Background/Purpose: Background: The clinical overlap of axial spondyloarthritis (axSpA) and Crohn’s disease (CD) has given rise to the hypothesis that these conditions may share pathophysiologic mechanisms. However, the relationship between each of these diseases and the “overlapping” condition of co-morbid CD and axSpA (OVR) remains unknown. Here, we sought to address this question by an initial unbiased immune profiling in the peripheral blood of individuals with axSpA, CD, and OVR relative to healthy controls (HC) using single-cell RNA sequencing followed by validation of potential cellular populations with flow cytometry.

Methods: Methods: Peripheral blood was collected from 71 subjects (18 HC, 26 CD, 17 axSpA, and 10 OVR) and peripheral blood mononuclear cells (PBMCs) isolated. Viable CD45+ PBMCs from two representative male patients in each group were isolated by flow sorting and underwent high-depth single-cell RNA sequencing (10X Genomics). Significance with respect to change in transcript abundance was set at P < 10^-6. Validation of findings was then performed by flow cytometry using PBMCs from the full cohort of 71 subjects. Flow data was analyzed in FloJo and GraphPad Prism, and significance was determined by Mann-Whitney-U test at P < 0.05.

Results: Results: Single cell RNA sequencing demonstrated an expansion of CD27+ Granzyme B+ memory (CD45RA-) T cells in our patients with axSpA and OVR. However, in CD, we observed an expansion of a similar memory T cell population expressing the innate-like T cell transcription factor PLZF, which we hypothesized to be gd T cells. B cell and monocyte populations in all disease groups had increased expression of genes within antigen presentation, inflammasome, and apoptotic pathways. Together, our sequencing data suggested upregulated cytolytic activity in the peripheral blood of individuals with axSpA, CD, and OVR patients. In order to validate these findings in our larger cohort, we utilized flow cytometry to interrogate the cytotoxic T cell population. We observed an increase in gd T cells in CD relative to HCs (P < 0.005).  In OVR, we saw a significantly decreased naïve T cell compartment (P< 0.005) compared to HC, CD, and axSpA, with a corresponding increase in effector memory (CCR7- CD45RA-) T cells (P < 0.005). The proportion of CD4 T cells bearing markers of cytotoxicity (Granzyme B+, NKG2a+) was significantly (P< 0.05) increased in OVR compared to HC, CD, and axSpA. The overall proportion of cytotoxic CD4s as fraction of total PBMCs in OVR was 1.3%, more than triple that found in the other groups (P< 0.05).

Conclusion: Conclusions: Altogether, our data suggest that the immunologic profile of individuals with OVR are distinct from both CD and axSpA. Specifically, we demonstrate an expansion of cytotoxic T cells in OVR relative to HC, CD, and axSpA, and the absence of the gd T cell expansion seen in CD. Given the association between cytotoxic CD4+ T cells and Th1 responses, these data may indicate a shift away from the Th17 dominant disease process found in axSpA. Future directions will validate these findings in an independent cohort of subjects as well as focus on elucidating the functional relevance of cytotoxic CD4 T cells in the pathophysiology of overlapping CD-axSpA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/expansion-of-peripheral-cytotoxic-t-cells-in-co-morbid-inflammatory-bowel-disease-and-spondyloarthritis/