RNA_seq analysis on Tfh and Treg before and after abatacept treatment

Analysis of transcriptome data identified 96, 64 and 3 genes that were differentially expressed (DE, adj. P value < 0.05) following abatacept treatment in Tfh, Treg and CD4+CD45RO+PD1+CXCR5- T cells, respectively. We determined if abatacept treatment altered the core transcriptional profiles of Tfh and Treg lineages by comparing gene expression of bulk sorted Treg and Tfh cells from cryopreserved PBMCs at baseline, week 24, and after abatacept withdrawal. Analysis of differentially expressed (DE) genes in Tfh was performed using Gene Ontology (GO) term enrichment analysis. This analysis revealed 9 significantly enriched GO categories, which were related to processes involved in cell division and proliferation. DE genes in the enriched GO categories showed a highly significant correlation in Tfh and Treg cells, further highlighting the similarity in the response of these two populations to abatacept. In contrast to Tfh and Treg, acatacept had a minimal impact on CD4+CD45RO+PD1+CXCR5- T cells. Overall design: Tfh, Treg and CD4+CD45RO+PD1+CXCR5- T cells populations were sorted from 30 different donors from blood draws at 3 different visits weeks 0, 24, and 52. 500 cells of each subset were sorted directly into SMARTseq v4 (Takara) lysis buffer to release RNA, and RT-PCR then used to generate cDNA. Sequencing libraries were constructed using a modified protocol of the NexteraXT DNA sample preparation kit (Illumina). Libraries were pooled and quantified by Qubit® Fluorometer (Life Technologies). For the Abatacept group, visit 0 was used as baseline and visit 7 represents the timepoint after abatacept treatment. The placebo group received placebo treatment first and then crossed over to abatacept treatment., hence we used v7 as baseline and visit 15 as the timepoint after abatacept treatment.