IL-33+ Stromal Cells in the Lymphoid Tissues Program Donor CD4+ T Cell Exhaustion That Blunts Subsequent IL-33+ Target Tissue-Driven Pathology but Not Graft Vs. Lymphoma Responses

Background: Graft vs. host disease (GVHD) remains a major complication of allogeneic hematopoietic stem cell transplantation (alloHSCT). To create space for donor stem cells and prevent their rejection, alloHSCT protocols rely on conditioning regimens involving chemotherapy and radiation. Conditioning causes tissue damage, which increases the tissue injury signal or "alarmin" interleukin (IL)-33 in fibroblastic reticular cells (FRC) of the secondary lymphoid organs (SLO). Mechanisms releasing IL-33 from its sequestration in the nucleus remain elusive, but free IL-33 directly stimulates donor CD4 T cells to prime IL-12-independent Type 1 T helper cell (Th1) differentiation and expansion. Targeting IL-33 early after alloHSCT limits GVHD in pre-clinical models. The gastrointestinal tract (GIT) also upregulates IL-33 in response to TBI and GVHD, but a direct role for local IL-33 in sustaining pathogenic donor responses is unclear. Our goal was to manipulate the IL-33 pathway in the SLO or GIT to better understand how stromal communications with donor T cells initiate and shape GVHD and graft vs. lymphoma (GVL) responses.

Methods: We compared donor T cells (plus or minus inducible deletion of the IL-33 receptor, ST2) for their ability to mediate GVHD vs. GVL (A20 lymphoma) in BALB/c recipients receiving total body irradiation (TBI) and CD45.1⁺ B6 T cell depleted bone marrow (TCD BM). To define the role for IL-33-derived from the SLO vs. the GIT, we assessed survival of B6 recipients deficient in IL-33 in FRCs (CCL19-CrexIl33^fl/fl) vs. those deficient in IL-33 in the epithelium of the GI tract (Vil-CrexIl33^fl/fl) receiving TBI and BALB/c T cells. To investigate if donor T cells mediate IL-33 release, we completed an ex vivo model using B6 St2^+/+, St2^-/-, and GzmB^-/- CD3 T cells co-cultured for 5 days with BALB/c TCD splenocytes and LN-derived FRCs that had been irradiated at 3500 cGy alone or with the IL-33 antagonist, sST2. Similar in vivo studies were conducted where the above donor B6 St2^+/+, St2^-/-, and GzmB^-/- CD3 T cells were transplanted into BALB/c recipients and assessed for GzmB and donor T cell expansion on day 5 post-alloHSCT.

Results: Ablating ST2 at days 10-14 post-transplant (after initial GVHD development) improved clinical scores and limited mortality. Further, sustained IL-33 signaling was not required for GVL activity. Mechanistically, late ST2 deletion was associated with increased Foxp3 expression and reciprocal Tbet decrease in donor CD4⁺ T cells from both SLO and GVHD target tissues. Sustained IL-33 signaling also maintained donor T cell TCF1 expression in SLO. Surprisingly, isolated deletion of FRC-derived IL-33 increased GVHD mortality in the CCL19-CrexIl33^fl/fl recipients. Mechanistic studies showing FRC-derived IL-33 stimulated CD4⁺ PD-1 expression and blunted the total number of CD4 and CD8 T effectors in the GIT at day 21 post-alloHSCT. Whereas, deletion of IL-33 in the gut epithelium in the Vil-CrexIl33^fl/fl recipients was protective and prolonged survival. RNAseq analysis suggested that IL-33 stimulates T cell granzyme B (GzmB) expression. GzmB deficient (Gzmb^-/-) donor T cells displaying reduced activation and expansion in vitro and in vivo, in a phenotype similar to ST2 deficient CD4 T cells. Consistent with the importance of GzmB in mediating IL-33 signals, antagonizing IL-33 had no impact on GzmB^-/- T cell responses similar to ST2 deficient CD4 T cells when compared to Gzmb^+/+, which failed to expand when IL-33 was sequestered.

Conclusions: Our data reveals that GzmB-mediated crosstalk between donor T cells and IL-33⁺ stroma orchestrates donor T cell identities and tunes local alloimmune responses after alloHSCT. Delayed deletion of ST2 signaling on donor T cells promotes survival through an upregulation of regulatory mechanisms in GVHD target tissues. Similarly, targeted deletion of IL-33 in the GIT provides protection from donor driven pathology. Whereas, targeted deletion of IL-33 from SLO FRC promotes GVHD mortality by down regulating intrinsic T cell exhaustion mechanisms in the SLO, which impacts later CD4⁺ T cell alloimmune responses to available IL-33 in target tissues, driving GVHD pathology. These data suggest distinct temporal and tissue specific roles for IL-33-driven programing of donor CD4⁺ T cells. In total, these data indicate that continual feedback between donor T cells and recipient stroma is central to the development and maintenance of GVHD.