Extended Data Fig. 5: iMo require CD69 to supply S1P to T cells in the dLN.

a, b, CFSE-labelled CD69-KO or littermate control CD69^low CD4 T cells were transferred i.v. into WT mice. The mice were injected s.c. with pIC or PBS, and the dLN were analysed 14h later. a, S1PR1 on the transferred cells, representative of the compilation in (b). b, Compilation of 3 experiments. LitCtl (PBS n = 6, pIC n = 6); CD69-KO (PBS n = 6, pIC n = 6). c, d, CFSE-labelled labelled CD69-KO or littermate control CD69^low CD4 T cells were transferred i.v. into CD69-KO mice. The mice were injected s.c. with pIC or PBS, and the dLN were analysed 14h later. c, S1PR1 on the transferred cells. d, Compilation of 3 experiments. LitCtl (PBS n = 6, pIC n = 6); CD69-KO (PBS n = 6, pIC n = 6). e, f, CD69-KO or littermate control mice were injected s.c. with pIC or PBS, and dLN were analysed 14h later. e, S1PR1 on CD8⁺ T cells. Controls are gated on CD69^low cells. f, Compilation of 3 experiments. LitCtl (PBS n = 4, pIC n = 7); CD69-KO (PBS n = 5, pIC n = 8). g, Dot plot of CD69 on CD11b⁺Ly6C⁺ iMo in the dLN of a WT mouse 14 h after pIC injection. CD69-KO iMo served as a negative staining control. h, Compilation of 4 experiments (n = 10). i–n, 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 to deplete CCR2-DTR⁺ cells. On d1, the chimaeras received CFSE-labelled CD69-KO lymphocytes i.v. On d2, the chimaeras were injected s.c. with PBS or pIC, and 14h later dLN were analysed. i, Percent of total CD45⁺ cells contributed by each genotype in dLN of the indicated chimaeras. Compilation of 3 experiments. LitCtl:LitCtl (PBS n = 4, pIC n = 4); CCR2^DTR:LitCtl (PBS n = 6, pIC n = 8); LitCtl:CD69-KO (PBS n = 5, pIC n = 5); CCR2^DTR:CD69-KO (PBS n = 5, pIC n = 6). j, Number of iMo in the dLN of the indicated chimaeras. Compilation of 3 experiments. LitCtl:LitCtl (PBS n = 7, pIC n = 6); CCR2^DTR:LitCtl (PBS n = 7, pIC n = 7); LitCtl:CD69-KO (PBS n = 5, pIC n = 5); CCR2^DTR:CD69-KO (PBS n = 5, pIC n = 6). k, Representative histograms of S1PR1 expression on CD69-KO CD4 T cells in the indicated chimaeras. l, 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. Compilation of 5 experiments. LitCtl:LitCtl (PBS n = 7, pIC n = 7); CCR2^DTR:LitCtl (PBS n = 10, pIC n = 12); LitCtl:CD69-KO (PBS n = 7, pIC n = 9); CCR2^DTR:CD69-KO (PBS n = 8, pIC n = 9). m, Representative histograms of S1PR1 expression on endogenous CD69-KO CD4⁺ T cells in the indicated chimaeras (top), and compilation of 5 experiments (bottom). LitCtl:CD69-KO (PBS n = 7, pIC n = 9); CCR2^DTR:CD69-KO (PBS n = 8, pIC n = 9). n, Representative histograms of S1PR1 expression on endogenous CD69-KO CD8⁺ T cells in the indicated chimaeras (top), and compilation of 2 experiments (bottom) LitCtl:CD69-KO (PBS n = 2, pIC n = 3); CCR2^DTR:CD69-KO (PBS n = 4; pIC n = 6). Data presented as mean values +/− SEM. Mann–Whitney two-tailed t-test.

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