Interferon Gamma (IFNγ)/STAT1 Signaling in Host Antigen Present Cells Suppresses MHC Class II-Dependent Presentation of Self-Antigens and Development of Graft Versus Host Disease (GVHD)

IFNγ signaling plays a critical role in the pathogenesis of GVHD. In this study, we observed that LPS-maturated bone marrow-derived dendritic cells (BMDCs) lacking IFNγ receptor (IFNγR, GRKO) or signal transducer and activator of transcription 1 (STAT1KO) had increased expression of major histocompatibility complex class II (MHC II), CD86, CD80, and enhanced allo-stimulatory capacity. This was further confirmed using fully MHC-mismatched bone marrow transplantation (BMT) studies. APC of GRKO or STAT1KO recipients had increased MHC II expression, which was associated with enhanced activation and expansion of donor CD4 and CD8 T cells and subsequently accelerated GVHD mortality compared to wild type (WT) controls. This increased GVHD mortality and increased MHC II expression on host APCs was further observed in the absence of recipient conditioning in the B6→CB6F1 mouse model. This was associated with increased presentation of host derived endogenous Eα52-68 peptide via I-Ab on recipient CD11c+ cells as detected by staining with the YA-e antibody. Furthermore, we could demonstrate that absence of IFNγR in BMDC promotes presentation Eα52-68 peptide and subsequently elicits pronounced activation, expansion and Th1 differentiation of TEa-TCR-tg CD4 T cells which recognize the Eα52-68 peptide presented by I-Ab. Next, we assessed the impact of this pathway on presentation of exogenous antigens. Interestingly, when lysate prepared from BALB/c splenocytes was incubated with BMDCs from B6 mice, Y-Ae expression on STAT1-/- BMDCs was significantly reduced compared to wild type BMDCs allowing us to hypothesize that IFNγ/STAT1 signaling may play an important role in promoting presentation of exogenous antigen while suppressing presentation of endogenous antigen. To further confirm this hypothesis, we used ovalbumin (OVA) as a second model antigen. To assess the impact of IFNγ/STAT1 signaling on presentation of exogenous antigen, WT, GRKO or STAT1KO BMDC were directly pulsed with OVA. To address the role in endogenous antigen presentation we studied act-mOVA-transgenic wildtype, act-mOVA.GRKO or act-mOVA.STAT1KO BMDCs. Transgenic OT-II CD4 T cells express a TCR specific for the OVA peptide 323-33 presented by I-Ab. The proliferation/activation of OT II T cells was monitored by flow cytometer as readout for effective Ag presentation. Our data demonstrated that IFNγR- or STAT1-deficient BMDCs loaded with exogenous intact OVA protein were compromised in promoting OT II proliferation. In contrast, responder OT-II CD4 T cells proliferated much more vigorously when stimulated with IFNγR/STAT1-deficient m-Act-OVA BMDCs compared to controls. We further observed significantly impaired OT-II cell proliferation in IFNγR or STAT1-deficient mice immunized with OVA indicating impaired presentation of exogenous antigens. However, OT-II CD4 T cells injected into lethally irradiated act-mOVA.STAT1KO transgenic mice proliferated more robustly and displayed increased Th1 differentiation compared to control mice when tested 3 days after OT II administration. We next started to assess several key factors (Ii [invariant chain, CD74], Cathepsin S [CTSS], H2-M, CIITA and MARCH1), known to be involved in the process of MHC class II antigen presentation and MHC II expression. We found retention of Invariant chain (CD74) expression as well as reduced CTSS and H2M expression in GRKO or STAT1KO BMDC following LPS-maturation. Furthermore, we observed significantly reduced lysosome formation/function in STAT1KO BMDCs compared to wild type BMDCs after LPS maturation. These data suggest that exogenous protein-derived peptide exchange in the MHCII compartment (MIIC) is impaired in STAT1KO BMDCs. Immature and LPS-maturated STAT1-/-BMDCs had significantly increased autophagy, which could explain enhanced endogenous Ag presentation since autophagy has been demonstrated to be critical in MHC II Ag presentation of cytoplasmic constituents. Finally, we found evidence of enhanced MHC II synthesis as supported by increased CIITA mRNA expression and conversely reduced MHC II degradation as indicated by reduced MARCH1 expression. In summary our data suggest that absence of IFNγR/STAT1 signaling in DC leads to abnormal surface MHC II turnover, promotes presentation of endogenous peptides and concomitantly impairs processing and presentation of exogenous antigens.