Peripheral CD4 +CD25 + regulatory T cells (T reg) block alloreactive host anti-donor T cells in canine mixed hematopoietic chimeras

Abstract

CD4+CD25+ Treg cells may be important immune regulators for the maintenance of immune tolerance after allogeneic hematopoietic cell transplantation (HCT). In the well-established canine model of nonmyeloablative HCT, stable mixed donor/host hematopoietic chimerism persists for the lifespan of the recipient following 2 Gray total body irradiation, dog leukocyte antigen (DLA)-identical bone marrow transplantation and combined postgrafting immunosuppression with mycophenolate mofetil and cyclosporine for 4 and 5 weeks, respectively. We hypothesized that CD4+CD25+ Treg are an important component of the active cellular mechanism of peripheral tolerance that maintains stable mixed chimerism in this HCT model. Previously, we showed that depletion of CD4+CD25+ Treg cells from mixed lymphocyte culture (MLC) significantly increased allo-specific proliferation in the DLA-mismatched setting. In the current study, we asked if depletion of CD4+CD25+ Treg from peripheral blood mononuclear cells (PBMC) obtained from dogs with stable mixed chimerism 1 to 4 years after nonmyeloablative HCT could generate minor histocompatibility antigen (mHAg)-specific, host anti-donor cytotoxic T lymphocytes (CTL). Responder PBMC from mixed chimeric dogs were cultured in a modified MLC with irradiated, CD34+ derived dendritic cells (DC) from the respective DLA-identical HCT donor at a 10:1 responder: stimulator ratio. On day 4 of MLC, CD4+CD25+ T cells were depleted by either immunomagnetic selection (Miltenyi) using the anti-CD25 monoclonal antibody (ACT-1) or the addition of 10−11M denileukin diftitox (DAB389IL-2, Ontak) for 18 hours. DAB389IL-2 is a fusion protein consisting of the ADP-ribosyltransferase domain of diphtheria toxin and IL-2. By flow cytometry analysis, depletion of CD4+CD25bright+ was 60%–66% with immunomagnetic selection and 70%–77% with DAB389IL-2. On day 5, culture media was changed to remove residual DAB389IL-2 in the respective flasks. A secondary MLC was established with CD4+CD25+ depleted responder cells and irradiated DC from the respective DLA-identical HCT donor. Peak proliferation of responder cells measured by 3H-thymidine incorporation was on day 4 of secondary MLC. There was a 54%–95% increase in proliferation of cells depleted of CD4+CD25+ T cells with immunomagnetic selection and a 121%–133% increase in proliferation of DAB389IL-2 treated cells compared with non-depleted responder cells. We evaluated mHAg-specific cytotoxic activity of the CD4+CD25+ depleted responder T cells by 51Cr release assay following 3rd MLC stimulation with respective DC. At a 10:1 effector: target ratio, there was 74%–82% specific lysis of the respective HCT donor cells by the CD4+CD25+ depleted responder T cells, while the non-depleted control responder cells had 4%–6% specific lysis. PCR analysis with informative microsatellite markers confirmed the alloreactive CTL were of host origin. In summary, depletion of CD4+CD25+ Treg from PBMC of mixed chimeras permitted the emergence of host anti-donor mHAg-specific CTL. These data suggest that mHAg-specific host anti-donor T cells persist in the peripheral blood of stable mixed chimeric dogs and that peripheral CD4+CD25+ Treg prevent alloreactive immune responses and maintain mixed chimerism. These results support the hypothesis that mixed hematopoietic chimerism established without T cell depletion relies on peripheral immune tolerance mechanisms.