Regulatory T Cell Derived Trefoil Factor 1 Drives Thymic Regeneration in Allogeneic Hematopoietic Cell Transplantation

T cell reconstitution is associated with less morbidity and mortality from infections, GVHD, and relapse after allogeneic hematopoietic cell transplantation (allo-HCT). T cell production after allo-HCT depends primarily on regeneration of a functional thymus. Our group has recently reported that regulatory T cells (Tregs) mediate thymic regeneration and T cell production after sublethal injury. While Tregs are known to confer protection from GvHD, their role in thymic regeneration during allo-HCT and the implicated molecular mechanisms are unknown. This study aims to reveal the role of Tregs and their products in thymic regeneration during allo-HCT.

Using an MHC-disparate, T cell depleted model of allo-HCT (C57BL/6J into BALB/cJ) we investigated the presence and origin of Tregs in the thymus pre- and post-transplantation (days 0, 1, 4, 7, 14, and 21). Host-derived Tregs demonstrated remarkable resistance to lethal irradiation (950 cGy) relative to other immune cells in the thymus. The frequency of Tregs increased significantly after injury (p=0.002), peaking at the nadir in thymic cellularity on day 7; and on this day, thymic Tregs were primarily of host origin (99.7 ± 0.30% of Tregs, p=0.002).

Host-derived thymic Tregs were phenotyped on day 7 post-allo-HCT by single cell RNA sequencing and compared to Tregs from other target organs of GvHD: spleen, bone marrow, liver, and skin (>50K CD4 T cells, >10K Treg cells). Thymic Tregs significantly upregulated anti-apoptotic genes after injury, such as genes related to IL-2 (Ecm1, Nrp1, Itgae, Ltb) and TGF-β signaling (Pmepa1, Skil, Tgfbr1, Smad7). Interestingly, thymic Tregs were also enriched for genes implicated in regeneration (Areg and Penk) including Trefoil factor 1 (Tff1); a secreted, lectin-like protein known to mediate epithelial barrier homeostasis and regeneration in other organs. Tff1 was significantly upregulated in thymic Tregs compared to Tregs from all other assessed organs after injury (Log2(FC)=1.91, p=0.0002). In the thymus, Tff1 was solely expressed by Tregs compared to both stromal and immune cell populations after injury. Additionally, the total protein concentration of TFF1 increased significantly in thymic lysates after injury (p=0.002), coinciding with the increase in Tregs in T cell-depleted allo-HCT.

To assess if TFF1 was a mediator of regeneration we created a knock-out (TFF1-KO) mouse model by CRISPR-Cas9, with genetic excision of the Tff1 locus. TFF1-KO mice had similar thymic cellularity before injury; however, thymic regeneration was significantly reduced on day 14 after allo-HCT compared to wild type (WT) littermates (p=0.0159). TFF1 deficiency led to a significant reduction in thymic epithelial cell (TEC) counts (p=0.0079) during thymic regeneration. Furthermore, regenerating TECs had less MHC-II expression (p=0.0159) in TFF1-KO mice, with a decrease in the number of double positive (DP) thymocytes (p=0.0159) relative to WT mice. This phenomenon was independent of early thymocyte development, since neither the addition of recombinant TFF1 to an in vitro thymic culture system, nor injury mediated regeneration in TFF1-KO mice led to altered early (double negative) thymocytes.

TFF1 protein concentration was abrogated in GvHD compared to T cell-depleted allo-HCT, coinciding with both prolonged reduction in thymic cellularity and a reduction in host-derived Treg numbers (p=0.002). Although Tregs were reduced in the GvHD thymus, the remaining Tregs maintained their transcriptional capacity to express Tff1. However, this expression of Tff1 in GvHD thymic Tregs was associated with transcriptional changes implicated in cellular stress and dysfunction. Specifically, high expression of genes implicated in cell cycle (Snrpg, Ppia, Ube2s) and unfolded protein responses (Eef2, Ddit4, Eif4a1).

Altogether, our findings reveal that a) injury resistant Tff1 expressing host-derived Tregs are increased in the thymus after allo-HCT, b) TFF1 mediates thymic regeneration by supporting thymic epithelial cell regeneration, and c) GvHD leads to depletion in host-derived thymic Tregs, reduced TFF1 levels and delayed thymic regeneration. Thus, TFF1 may be a novel therapeutic option for enhancing thymic regeneration in allo-HCT and GvHD.