Extended Data Fig. 1: Generation of TCR transgenic Env126 mice.

(a) Experimental design: WT mice were immunized with Env_126-138 peptide and polyI:C at three time points. At day 21 post immunization, splenocytes were isolated and restimulated ex vivo o/n with the cognate peptide before cell sorting. (b) Sorting strategy and frequency of IFN-ɣ producing CD4⁺ T cells after 4 h and o/n in vitro peptide restimulation and subsequent IFN-ɣ⁺ cell enrichment prior sorting. As a control, isolated splenocytes were cultured with an irrelevant Ag (CtrlAg, OVAII) and Concavalin A (ConA). (c) RNA encoding TCR α and β chains were electroporated into activated WT CD4⁺ T cells (2×10⁵ cells) and tested with Env_126-138 peptide via IFNγ ELISpot in the presence of peptide pulsed bone marrow-derived cells (BMDCs, 1×10⁴ cells). OVAII peptide was used as control (Ctrl-Ag). (d) Transgenic constructs maps generated after cloning of the Env_126-138-specific TCR alpha and beta chain sequences into cassette plasmids²¹. (Upper) Plasmid cassette (pTalphaCass) for TCRα gene expression comprises the Vα (variable alpha region) sequence of the specific Env_126-138 TCR (green), the the α constant region (blue) downstream enhancers components (black). (Lower) Plasmid cassette (pTbetaCass) for TCR β gene expression comprises the Vβ (variable beta region) sequence of the specific Env_126-138 TCR (orange), the β constant region (blue) downstream enhancers components (black). (e) MFIs of indicated markers on splenic WT or Env126 CD4⁺ T cells. (f) MFIs of CD44 and Ki-67 on ex vivo 4 h restimulated Env126 CD4⁺ T cells or WT CD4⁺ T cells in indicated conditions. WT unstimulated CD4⁺ T cells were included as a control (Ctrl). (g) MFIs of indicated markers on splenic polyclonal WT or Env126 CD4⁺ T cells after 48 h in vitro stimulation with indicated stimuli. WT unstimulated CD4⁺ T cells were included as a control (Ctrl). In (e-g) CD4⁺ T cells were identified as Live, CD45⁺, B220⁻CD19⁻, CD3⁺, CD8⁻CD4⁺. Env126 CD4⁺ T cells were identified as Live, CD45⁺, B220⁻CD19⁻, CD3⁺, CD8⁻CD4⁺Env-Tet⁺. (e-g) n = 3 (WT), 5 (Env126). Results are expressed as mean ± SEM and are representative of at least 3 independent experiments. P values were calculated with two-way ANOVA with Bonferroni post-test. ** P<0.01, *** P<0.001.