Donor-Derived Amphiregulin Drives CD4+ T Cell Expansion and Promotes Tissue Pathology after Experimental Allogeneic BMT

Amphiregulin (Areg), a ligand for the epidermal growth factor receptor (EGFR), contributes to cell proliferation and survival. Work in recent years has demonstrated that immune cells such as Tregs and ILC2s can produce Areg, leading to tissue repair after injury. In the bone marrow transplant (BMT) setting, clinical studies have reported that high concentrations of plasma Areg may predict for poor prognosis acute GVHD. However, impacts of Areg/EGFR signaling within the immune system are poorly understood. We thus investigated the role of T cell-derived Areg following allogeneic (allo)-BMT.

To evaluate Areg expression in donor T cells, we performed C57BL/6 into B6D2F1 (B6BDF1) 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 B6BDF1 allo-BMT using T cells from Areg^fl/fl x CD4-Cre (Areg^-/-) or Areg^fl/fl x Foxp3-Cre (Areg^ΔFoxp3) mice, lacking Areg in all T cells (Areg^-/-) or specifically in regulatory T cells (Areg^ΔFoxp3). 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 B6BALB/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 Areg^ΔFoxp3 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 Areg^ΔFoxp3 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 EGFR^fl/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.