Session: 701. Experimental Transplantation: Basic and Translational: Novel Mechanisms of Acute and Chronic Graft-versus-Host Disease
Hematology Disease Topics & Pathways:
Research, Translational Research
To evaluate Areg expression in donor T cells, we performed C57BL/6 into B6D2F1 (B6BDF1) MHC-mismatched allo-BMT and found that donor CD4+ T cells produced Areg post-transplant. We initially hypothesized that T cell-derived Areg may contribute to tissue repair after BMT. To investigate the effects of T cell-derived Areg in the transplant setting, we performed B6BDF1 allo-BMT using T cells from Aregfl/fl x CD4-Cre (Areg-/-) or Aregfl/fl x Foxp3-Cre (AregΔFoxp3) mice, lacking Areg in all T cells (Areg-/-) or specifically in regulatory T cells (AregΔFoxp3). Unexpectedly, recipients of Areg-/- T cells demonstrated reduced mortality compared to recipients of wild-type (WT) T cells (Figure, left). This observation was recapitulated in a distinct B6BALB/c allo-BMT model (not shown). Examining the effects of Areg deficiency at the tissue level, recipients of Areg-/- T cells demonstrated reduced GVHD pathology in liver, ileum, and colon ten days post-BMT, while recipients of AregΔFoxp3 T cells showed no difference compared to controls. Consistent with the GVHD histopathology, intestinal stem cell frequencies were substantially preserved in the small intestines from recipients transplanted with Areg-/- T cells compared to recipients receiving WT T cells or AregΔFoxp3 T cells. These data suggested that conventional T cell-derived Areg may contribute to tissue damage in GVHD.
To assess the mechanisms by which T cell-derived Areg contributes to GVHD severity, we analyzed donor T cells in spleen, mesenteric lymph nodes, and lamina propria post-BMT. Areg-/- donor CD4+ T cells demonstrated impaired expansion while donor CD8+ T cell expansion remained intact. Areg-/- donor CD4+ T cells also demonstrated a reduced Th1 response as evidenced by decreased IFNg expression. In addition, co-culture in vitro and co-transfer in vivo of WT and Areg-/- CD4+ T cells indicated that the reduced expansion of Areg-/- donor CD4+ T cells was cell-intrinsic and likely occurring in an autocrine Areg-dependent fashion. Notably, these observations were recapitulated following allo-BMT using EGFRfl/fl x CD4-Cre (EGFR-/-) donor T cells: recipients transplanted with EGFR-/- donor T cells demonstrated reduced donor CD4+ T cell expansion and improved survival post-transplant (Figure, right).
To further investigate the mechanisms driving Areg-dependent CD4+ T cell expansion, we compared WT and Areg-/-B6 T cells following ex vivo activation with anti-CD3 and anti-CD28 antibodies or activation with allo BALB/c-derived dendritic cells. Compared to WT CD4+ T cells, Areg-deficient CD4+ T cells exhibited reduced proliferation, which was restored by supplementation with recombinant Areg. Additionally, we found that naive CD4+ T cells increased Egfr and Areg mRNA expression upon T cell receptor stimulation. Moreover, Areg inhibition using anti-Areg neutralizing antibodies impaired proliferation of WT naive CD4+ T cells after activation, reducing their expansion upon activation ex vivo. To better understand how Areg was impacting activation-induced CD4+ T cell expansion, we examined downstream targets of EGFR signaling and observed that Areg inhibition resulted in impairment of Akt phosphorylation and reduced glucose uptake in naive CD4+ T cells following activation. Finally, despite the reduction in proliferation and GVHD pathogenicity, Areg-/- donor T cells demonstrated intact GVL activity against A20 lymphoma cells, resulting in an improved overall survival following BMT with tumor challenge.
In summary, we found that CD4+ T cell-derived Areg contributes to CD4+ T cell proliferation after activation, leading to increased T cell expansion, tissue damage, and GVHD-related mortality after allo-BMT. Careful inhibition of the T cell Areg/EGFR axis may represent a novel therapeutic target for prevention or amelioration of GVHD.
Disclosures: Vinci: ClearView Healthcare Partners: Current Employment. Hanash: Intellectual Property: Other: Holds intellectual property related to Interleukin-22 and GVHD; Evive Biotech: Other: Served as Co-PI of a clinical trial supported by Evive Biotech, Research Funding.
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