IFNγ-driven MHC class II expression by intestinal epithelial cells dictates local cytolytic Th1 cell differentiation and intestinal stem cell loss Free

Allogeneic hematopoietic stem cell / bone marrow transplantation (hereafter BMT) is a curative therapy for malignant and non-malignant hematological disorders. While hematopoietic reconstitution from a health donor and immunological graft-versus-leukemia (GVL) effects lead to the therapeutic benefits of BMT, graft-versus-host disease (GVHD) remains a procedural limitation. Acute GVHD induced by donor alloreactive T cells typically occurs in the skin, liver and GI tract, and gut GVHD in particular portends a high mortality.

Alloantigen presentation initiates the activation of donor CD4⁺ and CD8⁺ T cells, a prerequisite to generate GVHD. Host rather than donor-derived antigen presenting cells (APC) are the dominant subset in this process and amongst host APC, both hematopoietic and non-hematopoietic APC can generate CD4⁺ T cell-mediated GVHD. We have shown that MHC class II (MHC-II) expressed by host intestinal epithelial cells (IEC) promotes lethal gut GVHD. However, the molecular cues by which CD4⁺ T cells are invoked to mediate lethal gut GVHD at the epithelial interface after allogeneic BMT is unclear.

We compared GVHD induced by male antigen (H-Y) reactive TCR-transgenic CD4⁺ T cells (Marilyn T) in which MHC-II was deleted prior to or shortly after BMT by tamoxifen injection (day -10 to -6 versus day +4 to +8, respectively) in Villin CreER^T2IAb^fl/flmale recipient mice. Only the mice in which MHC-II was deleted prior to BMT were protected from lethal GVHD (11 % vs 58% survival in MHC-II non-depleted vs depleted pre-BMT (p = 0.0056); 0 % survival in both non-depleted and depleted post-BMT (p=0.85)). Consistent with this, we detected highly activated CD69⁺ donor CD4⁺ Marilyn T cells in the intestine of allogeneic but not syngeneic transplant recipients within 1 day of BMT.

The induction of MHC-II on IEC expanded perforin/granzyme expressing cytolytic donor Th1 whilst restraining Th17 differentiation locally within the ileum. MHC-II expression was absent in IEC of IFN-γ receptor (IFN-γR) deficient recipients (Ifnγr^–/–) and donor Marilyn T cell expansion in the ileum of these male recipients was attenuated after BMT. We next compared GVHD in mice in which we conditionally deleted the IFN-γR on IEC with tamoxifen in VillinCreER^T2Ifnγr^fl/fl recipients. The deletion of the IFN-γR or MHC-II specifically in IEC of recipients improved survival and reduced donor CD4⁺ T cell expansion (1.5 ± 0.2 [control], 0.2 ± 0.03 [MHC-II depleted] or 0.5 ± 0.05 [IFN-γR depleted] of Marilyn T (× 10⁶) in ileum, p<0.05), and cytolytic (perforin⁺ granzyme B⁺) Th1 differentiation (%IFN-γ⁺in Marilyn T: 52.4 ± 2.1 % [control], 40.8 ± 2.6 % [MHC-II depleted] or 44.5 ± 2.5 % [IFN-γR depleted], p<0.05) in the ileum. We confirmed these effects in a model of GVHD induced by polyclonal CD4⁺ T cells (Balb/c → B6).

The specific depletion of the IFN-γR on IEC suppressed both MHC-I and MHC-II expression. This led us to investigate the role of MHC-I by IEC in CD8⁺ T cell-mediated GVHD using Villin CreER^T2β2m^fl/flrecipient mice. However, in contrast to MHC-II deletion, deletion of MHC-I had no effect on GVHD severity, systemic cytokine production or donor CD8⁺ T cell expansion. We thus demonstrate that MHC-II but not MHC-I expression by IEC within the first three days of transplantation, and not thereafter, determined lethal GVHD.

In contrast to IEC, MHC-II expression by intestinal stem cells (ISC) was not required for the induction of GVHD in Lgr5-cre driven systems but MHC-II⁺ ISC were preferentially and progressively depleted in the ileum by locally expanding cytolytic Th1 cells. This effect required the induction of MHC II-expression on ISC by IFNg. In sum, direct IFNg-signaling in IEC is critical for the MHC-II-dependent early local generation of cytolytic Th1 differentiation that thereafter mediate MHC-II-dependent ISC deletion, demonstrating a critical interferon–MHC-II feed forward axis in the initiation of cytolytic Th1-dependent gut disease.