Background/Purpose: CD4⁺ T cells dominate focal lymphocytic infiltrates in Sjögren’s disease (SjD), but their differentiation states have remained unclear. Here, we used single cell (sc)RNAseq to discover CD4+ T cell states and spatial proteomics to confirm the findings.

Methods: CD4⁺CD45RA^– memory T cells were singly sorted from minor salivary gland biopsies of individuals meeting the 2016 ACR/EULAR criteria for SjD (n=6) and subjected to scRNAseq using SmartSeq2. Transcripts aligning to the genome with HISAT2 and expressed in >10% of cells were used for dimensionality reduction. Data from the first 10 principal components were visualized with UMAP. Gene set enrichment analysis (GSEA) was used to identify the clusters. Differentially expressed (DE) transcripts between clusters were assessed by MAST using genes expressed in >1% of cells. FFPE tissue sections from an independent set of SjD cases (n=49) and symptomatic individuals with no objective signs of SjD (n=15) were subjected to Nanostring spatial proteomic analysis, with regions of interest (ROI) segmented on CD45 or pan cytokeratin (PanCK) fluorescent signals. Data normalized to ROI area were used to evaluate relationships between proteins using Pearson’s correlations.

Results: Half of single cell cDNAs passed quality control (QC) and underwent library preparation and sequencing with PE150 reads. Of 402 sequenced cells, 385 passed QC (< 30% mitochondrial reads, avg expression >3). Clusters (CL) corresponded to central memory T helper (CL0), quiescent (CL1), activated CD4+ cytotoxic T lymphocyte (CTL) (CL2), early activated/stressed (CL3), and regulatory/T follicular helper (CL4) cells. CL0 and CL2 were enriched for CD4⁺ CTL precursor and effector signatures, respectively, by GSEA. Within Nanostring ROIs containing lymphocytic foci, GZMA and GZMB showed stronger correlative relationships with CD3 (r=0.724, p< 0.0001; r=0.640, p< 0.0001) compared to CD56 (r=0.462, p< 0.0001; r=0.449, p< 0.0001) in SjD cases. GZMA and GZMB were more strongly correlated with CD4 (r=0.711, p< 0.0001; r=0.512, p< 0.0001) than CD8 (r=0.523, p< 0.0001,r=0.221, p=0.003) in SjD cases. Correlations between CD4 and GZMA (r=0.432, p=0.004) or GZMB (r=0.185, p=ns) were weaker or absent in non-SS control ROIs containing lymphocytic clusters. CD4 in the CD45 segment of SjD cases correlated with markers of epithelial to mesenchymal cell transition (FAP-a) and tissue damage (CD95/Fas, PARP, BIM, p53, GZMA, GZMB) in the PanCK segment of matched ROIs, while similar relationships with CD8 were absent or weak.

Conclusion: Activated CD4⁺ CTL are present in minor salivary glands of SjD cases, and proteomic markers of cytotoxicity associate with CD3 and CD4 in regions containing focal infiltrates. GZMA, GZMB, and CD4 protein levels in CD45⁺ cells correlate with markers of tissue damage in epithelial cells near lymphocytic infiltrates, implicating CD4⁺ CTL in glandular pathology of SjD.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cd4-cytotoxic-t-lymphocytes-ctl-are-present-in-sjogrens-disease-minor-salivary-glands-and-associate-with-markers-of-epithelial-cell-damage-near-infiltrates/