IFN-γ/STAT1-Dependent Regulation Of Antigen Presenting Cell (APC) Function: Role In Graft-Versus-Host Disease (GVHD)

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that has diverse activities in mediating host defense and immunopathology and is involved in both innate an adaptive immunity. Furthermore, IFN-γ has been shown to be a key regulator of GVHD. STAT1 is the major downstream signal transducer for IFN-γ. IFN-γ has been shown to up-regulate MHC class II expression on macrophages and dendritic cells. In this study, we unexpectedly observed that STAT1-deficient mouse bone marrow-derived dendritic cells (BM-DCs) displayed significantly increased MHC class II, CD86, CD80, CD40 expression compared to wild type BM-DCs upon LPS stimulation. To determine whether these LPS-maturated STAT1^-/- BM-DCs are functional, freshly isolated pan-T cells from BALB/c (H2^d) mice were stimulated with either irradiated LPS-maturated STAT1^+/+ or STAT1^-/- BMDCs from 129Sv [H2^b] mice at different DC/Responder ratios for 5 days. Pan-T cell proliferation was measured using by 3H-incorporation or in separate assays by CFSE dilution. Both proliferation assays clearly demonstrated that STAT1^-/- BM-DCs were significantly more potent stimulators in Mixed Lymphocyte Reaction (MLR) assays compared to STAT1^+/+ BM-DCs. Further studies demonstrated that this increased surface MHC II expression was also observed in LPS-stimulated IFNγR^-/- BM-DCs or on wild type BM-DCs treated with neutralizing IFNγ Abs but not in LPS-treated IFNα-R^-/- BM-DCs.

STAT1 deficient DCs have been reported to be defective in IL-12 production upon stimulation with various Toll-like receptors (TLRs). However, we were able to observe that STAT1^-/- BM-DCs had increased CD40 expression after LPS maturation, and more importantly, STAT1 deficient BM-DCs had significantly increased IL-12 production upon treatment with CD40-ligand. These data suggest that STAT1 deficiency in LPS-stimulated DCs results not only in increased expression of MHC class II and costimulatory molecules, but also in enhanced CD40-dependent IL-12 production promoting generation of Th1 and Tc1 cell of wildtype responder cells.

To test the influence of IFNγ/STAT1 deficiency in host APC on GVHD induction, wild type or STAT1-deficient 129 mice (H2^b) underwent allogeneic Bone Marrow Transplantation (BMT) following lethal irradiation (1044 rad) receiving BALB/c spleen cells. 129.STAT1^-/- recipients had significantly accelerated GVHD mortality (MST 8 days vs. 5 days, log rank test p=0.004) compared with wild type recipients. In contrast, STAT1-deficient syngeneic controls did not show enhanced mortality or morbidity ruling out that the observed accelerated mortality following allogeneic BMT was due to conditioning toxicity. On day+4 post-BMT animals were sacrificed and splenocytes were analyzed by flow cytometry. We could observe a significantly enhanced expansion and activation of donor CD4 and CD8 T cells in STAT1^-/- recipients. Importantly, the increased MHC II expression was confirmed in host CD11b⁺ and CD11c⁺ cell populations. To clarify that STAT1 deficiency in host APCs do contribute to increased GVHD induction, we generated STAT1^-/-→STAT1^+/+, and STAT1^+/+→STAT1^+/+ radiation chimeric mice as recipients so that only the hematopoietic cells were STAT1 deficient. We again observed significantly increased GVHD mortality and increased activation of fully MHC-mismatched donor T cells in STAT1^-/-→STAT1^+/+ recipients following allogeneic BMT and GVHD induction. Similar results i.e. increased GVHD mortality, increased donor cell activation and increased MHC II expression on host CD11c⁺, B220⁺ cells were also found in IFNγR^-/- recipient mice when compared with control mice following full MHC-mismatched allogeneic BMT. In summary our data reveal previously unknown effects of IFNγ/STAT1 signaling on APCs function suggesting that LPS-driven maturation of APC’s is negatively regulated by IFNγ/STAT1 leading to enhanced expression of MHC class II, costimulatory molecules and responsiveness to CD40-ligation and subsequent IL-12 production in the absence of STAT1. Furthermore, our results explain the accelerated GVHD induction and demonstrate that IFN-γ/STAT1 signaling in host APC mitigates the development of GVHD.