Background/Purpose: For rheumatoid arthritis (RA), as with many other rheumatic diseases, the importance of determining which therapy will work best, early in disease, to prevent further progression, is an important area of research. Progress in treatment response has been limited, possibly due to the complex interplay between various cell types. As such, specific T-cell signatures, determined by single cell RNA-Seq (scRNA-Seq), could be predictive of future response to treatments such as anti-TNF biologic therapies. Our aim was therefore to determine the optimal study design and to assess the potential of scRNA-Seq to identify T-cell signatures under resting and stimulated conditions to inform future studies.

Methods: Primary CD4+ T-cells were either stimulated using anti-CD3/CD28 beads or subjected to the same conditions without stimulation for 4 hours. Single cells were isolated using the 10X Genomics Chromium Controller with a target recovery of 6000 cells. Each scRNA-Seq library was sequenced on 4 Illumina HiSeq 4000 lanes (~200K reads/cell) and processed using the cellranger pipeline. Further quality control and cluster analysis was performed using Seurat.

Results: For the unstimulated sample 5,586 cells were recovered and after quality control and filtering, 5,387 cells remained. Similarly, for the stimulated sample, 4,621 cells were recovered and 4,473 remained. This resulted in an average of 1,094 and 1,456 genes per cell. Similar clusters were seen after downsampling the stimulated dataset to 1 lane (~379M reads, ~82K reads/cell), suggesting that CD4+ T-cells are defined by large gene expression changes rather than subtle variations, consistent with protein expression data. Cluster exploration allowed the identification of several typical CD4+ T-cell populations, including naïve, helper and regulatory. Furthermore, alignment of the two conditions in Seurat, identified classical and non-classical markers of activation, such as CD69, CCR7, MYC and PIM3. Finally, the relative cluster location and the expression of indicative markers suggested evidence of a progression from a naïve cell state to an ‘active’ effector state.

Conclusion: This data has provided important insights into future study design and confirmed the potential of scRNA-Seq to identify T-cell signatures. Importantly, despite obvious expression changes, cluster identity was maintained between stimulatory conditions. This implies it is possible to directly compare scRNA-Seq expression profiles between patient samples showing different disease activity without confounding the conclusions and enable the use of scRNA-Seq to investigate its predictive potential in RA treatment response. We are therefore in the process of expanding this work to study patient samples and different cell types. For example we have already generated similar data for monocytes on 3 RA samples and 3 healthy samples.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/exploration-of-t-cell-signatures-following-tcr-stimulation-using-single-cell-rna-seq-to-inform-treatment-response-studies-in-rheumatoid-arthritis/