Donor T Cell Independent Mechanism Is Critical for Mediating Steroid Refractory Gvhd in Murine Models
Authors: Toubai Tomomi , Rossi Corrine , Tawara Isao , Liu Chen , Zajac Cynthia , Oravecz-Wilson Katherine , Peltier Dan , Sun Yaping , Fujiwara Hideaki , Riwes Mary Mansour , Henig Israel , Kim Stephanie , Suto Masahiro , Ishizawa Kenichi , Reddy Pavan 
Published: October 2019

Abstract
Acute graft-versus-host disease (GVHD) is a major complication of allogeneic cell transplantation (Allo-HCT). Steroids have remained the first line therapy for acute GVHD. However, 30-50% of patients develop steroid refractory GVHD (SR-GVHD) which shows extremely poor prognosis. The development of therapies for SR-GVHD is limited by an incomplete understanding of its pathophysiology because of the lack of clinical animal models. Here we generated SR-GVHD murine models by developing a major histocompatibility (MHC) matched, multiple minor histocompatibility antigens (miHAs) mismatched as well as an MHC mismatched haploidentical murine bone marrow transplantation (BMT) models and determined whether donor T cells are crucial role in mediating SR-GVHD.
Clinically, most allo-HCTs are human leukocyte antigen (HLA) matched. Therefore, we utilized the clinically relevant MHC matched multiple miHAs mismatched C3H.swàB6 model. Recipient B6 WT animalswere lethally irradiated and transplanted with splenic T cells along with bone marrow (BM) cells from either syngeneic B6 or allogeneic C3H.swdonors. Dexamethasone (DEX) was administered from day +7 to day +21 at a dose of 0.1mg/kg. We observed three phenotypes in response to DEX: 1) steroid refractory (REF), characterized by progressive GVHD with severe peak GVHD clinical scores, progressive weight loss and early mortality; 2) stable GVHD (ST), characterized by moderate GVHD clinical scores, moderate body weight loss, and minimal late mortality; 3) steroid responsive (RES) GVHD, characterized by near complete normalization of GVHD clinical scores, near complete normalization of weight loss, and no mortality. We were able to differentiate among these three phenotypes based on GVHD clinical score and body weight change on day +21 after allo-BMT which included14 days of DEX treatment. These criteria clearly separated steroid treatment animals into three groups based on mortality and resulted in significantly higher day +21 GVHD specific histopathological scores of GVHD target organs in REF relative to RES animals. To eliminate strain dependent factors, we used another BMT model, B6àB6D2F1 and found a similar differential response to steroid; albeit with a slightly different distribution amongst the groups and greater mortality in the ST and RES groups.
We next sought to determine whether steroid refractoriness can be modeled at a later time-point, to account for any contribution from radiation toxicity. To achieve this, we utilized the same C3H.sw→B6 model except that DEX was administered from day +21 to +35. We again obtained similar overall survival and GVHD specific histopathological scores in target organs as in our early steroid treatment model.
We next determined whether the steroid refractoriness correlated with changes in donor T cell, such as persistent activation, in REF animals. However, to our surprise, in both our early and late steroid treatment models, we found no difference in the proportion of donor activated, effector or memory T cells between the REF and RES groups. In addition, there was no difference in absolute number or percent of donor splenic T cells or their subsets including Tregsand exhausted T cells. Furthermore, there were no differences in serum inflammatory cytokines, donor intrahepatic lymphocytes, GI tract donor intraepithelial lymphocytes, or donor myeloid cells on day 7 or 14 of DEX treatment in the early and late steroid models, respectively.
Because target tissue intrinsic mechanisms are critical for initiating GVHD, we tested whether gut homeostasis related proteins were altered in SR-GVHD and found that the expression of CD324 (E-cadherin) in gut epithelial cells is significantly increased in SR-GVHD.
In conclusion, we established clinically relevant SR-GVHD murine models. To our surprise, donor T cell characteristics were not significantly different between groups treated with steroids. However, CD324 expression in gut epithelial cells is dramatically increased in refractory animals. These data suggest that donor T cell independent mechanisms may also contribute to steroid refractoriness more so than was previously considered.

Disclosures
Ishizawa: Chugai: Honoraria; Sanofi: Research Funding; Phizer: Research Funding; Celgene: Honoraria; Eisai: Honoraria; Janssen: Honoraria; Otsuka: Research Funding.