Extended Data Fig. 3: CD11b⁺LY6C^hiCCR2⁺ cells contribute to increased LN S1P.

a, Phenotype of cells infiltrating the dLN in WT mice 14h after pIC injection. Representative of 5 experiments. b, Phenotype of sorted iMo. Left panel, sort gate (Ly6C^hiCD11b^hi). Right top panel, staining of sorted cells for Ly6G and CCR2. Representative of 3 experiments. Right bottom panel, Wright-Giemsa stain of sorted cells. Representative of 2 experiments. c–h, C57BL/6 mice were injected i.p. with a depleting anti-Ly6C/G antibody on d0 and d2, or left untreated. On d1, the mice received CD69-KO CD45.1⁺ lymphocytes i.v. On d2, the mice were treated s.c. with PBS or pIC, and dLN were analysed 14h later. Compilation of 3 experiments. c, Representative flow cytometry plots. d, Number of iMo in the dLN (PBS n = 4, pIC n = 6, pIC anti-Ly6C/G n = 7). e, S1PR1 on CD69-KO CD8⁺ T cells. f, Compilation (PBS n = 4, pIC n = 5, pIC anti-Ly6C/G n = 7). g, Percent CD4⁺ T cells that were CD69⁺ (PBS n = 6, pIC n = 5, pIC anti-Ly6C/G n = 8). h, Percent CD8⁺ T cells that were CD69⁺ (PBS n = 4, pIC n = 5, pIC anti-Ly6C/G n = 7). i–m, Lethally irradiated C57BL/6 mice were reconstituted with a 1:1 mix of the indicated BM, and analysed 12-16 weeks later. On d0 and d2, the chimaeras were treated with DT. On d1, the chimaeras received CD69-KO CD45.1⁺ lymphocytes i.v. On d2, the chimaeras were injected s.c. with PBS or pIC, and 14h later the dLN were analysed. i, Percent of total CD45⁺ cells contributed by each genotype in dLN of the indicated chimaeras. Compilation of 5 experiments. LitCtl:LitCtl (PBS n = 6; pIC n = 6); CCR2^DTR:LitCtl (PBS n = 6; pIC n = 6); LitCtl:SPHK-KO (PBS n = 4; pIC n = 4); CCR2^DTR:SPHK-KO (PBS n = 6 pIC n = 11). j, Number of iMo in the dLN of the indicated chimaeras. Compilation of 3 experiments. LitCtl:LitCtl (PBS n = 6, pIC n = 6); CCR2^DTR:LitCtl (PBS n = 6, pIC n = 4); LitCtl:SPHK-KO (PBS n = 4, pIC n = 4); CCR2^DTR:SPHK-KO (PBS n = 6, pIC n = 5). k, Representative histograms of S1PR1 expression on CD69-KO CD4⁺ T cells in the indicated chimaeras. l, Representative histograms of S1PR1 expression on CD69-KO CD8⁺ T cells in the indicated chimaeras (top) and compilation (bottom) of S1PR1 expression on CD69-KO CD8⁺ T cells in the indicated chimaeras. Compilation of 3 experiments. LitCtl:LitCtl (PBS n = 5, pIC n = 5); CCR2^DTR:LitCtl (PBS n = 4, pIC n = 3); LitCtl:SPHK-KO (PBS n = 4, pIC n = 3); CCR2^DTR:SPHK-KO (PBS n = 5, pIC n = 6). m, Representative histograms (top) and compilation (bottom) of S1PR1 expression on endogenous CD4 T cells in the indicated chimaeras. For the compilation, the S1PR1 MFI of the CD69^low CD4⁺ T cells in each mouse in the PBS-treated group was divided by the mean S1PR1 MFI of the CD69^low CD4⁺ T cells in the PBS-treated group; the S1PR1 MFI of the CD69^high CD4⁺ T cells in each mouse of the pIC-treated group was similarly divided by the mean MFI of the CD69^low CD4⁺ T cells in the PBS-treated group. LitCtl:LitCtl (PBS n = 7, pIC n = 6); CCR2^DTR:LitCtl (PBS n = 6, pIC n = 6); LitCtl:SPHK-KO (PBS n = 4, pIC n = 4); CCR2^DTR:SPHK-KO (PBS n = 8, pIC n = 12). Compilation of 5 experiments. Bars represent mean +/− s.e.m. Mann–Whitney two-tailed t-test.

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