Session: 701. Experimental Transplantation: Basic Biology, Pre-Clinical Models: Hematopoietic Stem Cells and Alternate GVHD Tissues
Hematology Disease Topics & Pathways:
Diseases, Animal models, GVHD, cellular interactions, Biological Processes, Immune Disorders, Study Population, Clinically relevant, immune mechanism, inflammation, pathogenesis
To characterize stromal sources of Notch ligands early after allo-BMT, we combined a Ccl19-Cre transgene with a ROSA26-YFP Cre-activated allele in Dll4-mCherry BAC reporter mice. We observed combined Ccl19-Cre activity and Dll4-mCherry reporter expression in CD157hi fibroblastic reticular cells, MAdCAM1+ marginal reticular cells, CD21+ follicular dendritic cells, and a subset of MAdCAM1+ lymphatic endothelial cells. 12 hours after high intensity conditioning and allo-BMT, Dll4 mRNA and cell surface expression increased in fibroblastic but not in hematopoietic or endothelial cellular compartments. Dll4 upregulation required transfer of alloreactive T cells and was potentiated by the intensity of myeloablative conditioning, suggesting that fibroblastic stromal cells have increased availability of Notch ligands to drive GVHD after high-intensity conditioning.
To assess the functional impact of conditioning on the cellular sources of Notch ligands, we studied a parent-to-F1 allo-BMT model in which recipients are tolerant to donor-derived T cells, and the conditioning intensity can be titrated (C57BL/6 [H-2b] or BALB/c [H-2d] into BALB/c x C57BL/6 F1 [H-2d/b]). Cell-specific genetic loss of function systems combining floxed alleles for Dll1 and Dll4 with Ccl19-Cre+ and Mx-Cre+ transgenes allowed conditional deletion in SLO fibroblastic stromal cells or hematopoietic cells, respectively. Across all conditioning intensities tested, upregulation of core-2 O-glycosylation machinery in alloreactive T cells was critically regulated by Notch signals. Thus, induction of the core-2 glycoform of CD43 could be used as a sensitive surrogate readout of Notch signals received by alloreactive T cells. In models of allotransplantation with high-intensity (11 Gy), low-intensity (3 Gy), and no conditioning, fibroblastic stromal cells remained essential and hematopoietic cells were dispensable as sources of Notch signals driving both T cell core-2 O-glycosylation and lethal alloimmunity.
Because previous studies indicated that non-hematopoietic cells can serve as antigen-presenting cells in CD4+ T cell mediated GVHD, we tested if fibroblastic stromal cells could present MHC class II alloantigens in addition to Notch ligands. We used Ccl19-Cre+ crossed to mice with a floxed I-A allele to delete the cell-intrinsic ability of fibroblastic stromal cells to present MHC class II alloantigens. In multiple models of high-intensity conditioned CD4+ T cell mediated GVHD (BALB/c [H-2d] into BALB/c x C57BL/6 F1 [H-2d/b] and TEa TCR Tg into BALB/c x C57BL/6 F1 [H-2d/b]), fibroblastic stromal cells were dispensable as sources of alloantigen to drive T cell alloreactivity and lethal GVHD. Together, these results indicate that across varying conditioning intensities, specialized subsets of SLO fibroblastic stromal cells critically regulate T cell alloreactivity through presentation of Notch ligands independently of antigen presentation. Selective targeting of Delta-like Notch ligand expression on SLO fibroblastic cells may prevent T cell immunopathology while preserving other beneficial antigen-dependent T cell responses after both high-intensity conditioned allo-BMT and allotransplantation with reduced intensity conditioning.
Disclosures: Blazar: Kadmon Corporation, LLC: Consultancy, Research Funding. Siebel: Genentech: Employment.
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