disease (GVHD) in a high-risk group of elderly patients undergoing matched-sibling transplantation (Blood , 2005, 106:1123). However, it is possible that the concurrent removal of CD4+CD25+ regulatory T cells (Tregs) with alloactivated CD25+ T cells could precipitate more GVHD if depletion of alloreacting cells were incomplete. We, therefore, studied the recovery of Tregs in 16 patients receiving CD25-IT-treated stem cell grafts during the first 150 days after transplantation. Tregs were characterized by surface phenotyping for CD3, CD4, CD25 and CD27, intracellular staining for forkhead protein3 (foxp3) and quantitative real time PCR for foxp3 gene. We also measured Tregs in 13/16 donors (Don), 10/16 SD products and 13/16 patients pretransplant (Pre). Patients received a median of 1.0 (0.2–1.5) ×108/kg selectively depleted (CD25−) CD3+ T cells and a stem cell product containing 0.25 ×104/kg (0.1–5.0) residual unselected CD3+ T cells. Lymphocyte recovery was prompt with a median lymphocyte count of 613/μL (215–2883/μL) and median 97% donor T cell chimerism at day+30. Despite CD25-depletion, patients reconstituted sufficient levels of CD4+foxp3+ T cells at day+30. These CD4+foxp3+ T cells underwent further expansion and reached their highest median levels 150 days post transplantation (Figure A). Interestingly, all SD-products contained a significant fraction of CD4+foxp3+ Tregs that persisted after complete CD25-depletion. These cells were exclusively CD25− CD4+foxp3+ Tregs and represented 1.5–4.8% of the CD4+ cells in the SD product. Of note, the Treg content of the donor was associated with the risk of GVHD development posttransplant. Acute grade II GVHD was restricted to 5 patients whose donors had significantly fewer Tregs compared to those with no or grade I GVHD (Figure B). In this study we show efficient Treg reconstitution despite giving a CD25-depleted SD allograft. We identified a population of CD25− CD4+foxp3+ Tregs in the SD product. Such cells have been shown to exert regulatory function in mice. Our results suggest that Treg recovery following SD is derived from a major contribution of CD25− Tregs which escape the depletion process in addition to a residual fraction of CD25− and CD25+ Tregs delivered with the stem cell product. All these Treg fractions may undergo marked expansion post-transplant and provide additional GVHD protection if the removal of alloreacting T cells by the SD procedure were incomplete. [Display omitted]