Extended Data Fig. 5: Group 3 clones express common hallmarks of T follicular helper cells.

As shown in Fig. 3, comparative RNA-Seq analysis of Group 3 SAS and Group 1 ANT TCRrg cells. The SAS and ANT TCRs were cloned and expressed as TCRrg mice (see Methods). TCRrg T cells were purified from the pooled secondary lymphoid organs (SLOs) of primary TCRrg mice and subjected to bulk RNA sequencing (see Methods). Only biological samples containing ≥ 1 × 10⁵ TCRrg T cells were included for analysis (n = 4 mice per TCR). a, Gating strategy schematic for the fluorescence-activated cells sorting (FACS) of TCRrg T cells for RNA sequencing. TCRrg T cells were selected by gating on Dump^neg (B220^negCD11b^negCD11c^negF4/80^neg), followed by CD4⁺CD8β^neg, then by Thy1.1⁺CD45.1^neg. b, RNA-seq volcano plot depicting differential gene expression for Group 3 SAS and Group 1 ANT TCRrg cells. The –log₁₀ q-value versus log₂ fold-change is depicted for genes with average log₂ CPM ≥ −1. Blue and red dots denote genes over-represented in Group 1 ANT and Group 3 SAS TCRrg T cells, respectively, with FDR ≤ 0.05. Labels denote top genes that are over- or underrepresented, q ≤ 1 × 10⁻⁵⁰ (82 genes). Data are pooled from two independent experiments. q values were generated using edgeR (see Methods). c, Principal component analysis (PCA) of mRNA expression in Group 1 ANT and Group 3 SAS TCRrg T cells, presented as log-scaled normalized expression (log₂ CPM). Each dot corresponds to an independent biological sample. Green and magenta dots denote samples isolated from Group 1 ANT and Group 3 SAS TCRrg mice, respectively. n = 4 per TCR. Data are pooled from two independent experiments.