Single-Cell Transcriptional Survey of the Initiation, Propagation and Maintenance of the Intestinal Graft-Versus-Host Disease

Background:

Graft-versus-host disease (GVHD) is a life-threatening complication of hematopoietic stem cell transplantation (HSCT). The intestine not only is a primary target of immune attack, but also regulates the severity of systematic GVHD. Although the critical roles of immune cells in GVHD have been documented, the dynamic changes of cell populations, and cell-associated transcriptional signatures during immune attack and epithelial regeneration remain elusive.

Methods:

To better characterize disease progression, tissues were harvested from murine intestine suffering from GVHD (GVHD_D3, GVHD_D10, GVHD_D21), coupled with irradiated and repaired samples (BM_D3, BM_D10, BM_D21), and healthy control (CON). Each sample mixed with segments of proximal and distal small intestine from three mice. Tissues were dissociated into single-cell suspensions and processed using the 10x Genomics Chromium workflow. After data pre-processing and quality control, we profiled 69242 individual cells from 7 samples.

Results:

Uniform manifold approximation and projection (UMAP) analysis yielded 17 clusters, including 8 types of epithelial cells, 8 types of immune cells and 1 type of stroma cell.

In healthy mice, epithelial cells were highly enriched in murine small intestine, taking up 79.31% of total cells: differentiated enterocytes (EC) (59.31%) represented the largest cohort of epithelial cells, while intestinal stem cells (ISC), trans-amplifying (TA) cells and enterocyte progenitor (EP) only occupied very small portions. In mucosal immune system, the abundance of T cells (9.91%) and B cells (10.43%) was much higher than that of ILC, macrophage and neutrophils. Upon radiation and the development of GVHD, cell populations of murine small intestine dramatically changed. Three days after transplantation, the proportion of ECs dramatically dropped. On the contrast, ɣδ T cells and ILC rapidly accumulated in the damaged intestine. From day 3 to day 10, we observed a steep decrease in ɣδ T cells from samples of BM and GVHD group. At our last observation, BM resorted its epithelial components, while GVHD had a great abundance of T cells (41.73%) and a lower level of differentiated ECs (36.90%).

In epithelial components, a total of 44468 epithelial cells were subclustered them into 22 subsets. ISC serves as the production department, which was enriched in mitochondrial genes and genes regulating protein folding. The cluster TA_1 from D21 samples was enriched in pathway related to structure organization and BMP signals, which is required for the initiation of intestinal differentiation and morphogenesis. At day 3, a rapid expansion of subsets of TA_2 and Regenerative progenitors caught our attention. Metabolic pathways such as tryptophan catabolic process, arachidonic acid secretion, methionine metabolic process were highly correlated with wound healing and cell proliferation. EP_1 emerging at day 3 majored in material transportation. EP in samples from control and BM (EP_2) was able to repair DNA damage, while EP_3 that mostly detected in GVHD sample were involved in metabolic disorders, which may explain its deficiency in EC differentiation.

T cells and ILC were divided into 16 subsets. In addition to its well-known effects in host defense, several genes that expressed in γδT cells are essential for maintaining intestinal homeostasis. The Tgfb3-Vegfa+ subset regulated microvillus organization and intestinal absorption; the Tgfb3-Vegfa- subset participated in sensory and ciliary movement. At day3, the above clusters undergone a rapid expansion, suggesting an enhanced protective role of γδT cells in the early period of transplantation. We also identified different subtypes of T cells between BM and GVHD samples. Two clusters of cycling CD8+ T cells were more enriched in GVHD samples, which also express high levels of cytotoxic markers. While in BM, we found two clusters of anergic T cells without expression of markers of naïve, memory, effector and exhaustion. The double positive T cells in GVHD may regulate immune infiltration, while the CD4+ Th2-like cell in BM may link to immunoglobin production and Treg differentiation.

Conclusions:

Single-Cell transcriptional analysis revealed that different cell populations between BM and GVHD. T cells in GVHD were highly-proliferative and cytotoxic, and epithelial cells in GVHD had metabolic disorder and were resistant to enterocyte differentiation.