Multiplexed single cell RNA/AbSeq sequencing of ex vivo Treg and ex-Treg from different time points
Purpose: identify differentially expressed genes between ex-Treg obtained 5, 10 or 15 days post-adoptive transfer into a lymphopenic environment compared to ex vivo Treg in order to identify Treg subset with heightened instability. Method: Treg from Foxp3YFP-Cre Rosa26RFP (PMID: 18387831, PMID: 17171761) were co-injected with congenically-disparated naive T cells in a 1:1 ratio into Rag1KO mice and ex-Treg were sorted at 15 days, 10 days or 5 days post-transfer. As a control, ex vivo Treg were sorted from Foxp3YFP-Cre Rosa26RFP (d0 sample). Next, samples were stained with multiplexing antibodies using the BD Single-Cell Multiplexing Kit (BD biosciences), then pooled and stained with the following  oligonucleotide-conjugated antibodies: anti-CD25 (PC61), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-GITR (DTA-1), anti-CD4 (RM4-5), anti-TCRb (H57-597), anti-CD69 (H1.2F3), anti-TIGIT (1G9), anti-CD95 (Jo2), anti-CD122 (TM-1ß), anti-Tim-3 (5D12), anti-CCR7 (4B12), anti-CD103 (M290), anti-CD279 (J43), anti-CD274 (MIH5), anti-CD233 (C9B7W), anti-CD71 (C2), anti-CD278 (7E7G9), anti-ITGB7 (M293), anti-CD137 (1AH2), anti-CD40 (3/23), anti-CD3e (145-2C) from BD Biosciences. Single-cell capture and cDNA library preparation was performed using the BD Rhapsody Single-cell analysis system (BD Biosciences), according to the manufacturer's instructions, using a custom gene panel (591 genes). Sequencing was performed on an Illumina NextSeq500 instrument using a Mid-Output kit v2.5 (150 cycles, paired-end). Sample demultiplexing, barcode processing, alignment, filtering, UMI counting were done using the standard BD Biosciences Rhapsody analysis pipeline on Seven Bridges (www.sevenbridges.com). Result: In this study, we showed that ex-Treg downregulate Treg core-specific genes immediatly upon adoptive transfer into a lymphopenic environment. Further, we identified a Treg population enriched for unstable Trg clones. Conclusion: Our study was able to identify a Treg subset enriched for unstable Treg with plastic potential. This Treg subset appears highly enriched for naïve peripheral-induced Treg. Overall design: ex vivo Treg and ex-Treg 5, 10 or 15 days post-adoptive transfer into Rag1KO mice were sorted, stained for multiplexing antibodies and stained for Abseq antibodies. Cell capture and single cell RNA sequencing was performed for a custom gene panel using the BD Rhapsody and  Illumina NextSeq500 instruments.