Protection of Mice from Acute Graft-versus-Host Disease Requires CD28 Co-stimulation on Donor CD4+ Foxp3+ Regulatory T Cells

Abstract

Acute graft-versus-host disease (aGvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell plus T cell transplantation (allo-HSCT). In this study, we investigated the requirement for CD28 co-stimulation of donor CD4+ conventional (CD4+CD25−Foxp3−, Tconv) and regulatory (CD4+CD25+Foxp3+, Treg) T cells in aGvHD using tamoxifen-inducible CD28 knockout (iCD28KO) or wild-type (wt) littermates as donors of CD4+ Tconv and Treg. In the highly inflammatory C57BL/6 into BALB/c allo-HSCT transplantation model, CD28 depletion on donor CD4+ Tconv reduced clinical signs of aGvHD, but did not significantly prolong survival of the recipient mice. Selective depletion of CD28 on donor Treg did not abrogate protection of recipient mice from aGvHD until about day 20 after allo-HSCT. Later, however, the pool of CD28-depleted Treg drastically declined as compared to wt Treg. Consequently, only wt, but not CD28-deficient, Treg were able to continuously suppress aGvHD and induce long-term survival of the recipient mice. To our knowledge, this is the first study that specifically evaluates the impact of CD28 expression on donor Treg in aGvHD. Moreover, the delayed kinetics of aGvHD lethality after transplantation of iCD28KO Treg provides a novel animal model for similar disease courses found in patients after allo-HSCT.

During acute graft versus host disease CD28 deletion in donor CD8+, but not CD4+, T cells maintain antileukemia responses in mice

“…A–C). We further observed a transient reduction in CD28 expression on wt CD8 + T cells, similar to what we had previously described for CD4 + T cells . Consequently, on day 3 after transplantation, we only observed a slight additional reduction in CD28 expression when we compared iCD28KO and wt CD8 + T cells (Fig.…”

“…To investigate if late‐onset aGvHD correlates with enhanced antitumor activity, we selectively deleted CD28 in donor Treg cells in mice treated in parallel to those shown in Figs. and A.…”

“…We depleted T cells from single cell suspensions of BM cells or splenocytes as described . Similarly, CD4 + and CD8 + T cells were depleted from single cell suspensions by first staining the cells with biotinylated αCD4 (1:100, Biolegend) or αCD8 antibody (1:100, BD Bioscience) for 15 min at 4°C and depleting antibody‐labeled cells with anti‐biotin beads (1:10, 15 min, 4°C, Miltenyi).…”

“…However, the requirement for CD28 costimulation for T‐cell activation is not absolute, as CD28 KO mice are still able to mount adaptive immune responses against pathogens . In different mouse models, CD4 + T‐cell‐driven aGvHD was reduced but not abrogated when the CD28 costimulatory signal in donor T cells was disrupted by genetic deletion or by systemic blockade of the CD28 molecule with antibodies . Moreover, interfering with CD28 costimulation also inhibited aGvHD when CD4 + and CD8 + T cells were transplanted .…”

“…We have previously established a mouse model of aGvHD that allows to delete CD28 in either donor CD4 + conventional T cells (Tconv cells) or regulatory T cells (Treg cells) after transplantation . We now used the same approach to investigate the effect of CD28 deletion in different donor T‐cell subsets on the GvL response and asked if CD28 costimulation of CD8 + T cells contributes to the severity of aGvHD.…”