Minor Histocompatibility Antigens Cross-Reactive Against Gut-Tropic Viral Epitopes Facilitate Acute GvHD of the Gut

Severe manifestations of acute gastrointestinal (GI) GvHD portend a poor prognosis after allo-HCT. Therefore, new tools to predict – and possibly mitigate – GI aGvHD risk are urgently needed. In HLA-matched allo-HCT, T cell alloreactivity is directed against minor histocompatibility antigens (mHAgs), for the identification of which we have previously developed an analytic framework based on polymorphism detection by whole exome sequencing (WES) of germline DNA from donor-recipient (D-R) pairs (Cieri, Nat Biotechnol 2024). Building on the clinical observation that viral reactivations often coincide with the development of GvHD, we hypothesized that sequence homology of mHAgs with gut-tropic viral epitopes (ADV, CMV and EBV) may contribute to GI aGvHD pathophysiology.

To quantify the extent of cross-reactivity across the patient-specific mHAg landscape, complete viral proteomes for ADV, CMV and EBV were retrieved from UniProt to create in silico all possible 8-11mers (n = 1,108,265) that were then subjected to HLA class I epitope prediction, resulting in a catalog of 195,081 viral epitopes across 129 HLA class I alleles. Predicted mHAgs from WES analysis of 520 HLA-matched D-R pairs (220 MRD, 300 URD) were first filtered for expression in GI-resident non-hematopoietic cells, as identified by single cell RNASeq datasets of GI tissue from healthy and post-transplant individuals. Homology with predicted viral epitopes was defined by either: (i) sliding window, i.e., mHAg and viral antigen sharing ≥6 consecutive amino acids; or (ii) skipping window, i.e., Levenshtein distance between mHAg and viral antigen ≤2 amino acids, irrespective of position. Predicted mHAgs were considered cross-reactive only if displaying homology with viral epitopes presented on the same HLA restriction.

Across the MRD cohort, the median number of cross-reactive GI-specific mHAgs was 37 (range: 13 – 87). Patients who developed severe GI aGvHD (grade III-IV) had a higher load of cross-reactive GI-specific mHAgs than patients who did not (P = 0.038). We considered the impact of diagnosis, prognostic risk score, status at transplant, conditioning regimen, graft source, donor age and CMV status against median cross-reactive mHAg load on risk for GI aGvHD. Only cross-reactive mHAg load > median was associated with increased risk of developing severe GI aGvHD (HR = 4.96, 95% CI: 1.13 – 19.44, P = 0.03). Preliminary analysis of an independent cohort of 300 HLA-matched URD D-R pairs confirmed the association of cross-reactive mHAg load with severe GI a GvHD (P = 0.018).

Our findings reveal cross-reactivity against gut-tropic viruses as a key contributor to GI aGvHD pathophysiology. Molecular characterization of D-R pairs to quantify cross-reactivity provides a promising tool for pre-transplant prognostication that could facilitate the incorporation of preventative strategies (such as vedolizumab) in high-risk patients.