Extended Data Fig. 4: uLIPSTIC labeling in inducible Cre lines in fully endogenous models.

(A–D) uLIPSTIC labeling of Treg cell interactions in the steady-state pLN. (A) Cellular interactome of Tregs at steady-state. Rosa26^uLIPSTIC/WT.Foxp3^CreERT2/Y experimental mice and Rosa26^WT/WT.Foxp3^CreERT2/Y controls were given tamoxifen and administered LIPSTIC substrate in the footpad 2 days later. Left, flow cytometry plots show uLIPSTIC labeling in selected immune populations in control (top) and experimental (bottom) mice. The presence of residual labeling in B cells is an artifact common to uLIPSTIC and to other flow-cytometry based methods aimed at identifying rare B cell populations, likely due to B cell receptor-dependent binding of detection components by polyclonal B cells. Right, quantification of the proportion of all labeled cells belonging to each major immune population in control (SrtA^–) or experimental (SrtA⁺) mice. Data for three mice per condition from one experiment, bar plots show mean ± SEM. (B-D) Treg cells interact with mDCs to a greater extent than conventional CD4⁺ T cells. (B) To test if enhanced interaction with mDCs is a specific feature of Treg cells or a general feature of all CD4⁺ T cells, we titrated the dose of tamoxifen in Rosa26^uLIPSTIC/+.CD4-CreERT2 mice to achieve a similar percentage of SrtA-expression among total CD4⁺ T cells as in Rosa26^uLIPSTIC/+.Foxp3^CreERT2/Y mice. (C) At a dose of 0.3 mg of tamoxifen, Rosa26^uLIPSTIC/+.CD4-CreERT2 mice showed SrtA expression in a small number of Treg cells (left), with most SrtA⁺ cells observed in CD4⁺ conventional T cells (center) and overall numbers of SrtA⁺ cells among total CD4⁺ T cells that were comparable with those of Rosa26^uLIPSTIC/+.Foxp3^CreERT2/Y mice treated with 10 mg tamoxifen (right). (D) When numbers of Treg and CD4⁺ conventional donor cells are equalized, acceptor mDCs show stronger interaction with Treg cell partners. For (C) and (D), data from two independent experiments with each symbol representing one mouse, P-values were calculated using two-tailed Student’s tests. (E–G) Kinetics of tamoxifen-driven recombination of the Rosa26^uLIPSTIC allele according to cell type. (E) SrtA expression in the highly proliferative mesenteric lymph node GC B cells of Rosa26^uLIPSTIC/WT.Aicda^CreER/WT mice was assessed at different timepoints after tamoxifen administration. The fraction of recombined cells plateaus at 24 h post-tamoxifen administration (hpt), while SrtA protein expression is still increasing by 96 hpt. (F) Labeling of GC B cell interacting partners can be detected as early as 12 hpt, increasing thereafter according to SrtA expression levels. (G) In contrast, SrtA expression in quiescent naïve (PD-1^–CXCR5^–CD69^–) CD4⁺ T cells in Rosa26^uLIPSTIC/WT.Cd4-CreERT2 mice increased at a slower rate than, reaching >80% positive cells only at 96 hpt. For (E), (F) and (G), each plot used three mice per condition from one experiment, P-values were calculated using two-tailed Student’s tests.

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