Anti cd8 53 6.7

For isolation of T_H1 cells and T_FH cells, splenocytes from Mettl3^fl/flCd4-Cre mice and their control littermates on day 8 post viral infection were subjected to depletion of cells positive for lineages markers by using biotin-conjugated antibodies (anti-B220 (RA3-6B2; 1:100), anti-CD8 (53-6.7; 1:100), anti-Gr.1 (RB6-8C5; 1:100), anti-CD11b (M1/70; 1:100), anti-CD11c (N418; 1:100), anti-TER119 (TER-119; 1:100), and anti-CD49b (DX5; 1:100); all from Thermo Fisher Scientific) coupled to Dynabeads M-280 Streptavidin (Invitrogen), followed by surface stained. CD44⁺SLAM^hi T_H1 cells and CD44⁺SLAM^lo T_FH cells were sorted with a FACSAria II cell sorter (BD Biosciences) with FACSDiva software (v7.0) and subsequently lysed with TRIzol Reagent (Life technologies). Total RNAs were extracted and then subjected to Annoroad (Beijing, China) for library construction and RNA sequencing. The qualities of clean reads were assessed by FastQC (v0.11.5). Then the reads were mapped to mouse genome mm10 (version M17) using TopHat (v2.1.1). The read counts of all genes were estimated by HTseq (v0.6.1) and differentially expressed genes were identified by DESeq2 (v1.18.1). TPM, FPKM, and RPKM were calculated, and upregulated or downregulated genes in Mettl3^fl/flCd4-Cre T_FH or T_H1 cells were identified by expression changes ≥ 2-fold and FDR < 0.01.

Cells were incubated with Fc Block (CD16/32, BD Biosciences, San Jose, CA), stained with antibodies, and then fixed with 2% PFA. Samples were acquired on the FACS Canto II (BD Biosciences) and analyzed using FlowJo (TreeStar, Ashland, OR). Dead cells were excluded using the eBioscience Fixable Viability Dye eFluor® 506 (Thermo Fisher Scientific, Waltham, MA). The following antibodies were used: anti-CD8 (53-6.7) and anti-TCR-γδ (ebioGL3) from Thermo Fisher Scientific; anti-CD4 (GK1.5), anti-CD19 (6D5), anti-CD11b (M1/70), and anti-CD45 (30-F11) from BioLegend, San Diego, CA; and anti-TCR-β (H57-597) from BD Biosciences. APC-conjugated mouse CD1d tetramers loaded with glycolipid PBS-57 (CD1d-tet) and an unloaded tetramer comprised of only the glycolipid PBS57 were obtained from the tetramer facility of the National Institutes of Health (NIH).