Integrated transcriptomic and TCR profiling reveals local immune dysregulation of T cells in Gl aGvHD

Patients were prospectively recruited between 2021 and 2023 after approval by the ethics committee of the Technische Universität Dresden (BO-EK-11012020) and provision of written informed consent. Enrollment took place at the University Hospital Dresden, Germany. Eligible patients were ≥ 18 years old and had received their first allo-HCT for a hematologic malignancy. Detailed information regarding material and methods can be viewed as supplement. Nineteen patients undergoing endoscopy for suspected GI aGvHD were included (Suppl. Table 1 , Suppl. Fig. S1 ). Histopathology identified 12 with GI aGvHD and 7 without GI involvement. In blood circulating T cells, 9 genes were differentially expressed between groups, without clear functional clustering (Suppl. Fig. S2A ). Gene set enrichment analysis showed negative enrichment of pathways such as graft-versus-host disease, NK cell cytotoxicity, and antigen processing in GI aGvHD patients (Suppl. Fig. S2B ). In contrast, gastrointestinal T cells showed pronounced changes: 786 genes were differentially expressed (741 up-, 45 downregulated, Fig. 1a ). These were not linked to the predefined GvHD pathway but were associated with diverse processes including adhesion, signaling, and pathogen-related pathways (Suppl. Fig. S2C ). a Gene expression in Biopsy samples: aGvHD vs non-GI aGvHD - Volcano plot of differentially expressed genes in and gastrointestinal T cells at the time of endoscopy of GI aGvHD versus non-GI GvHD patients. Genes with |log2 fold change, log2FC | > 1.5 and q < 0.1 (10% false discovery rate) were considered as significantly differentially expressed. b GI aGvHD associated genes from Biopsy in Blood - Categories delineating gene expression patterns of potential GI aGvHD associated genes from gastrointestinal biopsies in peripheral blood samples, along with genes falling into each category. The x-axis represents the log2 fold change in GI aGvHD gastrointestinal T cells compared to non-GI GvHD gastrointestinal T cells (−8 to 8, left to right), while the y-axis indicates the log2 fold change in blood-circulating T cells (−5 to 5, bottom to top). Genes with |log2 fold change, log2FC | > 1.5 in both gastrointestinal and blood-circulating T cells and q < 0.1 in gastrointestinal T cells are included. c TRA repertoire diversity in Biopsy and Blood: aGvHD vs non-GI aGvHD - Bulk TCR sequencing of sorted CD3 + T cells showing percentage of reads from clonotypes with read frequency > 1% (y-axis) and inverse Simpson Diversity index (y-axis) for TRA chain. Comparisons were done between GI aGvHD (red) and non-GI GvHD (gray) patients across gastrointestinal biopsies (Biopsy) and corresponding blood samples (Blood). Significant differences are denoted by * (0.01–0.05), ** (0.001–0.01), *** (0.001–0.0001), and **** (< 0.0001) using Mann-Whitney U tests. The median with interquartile range is indicated for each. d Percentage of TRA repertoire occupied by MelanA specific clonotypes in Biopsy and Blood: aGvHD vs non-GI aGvHD - Violin plots showing percentage of MelanA reactive TRA clonotypes within the total TRA repertoire (y-axis) compared between GI aGvHD (red) and non-GI aGvHD (gray) patients for gastrointestinal biopsies and peripheral blood samples. Significant differences are denoted by * (0.01–0.05), ** (0.001–0.01), *** (0.001 to 0.0001), and **** (< 0.0001) using Mann-Whitney U tests. e Aberrant MelanA expression (brown) in gastrointestinal capillary endothelia of GI aGvHD biopsy from Patient Tx009. Immunohistochemical staining with monoclonal mouse anti-human MelanA antibody (Clone A103) on biopsy obtained via colonoscopy (Colo). Scale bar: 50 µm. Comparison of biopsy and blood data revealed that most transcriptional changes were tissue-specific: 728 genes were altered only in biopsies, while 22 genes were similarly regulated in both compartments, mainly involving cytokine and chemokine signaling (e.g., CCR9, CXCL13, CD70) (Fig. 1b , Suppl. Figs. S2D , S2E ). TCR repertoire analysis showed lower diversity in gastrointestinal tissue compared to blood in both groups ( P < 0.01). However, GI aGvHD biopsies displayed higher diversity than non-GI GvHD, along with fewer dominant clonotypes (Fig. 1c , Suppl. Fig. S3A ). Analysis of TCR clonotypes identified a large number unique to GI aGvHD (Suppl. Fig. S3B ). Among known antigen-specific clonotypes, viral targets predominated, but a subset recognized human antigens (Suppl. Fig. S3D ). Notably, MelanA-specific clonotypes were the most abundant among human antigen-specific TCRs, especially in GI aGvHD biopsies (Suppl. Fig. S3E , S3F , S4 ). MelanA-specific clonotypes were present in 68% of GI aGvHD biopsies but less frequent in non-GI samples. Their relative abundance was higher in GI aGvHD tissue, while blood levels were similar across groups (Fig. 1d , Suppl. Fig. S3F , S4 ). Immunohistochemistry showed heterogeneous MelanA expression in gut tissue (Fig. 1e , Suppl. Fig. S5 , Suppl. Table 2 ). In selected cases, MelanA expression co-occurred with MelanA-specific TCRs, suggesting a potential role in disease, though this pattern was not consistent across all patients (Suppl.Fig. S5 , Suppl. Table 2 ).