Extended Data Fig. 4: In vitro stimulation assay using SFB-specific TCR^Tg naïve CD4 T cells, BMDCs, and synthesized peptides.

(A) Schematic representation of an in vitro naïve TCR^7B8 T cell stimulation assay using BMDCs and synthetic peptides. Synthesized peptides were mixed with 2-4 × 10⁴ BMDCs induced from the BM of CD45.1/CD45.1 C57BL/6 mice together with LPS in 96-well round-bottom plates. After 24 h, equal numbers of magnetically sorted naïve CD4 T cells from TCR^7B8 or TCR^1A2 animals on the CD45.2/CD45.2 Rag2^−/− background were added together with recombinant IL-2. After an additional 24 h, all cells were collected and stained with anti-CD25, anti-CD69, anti-CD45.2, anti-TCRβ, and DAPI. For 72 h culture experiments, naïve CD4 T cells were labeled with CFSE just prior to co-culture with BMDCs. After 72 h, cells were stained with anti-CD25, anti-CD69, anti-CD45.2, anti-TCRβ, and Aqua. After fixation, cells were additionally stained with anti-Nur77 and anti-Ki67 and were then analyzed via FACS. (B) The approach used to gate on CD25/CD69 double-positive proliferating TCR^Tg CD4 T cells at 24 h following co-culture. (C) Other proliferation and activation makers expressed on CD4 T cells were analyzed at 72 h following co-culture. CFSE-high CD25-negative non-proliferating T cells are highlighted with a black rectangle in non-peptide treated wells and with a green rectangle in the 2.5 nM peptide well. Effectively proliferating cells were defined as CD25+ cells exhibiting CFSE dilution, and these cells were gated with a red square and further assessed for CFSE dilution, CD25, Ki67, and Nur77 expression (bottom).