Fate challenge purges the regulatory T cell repertoire of unstable clones - single cell RNA sequencing tables S.Junius
paper: "Fate challenge purges the regulatory T cell repertoire of unstable clones" Steffie Junius, Pierre Lemaitre, Adamantios V. Mavrogiannis, Oliver Burton, Adrian Liston, Susan M. Schlenner 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‑1β), 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 Treg 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.
Steps to reproduce
protocol: At day 5, day 10 and day 15, recipient mice were euthanized and spleen and lymph nodes were processed and FACS sorted for ex-Treg. As a control ex vivo Treg were sorted from Foxp3YFP-Cre Rosa26 fl STOP fl RFP mice (day 0 sample). Next, samples were stained using the with sample tag antibodies (BD Single-Cell Multiplexing Kit, BD Biosciences). Afterwards, samples were pooled and stained using the following oligonucleotide-conjugated antibodies according to manufacturing instructions: anti CD25 (PC61), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-GITR (DTA-1), anti-CD4 (RM4-5), anti TCRbeta (H57-597), anti CD69 (H1.2F3), anti TIGIT (1G9), anti CD95 (Jo2), anti CD122 (TM 1β), 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 CD3a (145-2C) from BD Biosciences. Single-cell capture and cDNA library preparation was performed using the BD Rhapsody single-cell analysis system (BD Biosciences). Libraries were constructed according to manufacturers recommendation (BD Biosciences, Doc ID: 214508 Rev 3.0). Briefly, captured mRNA was amplified with a custom primer panel and with specific primer for the BD AbSeq and Sample tag antibodies. The Sample Tag and BD AbSeq PCR products were separated from the mRNA targeted PCR products by double-sided size selection with Agencourt® AMPure® XP magnetic beads. The BD AbSeq and Sample Tag PCR products underwent a separate index PCR from mRNA products with library index primers. BD Rhapsody™ mRNA targeted PCR products and Sample Tag PCR products underwent a second PCR amplification followed by an index PCR with library index primers. The raw data were processed through the BD Rhapsody pipeline. All steps in details are documented here: https://scomix.bd.com/hc/article_attachments/360048823252/23-21713-01_Rhapsody_Bioinfo_Handbook-ruo.pdf Briefly, the raw data were filtered from low quality reads based on average Phred score <20. R1 reads were used to determine the cell entity and molecule count. R2 reads were aligned to BD custom reference to define mRNA origin. Information about cell label, molecule and mRNA target was combined from R1 and R2. The molecule count was corrected by applying RSEC and DBEC correction for all genes that reached sequencing saturation >4. The residual cell labels were filtered based on cumulative number of mRNA reads in cells.