A Spatial Atlas of Human Gastro-Intestinal Acute Gvhd Reveals Epithelial and Immune Dynamics Underlying Disease Pathophysiology
Authors: Milo Idan , Azulay Nofar , Bussi Yuval , Ben Uri Raz , Keidar-Haran Tal , Eldar Michal , Elhanani Ofer , Harnik Yotam , Yakubovsky Oran , Nachmany Ido , Salame Tomer , Bertheau Philippe , Michonneau David , Socie Gerard , Keren Leeat 
Published: December 2024

Intro
Acute graft versus host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (aHSCT). Alloreactive donor T cells are driving factor in GVHD development in the gut, but knowledge of the role that additional donor and host cells play in this process is limited. Here, we leveraged Multiplexed Ion Beam Imaging by Time of Flight (MIBI-TOF), to generate comprehensive spatial maps of healthy and GVHD-afflicted duodenum at single cell resolution.
Methods
We profiled biopsies taken from 57 GVHD patients and 10 healthy controls and measured by MIBI-TOF the spatial expression of 40 proteins, designated to delineate distinct epithelial cells, stromal cells and immune cells in the gut. Overall, we acquired 256 high-dimensional images, each depicting the spatial expression of forty proteins in situ. To map single cell identity and function we segmented individual cells and classified them using a deep-learning pipeline. Altogether, we identified 32 cell populations, including populations of epithelial cells (enterocytes, goblet, endocrine, and Paneth cells), various immune populations among them CD4 and CD8 T cells, T regulatory cells (Tregs), plasma cells, B cells, neutrophils, macrophages, mast cells and several stromal populations.
Results
First, we interrogated the composition and spatial distribution of immune cells in normal duodenum. We found that plasma cells, macrophages and CD4+ T cells were the most frequent immune cell types, with prevalences of 30%, 27% and 20%, respectively. Following them were CD8+ T cells (8%), other cells, such as neutrophils and Tregs appeared at low prevalence (&lt; 2% each). The lamina propria was enriched with plasma cells, macrophages and CD4+ T cells, whereas CD8+ and double negative T cells were the predominant populations inside the epithelium.
Then we explored the effects of GVHD on the structure of the gut epithelium. GVHD patients exhibited increased fibrosis, alterations in crypt morphology, loss of Paneth cells, and accumulation of endocrine cells in the crypts. These alterations were correlated with pathological staging. To investigate the local immunological processes in GVHD, we analyzed the immune composition in GVHD patients and controls. We found that GVHD was characterized by a significant decrease in plasma cells and CD4+ T cells. Patients with severe clinical gut staging disclosed infiltration of additional immune cells including neutrophils, Tregs, macrophages and CD8+ T cells. To evaluate the consistency of these trends across patients, we clustered all samples according to their immune composition. We found that samples from healthy duodenum clustered together, while GVHD patients showed a breakdown of this organization, clustering into distinct immunological subtypes. The different subtypes were characterized by enrichments of distinct cell types, including CD8+ T cells, neutrophils and macrophages. Some subtypes were associated with specific clinical parameters for example a subgroup enriched with macrophages and CD8+ T was associated with overall worse prognosis. Altogether, we found that GVHD, and especially clinically severe GVHD, is characterized by a breakdown of immune homeostasis, with a reduction in plasma cells, and increase in distinct immune cells, including macrophages, neutrophils and T cells.
Moreover, we also observed intra-patient heterogeneity in the prevalence of different immune cells in the vicinity of individual crypts. Interestingly, crypts with dense accumulation of immune cells were the most deteriorated, while other crypts in the same biopsy were intact, with little immune infiltration. These results highlight the importance of spatial analysis that can uncover short-scale immune interactions and suggests local inflammation as a defining driving force of disease progression.
Finally, we used DNA fluorescent in situ hybridization against the X and Y chromosomes to uncover the host, or donor, origin of immune cells in samples from cross-gender aHSCT. Notably, we found that some host T cells, and plasma cells persist in the gut for months after immune ablation.
Conclusions
Overall, this spatial atlas of healthy and GVHD-afflicted duodenum reveals non-canonical immune landscape and heterogeneity underlying disease pathophysiology in humans, that could lead to potential implications for clinical diagnosis and therapeutics.