Alloactivation of Naïve CD4+CD8-CD25+T Regulatory Cells: Expression of CD8α Identifies Potent Suppressor Cells That Can Promote Transplant Tolerance Induction.

Nirupama D Verma,Paul Wilcox,Suzanne J Hodgkinson,Catherine M Robinson,Alexandra Sharland,Nicole Carter,Masaru Nomura,Karren M Plain,Bruce M Hall,Giang T Tran,Chaunmin Wang,G Alexander Bishop

doi:10.3389/fimmu.2019.02397

Abstract

Therapy with alloantigen-specific CD4+CD25+ T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are not alloantigen-specific and are weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells in the presence of rIL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rβ2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined additional markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4+CD25+Foxp3+Treg. After culture of naïve DA CD4+CD8−CD25+T cells with rIL-2 and PVG alloantigen, or rIL-2 without alloantigen, CD8α was expressed on 10–20% and CD8β on <5% of these cells. These cells expressed ifngr and Il12rb2. CD8α+ cells had increased Ifngr that characterizes Ts1 cells as well was Irf4, a transcription factor induced by TCR activation. Proliferation induced by re-culture with rIL-12 and alloantigen was greater with CD4+CD8α+CD25+Treg consistent with the CD8α+ cells expressing IL-12R. In MLC, the CD8α+ fraction suppressed responses against allogeneic stimulators more than the mixed Ts1 population, whereas the CD4+CD8−CD25+T cells were less potent. In an adoptive transfer assay, rIL-2 and alloantigen activated Treg suppress rejection at a ratio of 1:10 with naïve effector cells, whereas alloantigen and rIL-2 activated tTreg depleted of the CD8α+ cells were much less effective. This study demonstrated that expression of CD8α by rIL-2 and alloantigen activation of CD4+CD8−CD25+Foxp3+T cells was a marker of activated and potent Treg that included alloantigen-specific Treg.

Highlights

Induction of tolerance to transplanted tissue is a highly desirable goal that has been achieved in a limited number of clinical protocols, reviewed [1]
Enriched CD4+CD8−CD25+T regulatory cells (Treg) were prepared from lymph node and spleen cells of naïve DA rats by depletion of CD8α+ and B cells followed by positive selection of CD25+ cells with an antiCD25 Monoclonal Antibodies (mAb) to produce fresh thymic Treg (tTreg) (Figure 1A)
After culture with media alone or PVG alone there was a reduction in CD4+CD25+cells (a) and CD4+Foxp3+ cells which fell to about 30% (b), consistent with tTreg not surviving without rIL-2

Summary

Introduction

Induction of tolerance to transplanted tissue is a highly desirable goal that has been achieved in a limited number of clinical protocols, reviewed [1]. Multiple potential mechanisms may underpin alloantigen-specific transplant tolerance, while T regulatory cells (Treg) contribute tolerance induction and maintenance, reviewed [2]. Suppressor T cells were first identified in the early 1970’s [9] and characterized as CD8+I-J+ T cells [10, 11]. CD4+ not CD8+T cells transfer alloantigenspecific tolerance [5, 8, 13] while retaining the capacity to effect third-party rejection [5, 6, 13,14,15]. Transfer of alloantigen-specific tolerance is dependent upon short-lived cells expressing CD25, the IL-2 receptor alpha chain [8, 13, 15, 22]. Alloantigen-specific CD4+CD25+Treg mediate tolerance to autoantigens, tumors, and infectious agents [23]

Methods

Results

Conclusion