Donor XCR1⁺ and CD11b⁺ Dendritic Cells Determine the Severity of Th1 and Th17-Dependent Acute Gvhd within the Gastrointestinal Tract

Acute graft-versus-host disease (GVHD) remains a major complication of allogeneic stem cell/ bone marrow transplantation (hereafter referred to as BMT) and severe disease in the gastrointestinal (GI) tract is the principal determinant of early mortality. Although recipient antigen presenting cells (APC) are crucial for the induction of GVHD, donor graft-derived CD103⁺ CD11b^neg conventional dendritic cells (cDC) also modulate the severity of disease in the colon (Koyama, M. et al. J Exp Med. 2015;212(8):1303-21). More recently, donor plasmacytoid dendritic cells (pDC) have been proposed to both augment and attenuate acute GVHD, creating significant mechanistic ambiguity. We delineated donor DC subsets in the GI tract and lymphoid organs (ileum or colon; epithelial layer or lamina propria, together with mLN and spleen) after allogeneic BMT (T cell replete [GVHD] vs T cell depleted [non-GVHD] in a B6 → B6D2F1 system). Since XCR1, a chemokine receptor, is exclusively expressed by the CD8⁺/CD103⁺ cDC subset, we utilized B6 XCR1 reporter (XCR1-venus) mice as donors. Donor pDC were prominent in the ileum but not colon epithelium of non-GVHD mice compared to those of GVHD mice (P=0.0007, 0.61 ± 0.043 ×10⁶ in non-GVHD, 0.082 ± 0.045 ×10⁶ in GVHD), whereas donor CD103⁺ cDC were significantly increased in the mLN of GVHD mice (P=0.0027, 0.020 ± 0.0027 ×10⁶ in non-GVHD, 0.058 ± 0.011 ×10⁶ in GVHD) (mean ± sem). In addition, we confirmed that, with the exception of spleen, donor XCR1 expression after transplant was exclusively restricted to CD103⁺CD11b^neg cDC. We next utilized XCR1-DTR mice and Clec4c-DTR mice in which the DTR is expressed under the promoter of XCR1 or Clec4c respectively, to permit isolated CD103⁺ cDC and pDC depletion by diphtheria toxin (DT) administration. To assess GVHD induced by donor APC antigen presentation only, lethally irradiated BALB/c mice (H-2^d) were transplanted with T cell depleted (TCD) bone marrow from wild-type B6 (B6.WT), XCR1-DTR or Clec4c-DTR mice with 1.5 ×10⁶ Rag1^-/- TEa transgenic T cells which react to the BALB/c-derived I-E^d peptide presented on donor B6-derived MHC class II (I-A^b). DT was administered to donor mice on day -2, -1 and alternate days to recipients from day +3 to day +21. Donor XCR1⁺ cDC depletion but not pDC depletion attenuated lethal GVHD compared to non-depleted controls (P = 0.0013; B6.WT vs XCR1-DTRvenus, P = 0.2098; B6.WT vs Clec4c-DTR), consistent with XCR1⁺ cDC being the major determinant of aGVHD pathology. XCR1⁺ cDC depletion resulted in a reduction of alloantigen expressing donor cDC in the mLN quantified by the YAe antibody (which binds to a I-E^d-derived peptide loaded on I-A^b). When luciferase-expressing TEa T cells (TEa^luc+) were transferred into the BALB/c recipients of B6.WT or XCR1-DTR BM and B6.WT T cells on day 12, TEa expansion was decreased in the GI tract (P < 0.05; mLN and GI tract) of XCR1⁺ cDC depleted recipients. The depletion of of XCR1⁺ cDC profoundly attenuated integrin α4β7 expression (P < 0.0001; 49.1% vs 18.4%; B6.WT vs XCR1-DTRvenus), and the generation of interferon-γ secreting (IFN-γ) (P < 0.05) TEa T cells. Aldehyde dehydrogenase 1A (ALDH1A) expression by donor and host DC is known to modulate GVHD (Thangavelu G, et al. J Immunol, 2019, 202: 2795-2805) and we thus examined the effect of a pan-ALDH1A (ALDH1A1, 1A2 and 1A3) inhibitor, WIN18,446, on this axis. We found that oral WIN18,446 improved survival (P = 0.017, 18.8 vs 62.5 % at day 70; Control vs WIN18,446) in the context of reduced donor cDC expansion and downstream TEa T cell expansion. However these effects were mediated by inhibition of CD11b⁺ cDC subsets (CD11b⁺CD103⁺ and CD11b⁺CD103^neg) subsets rather than XCR1⁺ cDC, resulting in the inhibition of IL-17 secreting TEa T cells in the GI tract. We examined the effect of WIN18,446 after transplantation of wild-type or Aldh1a1^-/- bone marrow and noted that the effects on TEa expansion and Th17 differentiation were ALDH1A1-dependent. These findings highlight the limited role of pDC in the pathophysiology of gut GVHD and identify differential and dominant roles for XCR1⁺ and CD11b⁺ donor cDC in controlling Th1 and Th17 mediated gut aGVHD. Pharmacological prevention of gut GVHD should thus target both cDC subsets.

Amory:Celldex: Research Funding; NEXT: Consultancy; Ferring Pharmaceuticals: Research Funding. Hill:iTeos Therapeutics: Consultancy; Compass Therapeutics, Syndax Pharmaceuticals, Applied Mo- lecular Transport, Serplus Technology, Heat Biologics, Laevoroc Oncology, iTeos Therapeutics, Genentech, and CSL Behring.: Research Funding; Commonwealth Serum Laboratories (CSL): Consultancy; Generon Corporation, NapaJen Pharma, iTeos Ther- apeutics, and Neoleukin Therapeutics: Consultancy; Cynata Therapeutics: Consultancy; NapaJen Pharma: Consultancy; CSL Behring: Consultancy; Neoleukin Therapeutics: Consultancy.