CD27/CFSE-Based Ex Vivo Selection of Highly Suppressive Alloantigen-Specific Human Regulatory T Cells

TLDR

CD4+CD25+ regulatory T cells are essential for immune regulation and hold therapeutic promise for preventing transplant rejection, allergy, and autoimmune disease, but their efficacy hinges on antigen specificity. The study aims to develop an ex vivo CFSE‑based sorting method to isolate and expand highly potent, alloantigen‑specific Tregs, thereby reducing the total cell dose required for therapy. Freshly isolated CD4+CD25+ Tregs were labeled with CFSE, stimulated with alloantigen plus IL‑2/IL‑15, and the dividing, low‑CFSE cells were sorted and further distinguished by CD27 expression. Sorting CD27+ and CD27− Tregs yielded two highly suppressive alloantigen‑specific subsets that inhibited both naive and memory T cells, with CD27+ cells also suppressing ongoing responses, demonstrating the method’s capacity to generate potent, antigen‑specific Tregs for therapy.

Abstract

Abstract Naturally occurring CD4+CD25+ regulatory T cells (Treg) are crucial in immunoregulation and have great therapeutic potential for immunotherapy in the prevention of transplant rejection, allergy, and autoimmune diseases. The efficacy of Treg-based immunotherapy critically depends on the Ag specificity of the regulatory T cells. Moreover, the use of Ag-specific Treg as opposed to polyclonal expanded Treg will reduce the total number of Treg necessary for therapy. Hence, it is crucial to develop ex vivo selection procedures that allow selection and expansion of highly potent, Ag-specific Treg. In this study we describe an ex vivo CFSE cell sorter-based isolation method for human alloantigen-specific Treg. To this end, freshly isolated CD4+CD25+ Treg were labeled with CFSE and stimulated with (target) alloantigen and IL-2 plus IL-15 in short-term cultures. The alloantigen-reactive dividing Treg were characterized by low CFSE content and could be subdivided by virtue of CD27 expression. CD27/CFSE cell sorter-based selection of CD27+ and CD27− cells resulted in two highly suppressive Ag-specific Treg subsets. Each subset suppressed naive and Ag-experienced memory T cells, and importantly, CD27+ Treg also suppressed ongoing T cell responses. Summarizing, the described procedure enables induction, expansion, and especially selection of highly suppressive, Ag-specific Treg subsets, which are crucial in Ag-specific, Treg-based immunotherapy.