Fate challenge purges the regulatory T cell repertoire of unstable clones - supplementary tables S.Junius et al.
paper: "Fate challenge purges the regulatory T cell repertoire of unstable clones" Steffie Junius, Adamantios V. Mavrogiannis, Pierre Lemaitre, Margaux Gerbaux, Frederik Staels, Vanshika Malviya, Oliver Burton, Raul Yhossef Tito Tadeo, Jeroen Raes, Joost P. M. van Meerwijk, Stephanie Humblet-Baron, Adrian Liston*, Susan M. Schlenner* Folder single-cell RNA sequencing - Fig. 4: 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 was 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 and conclusion: In this study, we showed that ex-Treg downregulate Treg core-specific genes immediately upon adoptive transfer into a lymphopenic environment. Further, we identified a naïve-like Treg population enriched for unstable Treg clones. Folder microbiome sequencing- Fig S4: Purpose: Analyze diversity in microbial communities within fecal samples of SPF housed mice and SPF mice co-housed with pet store mice (CoH) method: Microbiome sequencing was performed as previously described in Pasciuto et al. (PMID: 32702313) result and conclusion: The microbial communities were different in SPF mice compared to CoH mice; 19 % of the variation on the microbial composition can be explained by this grouping (adonis test, R2 = 0.18791, p = 0.001). Twelve genera were identified with differences in relative abundance, seven enriched and five depleted in CoH mice (q-values < 0.1, Wilcoxon test).
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Fig 4/ Fig S10: 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-Foxp3 cells. 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 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). For more information please refer to the paper Fig S4: protocol: Foxp3YFP-Cre RosaRFP mice were either SPF-housed (n=13) or co-housed with wild-exposed pet store mice for 8 weeks (n=9). The microbial diversity of caecal contents in specific pathogen-free (SPF) and wild-cohoused (CoH) mice was compared through 16S rRNA analysis. For more information please refer to the paper Fig S5: Mathematical modeling of ex-Foxp3 kinetics after transfer is consistent with a stable Treg source. Results of mathematical modeling of active Foxp3 deconversion from previously stable Tregs at the point of transfer (t = 0) vs uninterrupted transient Foxp3 expression in non-Tregs. For more information please refer to the paper