*Purpose: Chronic rejection (CR) after organ transplantation (Tx) manifests as function-limiting allograft fibrosis and vasculopathy. Current immunosuppressants fail to prevent CR and insights into the mechanisms leading to CR are needed to generate effective therapies. The development of fibrosis during CR is similar to that observed in failed tissue repair, which lead us to hypothesize that dysregulated repair processes may be involved in CR. Amphiregulin (Areg) is a growth factor secreted by innate and adaptive immune cells in response to injury and initiates repair processes. Treg-secreted Areg has recently been implicated in muscle, epithelium, and nerve repair. Our purpose was to define: 1. how Tx-related stimuli (MHC/Alloantigen (AlloAg), co-stimulatory molecules, and local cytokines) shaped Treg and CD4⁺ T cell secretion of Areg in response to the injury-derived signal, IL-33 and 2. how Treg-secreted Areg-impacted CR.

*Methods: How ex vivo TCR, CD28, and cytokine receptor signaling regulated T cell Areg secretion ex vivo was defined using small molecule pathway inhibitors and Areg ELISA. In related studies, B6 mice transgenic for a TCR recognizing a BALB/c-derived AlloAg (Ea_52-68) presented on I-A^b were administered IL-33 or Ea_52-68 peptide alone, or combined, before CD4⁺ T cell isolation 7 days later. Areg secretion by isolated CD4⁺ during 4 days of culture with IL-33 or AlloAg was measured by ELISA. The impact of Treg and Areg on fibroblast proliferation, survival, and migration was assessed ex vivo using an IncuCyte Live-Cell Imaging Systems. B6 (H2^b) Foxp3-YFP-CrexAreg^fl/fl and Foxp3-YFP-Cre mice received heterotopic Bm12 (H2-Ab1^bm12) heart transplants. At days 14 or 100 post heart (H)Tx, isolated grafts underwent H+E, Trichrome, and IF staining.

*Results: Exposure to MHC/AlloAg limited the capacity of alloreactive Treg and CD4⁺ cells to secrete Areg in response to IL-33. Using the P38 inhibitor SB203580 and NF-kB inhibitors MG132 and TCPA-1 we established that both NF-kB and p38 are critical CD4⁺ T cell secretion of Areg. Surprisingly, the specific deletion of Areg from Treg provided protection against Bm12 heart Tx fibrosis. This was consistent with our observation that Areg drives the migration and activation of fibroblasts into wound sites in vitro.

*Conclusions: Our in vitro and in vivo studies suggest that Treg secreted Areg acts directly on fibroblasts to contribute to CR after HTx. Our data establishing that TCR signaling limits CD4⁺ T cell Areg secretion suggest that a dysregulated Treg tissue repair response may arise in the graft when damage signals persist in the absence of cognate MHCII or immunosuppressive drugs block T cell TCR signaling.

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