Human Gastro-Intestinal Graft-Versus-Host Disease Is Mediated By Retinoic Acid-Responsive CD8+ Effector T-Cells Under IL-23 Polarising Conditions
Authors: Ball Jennifer A , Clear Andrew James , Calaminici Maria , Stagg Andrew , Lindsay James , Gribben John G. , Davies Jeff 
Published: April 2021

Abstract
Background: Gastro-intestinal (GI) graft-versus-host disease (GvHD) results in significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation (AHSCT). Retinoic acid (RA), a metabolite of vitamin A, has diverse effects on immune cells. RA signaling promotes immune tolerance under steady state conditions. However in pro-inflammatory conditions RA increases Th1, Th17 CD4+ and Tc1 CD8+ T cell responses and is implicated in the pathogenesis of autoimmune inflammatory bowel disease. Furthermore, RA exposure and RA-receptor alpha (RARα) signaling in allogeneic T cells potentiates GI-GvHD in mice. Thus RA and cytokines that influence the effect of RA on T cells may represent potential therapeutic targets for GI-GvHD. However, the impact of RA at a tissue level in human GI-GvHD is unknown. We therefore investigated the role of RA-responsive T cells in human GvHD in vivo and human allogeneic T-cell responses in vitro .
Methods: We scrutinized GI biopsies from 47 patients after AHSCT (36 with and 11 without GI-GvHD confirmed by conventional histological criteria). We also analysed skin biopsies from 11 AHSCT patients. We used conventional immunohistochemistry and a novel deep phenotyping method using sequential staining, stripping and re-probing on the same fixed embedded biopsy to determine cellular co-expression of multiple markers to identify RA-responsive T cells in human GvHD biopsies at sites of GvHD damage. Finally, we used an HLA-mismatched allogeneic mixed lymphocyte co-culture (MLRs) system, combining CFSE dye dilution and multi-parameter flow cytometry to enable the phenotyping of alloproliferative T cells after manipulation of RA signalling.
Results: Firstly we observed thatthe number of cells expressing high levels of cytoplasmic RA binding proteins I/II (a surrogate marker of RA) was increased in gut biopsies in patients with GI-GvHD versus controls (median 155 vs 43 cells/mm2, p=0.056). Importantly the number of cells expressing RARα was also significantly increased (median 1315 vs 544 cells/mm2, p=0.001) consistent with RA-responsiveness. Crucially the number of RARα+ cells correlated with clinical stage of acute GI-GvHD with higher numbers in patients with severe (stage 3-4) versus less severe (stage 1-2) disease.
Having demonstrated that RA-responsive cells were associated with both occurrence and severity of GI-GvHD we went on to identify potential RA-responsive T cell subsets. Additionally, as IL-23 and IL-33 direct RA-programming of T cells to either pro-inflammatory or tolerogenic phenotypes we also measured cellular expression of these cytokines and their receptors. CD8+ T cells co-localized with RARα+ cells and were present in significantly increased numbers in gut biopsies from patients with GI-GvHD, as were Tbet+ Th1/Tc1 cells. In contrast, numbers of RORγ+ Th17 cells were unchanged and CD4+ cells were decreased. IL-23p19+ and IL-23R+ cell numbers were significantly increased in GI-GvHD biopsies, whereas IL33R (ST2+)cells were unchanged.
In order to determine if the RA-responsive, CD8+ and IL-23R+ cells represented a single effector subset, we used sequential stripping/re-probing to deep phenotype the T cells. We identified a unique single CD8+ T-cell population that co-expressed RARα+, T-bet and IL23R, present in significantly increased numbers in gut biopsies from patients with GI-GvHD versus controls. This is consistent with a RA-responsive, IL-23-dependent, inflammatory Tc1 effector cell mediating tissue damage in human gut GvHD. Importantly, there was no increase in this T cell subset in skin biopsies of skin GvHD patients demonstrating tissue-specificity.
Finally we determined the impact of exogenous RA exposure on T cell alloresponses in MLRs. RA significantly increased the proportion of alloproliferative effector CD8+ T cells co-expressing Tbet and gut-homing molecules, confirming that RA can directly potentiate human alloreactive CD8 T cell responses with capacity to home to the GI tract.
Conclusions: This is the first data to demonstrate a role for RA at a tissue level in human GI-GvHD. Furthermore we have identified a gut specific CD8+ T cell subset which co-expresses RARα, T-bet, and IL-23R localised in areas of GI-damage likely to represent the RA-responsive effector cell. Therapeutic blockade of IL-23R could target this cellular response to prevent or treat GI-GvHD.

Disclosures
Gribben: Acerta: Honoraria; Janssen: Honoraria; Kite: Honoraria; Celgene: Honoraria; TG Therapeutics: Honoraria; Karyopharm: Honoraria; Pharmacyclics: Honoraria; Genentech/Roche: Honoraria; Abbvie: Honoraria.