Background/Purpose:  In rheumatoid arthritis (RA), genome-wide association studies have identified over 100 risk alleles. A major challenge is identifying causal genes in RA risk loci. As RA risk loci are enriched with many genes expressed in CD4⁺ memory T cells, our objective was to determine which risk-associated genes regulate T cell cytokine expression and dissect relevant biologic pathways important in RA development.

Methods:  A lentiviral small hairpin RNA (shRNA) library targeting ~40 RA risk genes was generated. Each RA risk gene was targeted by 5-8 shRNA clones. The shRNA was used to disrupt expression of RA risk genes in primary human CD4⁺ memory T cells, which were negatively isolated from healthy donor peripheral blood mononuclear cells. Primary human CD4⁺ memory T cells underwent infection, selection and then T cell receptor activation to promote cytokine expression. Enzyme-linked immunosorbent assay (ELISA) was used to measure expression of IL-10, IL-13, IL-17A and IFN- γ in culture media. Generation of relative cytokine level per cell number, line of best fit and additional analysis was performed using SAS v9.3. Rank order system was generated to identify genes that modulated cytokine expression in comparison to control shRNA. Genes identified in the primary screen were reassessed in a secondary screen and then further validated.

Results:  DDX6, a member of the DEAD-box helicase and post-transcriptional regulator family, was identified in our screen as a negative regulator of IL-10, IL-13, IL-17A and IFN-γ expression in activated CD4⁺ T cells. DDX6 silencing in CD4⁺ T cells increased cytokine expression, validating the screen findings. In addition, DDX6 mRNA levels were elevated in resting CD4⁺ T cells but decreased when CD4⁺T cells are functionally activated. Loss of expression inversely correlated with cytokine expression. We then demonstrated that DDX6 (p54/Rck) physically interacts with cytokine mRNA during transcriptionally quiescent states using RNA immunopreciptation.

Conclusion: Our experimental approach provides a systematic strategy to functionally differentiate candidate RA susceptibility genes based on relevant biological pathways. Specifically, we identified DDX6 as an important regulator of effector function of primary human CD4⁺ T cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-functional-genomic-screen-of-rheumatoid-arthritis-risk-genes-in-primary-human-t-cells-reveals-ddx6-as-a-negative-modulator-of-cytokine-expression/