Early production of donor-derived CD4+ CD25+ regulatory T cells in patients given hematopoietic stem cell transplants

Abstract

In previous studies using murine models of BMT/DLI therapy, we found that donor-BM-derived CD4+ regulatory T cells (T-REG) produced in the host thymus were responsible for suppression of DLI-associated GVHD. Based on our mouse data, we hypothesized that failure of the host thymus to produce T-REG cells after allogeneic BMT in humans would result in an increased risk of GVHD after DLI therapy and an increased risk of chronic GVHD. Here, we sought to identify, expand and functionally characterize T-cells over time in patients given hematopoietic stem cell transplants (HSCT). Peripheral blood (PB) was collected from 19 adult and 8 pediatric patients at 2, 4, 6 and 12 mo. post-transplant. T-REG cells were identified by staining ficoll-separated PB cells for coexpression of CD4 and CD25. T-REG cells were defined phenotypically as CD4+ cells with high levels of CD25 staining. 88% (22/25) of patients analyzed at 2 mo. had detectable levels of CD4+ CD25+ cells. These cells persisted over time; only two patients with detectable T-REG cells at 2 mo. lost them by 6 mo. Some, but not all, CD4+ CD25+ cells had high TREC levels suggesting that they were recent thymic emigrants. We purified CD4+ CD25+ cells and CD4+ CD25- cells by cell sorting when sufficient PB was available and expanded them ex vivo using anti-CD3/anti-CD28 conjugated Dynal beads and rIL-2. The expanded CD4+ CD25+ cells were confirmed as being of donor-origin by VNTR analysis. Most were functional as shown by their ability to suppress T cell proliferation when titrated into MLR assays. Expanded CD4+ CD25+ cells from 5 of 8 patients suppressed proliferation of T cells from the original HSC donor (i.e., autologous T cells) by 26–94% (R : T-REG = 1:1). When donor T cells were not available, unrelated T cells were used in the MLR assays. Ex vivo expanded cells from 6 of 7 patients suppressed the proliferation of the allogeneic responder T cells by 34–77% (R : T-REG = 1:1). Our results indicate that functional CD4+ CD25+ regulatory T cells of donor origin are present early after HSCT in most patients. The patients, tissue source and histocompatibility of the HSCs used in this study were heterogeneous. Determining whether the de novo generated T-REG cells are sufficient to mitigate GVHD will require a more homogeneous patient group and longer follow-up. Adoptive transfer of T-REG cells isolated from the HSCT donor and expanded ex vivo may be necessary to more effectively modulate GVHD.