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Monocyte-derived S1P in the lymph node regulates immune responses

Abstract

The lipid chemoattractant sphingosine 1-phosphate (S1P) guides cells out of tissues, where the concentration of S1P is relatively low, into circulatory fluids, where the concentration of S1P is high1. For example, S1P directs the exit of T cells from lymph nodes, where T cells are initially activated, into lymph, from which T cells reach the blood and ultimately inflamed tissues1. T cells follow S1P gradients primarily using S1P receptor 1 (ref. 1). Recent studies have described how S1P gradients are established at steady state, but little is known about the distribution of S1P in disease or about how changing levels of S1P may affect immune responses. Here we show that the concentration of S1P increases in lymph nodes during an immune response. We found that haematopoietic cells, including inflammatory monocytes, were an important source of this S1P, which was an unexpected finding as endothelial cells provide S1P to lymph1. Inflammatory monocytes required the early activation marker CD69 to supply this S1P, in part because the expression of CD69 was associated with reduced levels of S1pr5 (which encodes S1P receptor 5). CD69 acted as a ‘stand-your-ground’ signal, keeping immune cells at a site of inflammation by regulating both the receptors and the gradients of S1P. Finally, increased levels of S1P prolonged the residence time of T cells in the lymph nodes and exacerbated the severity of experimental autoimmune encephalomyelitis in mice. This finding suggests that residence time in the lymph nodes might regulate the differentiation of T cells, and points to new uses of drugs that target S1P signalling.

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Fig. 1: S1P levels increase in the dLN after pIC injection.
Fig. 2: iMo supply lymph node S1P.
Fig. 3: CD69 promotes iMo S1P secretion in part by repressing S1pr5 expression.
Fig. 4: iMo supply S1P in the dLN in EAE.

Data availability

RNA sequencing data are available from the NIH/NCBI as Gene Expression Omnibus data set GSE139006. All other data will be available from the authors on reasonable request. Source data are provided with this paper.

Code availability

The ImageJ macro used for the localization of iMo is provided in the Supplementary Information.

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Acknowledgements

We thank members of the Schwab laboratory, J. Cyster, D. Littman and K. Narasimhan for discussions; E. Pamer for the CCR2-DTR mice; and the NIH Tetramer Core Facility for the MOG/Ab and hCLIP/Ab tetramers. This work was supported by NIH grants R01AI085166 and R01AI123308, and the Blood Cancer Discoveries Grant Program sponsored by the Leukemia & Lymphoma Society, the Mark Foundation for Cancer Research and The Paul G. Allen Frontiers Group (to S.R.S.). NYU’s core facilities were supported in part by NIH grants P30CA016087 to the Laura and Isaac Perlmutter Cancer Center and NCRR S10RR023704-01A1. This work is dedicated to Nilabh Shastri.

Author information

Authors and Affiliations

Authors

Contributions

A.B. designed, performed and conducted all of the experiments, analysed and interpreted the data, and wrote the manuscript. S.B. and V.S.C. performed the experiments. A.K.-J. analysed the RNA sequencing data. M.C. analysed the imaging data. S.R.S. designed the experiments, interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Susan R. Schwab.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Stephen Jameson, Ellen Robey and Hai Qi for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 S1P increases in the dLN after pIC injection.

a, CD69-KO lymphocytes (from LN) were incubated ex vivo with the indicated concentrations of S1P for 16h, and surface S1PR1 on CD4+ T cells was measured by flow cytometry. Average of triplicates +/− SEM in 1 experiment. bi, Experiment design and animals as in Fig. 1a–c. Compilation of 4 experiments for all panels except (h), which compiles 3 experiments. b, Absolute MFI of S1PR1 for the cells shown in Fig. 1c. PBS (n = 8); pIC (n = 9). c, Percent CD69+ among endogenous (WT) CD4+ T cells in dLN. PBS (n = 8); pIC (n = 11). d, Relative number of the indicated CD4+ T cells in dLN. The number of endogenous CD4+ cells in the dLN of each mouse was divided by the mean number of endogenous CD4+ cells in the PBS-treated group; the number of transferred CD69-KO CD4+ cells in each mouse was similarly divided by the mean number of CD69-KO CD4+ transferred cells in the PBS-treated group. PBS (n = 9); pIC (n = 11). e, (left) Representative histograms of S1PR1 on transferred CD69-KO, endogenous CD69low, and endogenous CD69high CD4 T cells in PBS- or pIC-treated mice. (right) Representative dot plots of CD69 vs S1PR1 on endogenous CD4 T cells in PBS- or pIC-treated mice. Representative of the data compiled in (f). f, The S1PR1 MFI of endogenous CD69low or endogenous CD69high CD4+ cells in each mouse divided by the mean S1PR1 MFI of endogenous CD69low CD4+ cells in the PBS-treated group. PBS (n = 8); pIC (n = 9). g, Representative histograms of surface S1PR1 on endogenous (WT) CD69low or transferred CD69-KO CD8+ T cells in the dLN. FTY720-treated mouse served as a negative control. h, The S1PR1 MFI of the endogenous CD69low CD8+ cells in each mouse was divided by the mean S1PR1 MFI of the endogenous CD69low CD8+ cells in the PBS-treated group; the S1PR1 mean fluorescence intensity (MFI) of the transferred CD69-KO CD8+ cells in each mouse was similarly divided by the mean MFI of the transferred CD69-KO CD8+ cells in the PBS-treated group. PBS (n = 7); pIC (n = 6). i, Relative number of the indicated CD8+ T cells in the dLN. PBS (n = 9), pIC (n = 9). j, Experiment design as in Fig. 1a. Percent CD69+ among total CD4+ (n = 11), Treg (CD4+ Foxp3+) (n = 9), CD8+ (n = 12), NK (NK1.1+CD3-) (n = 5), and NKT (NK1.1+CD3+) (n = 5) cells in the dLN of pIC-treated mice. Compilation of 4 experiments. k, CD69-KO Sensor+ T cells were transferred i.v. into WT recipients. 24h later, mice were treated s.c. with PBS or pIC. 14h later, dLN were analysed by confocal microscopy. Representative section, showing T zone, B follicles, and subcapsular sinus (SCS). Arrows indicate transferred cells. Scale bar, 100 μm. Representative of 5 experiments, PBS n = 7, pIC n = 8. Data are presented as mean values +/− SEM. Mann–Whitney two-tailed t-test.

Source data

Extended Data Fig. 2 S1P increases in the dLN of BM chimaeras after pIC injection.

(a-j) C57BL/6 mice were lethally irradiated, reconstituted with a 1:1 mix of CD45.2+ WT and CD45.1+ CD69-KO BM, and allowed to recover for 12-16 weeks. The chimaeras were injected s.c. with PBS or pIC, and dLN analysed 14h later. Compilation of 3 (for CD8 analysis) - 4 (for CD4 analysis) experiments. a, Experiment design. b, S1PR1 on WT CD69low (left) and CD69-KO (right) CD4+ T cells. c, Compilation. The S1PR1 MFI of the WT CD69low CD4+ T cells in each mouse was divided by the mean S1PR1 MFI of the WT CD69low CD4+ T cells in the PBS-treated group; the S1PR1 MFI of the CD69-KO CD4+ T cells in each mouse was similarly divided by the mean MFI of the CD69-KO CD4+ T cells in the PBS-treated group. Each point represents one mouse. PBS (n = 11), pIC (n = 13). d, Percent CD69+ among WT CD4+ T cells. PBS (n = 11), pIC (n = 13). e, Relative number of the indicated CD4+ T cells in the dLN. The number of CD4+ WT cells in the dLN of each mouse was divided by the mean number of CD4+ WT cells in the PBS-treated group; the number of CD69-KO CD4+ cells in each mouse was similarly divided by the mean number of CD69-KO CD4+ cells in the PBS-treated group. PBS (n = 9), pIC (n = 11). f, S1PR1 expression on CD69low WT or CD69-KO CD8+ T cells. g, Compilation, as in (c). PBS (n = 9), pIC (n = 10). h, Percent CD69+ among WT CD8+ T cells. PBS (n = 9), pIC (n = 10). i, Relative number of the indicated CD8+ T cells, as in (e). PBS (n = 9), pIC (n = 10). j, RT-qPCR for S1pr1, normalized to Hprt. Compilation of 3 experiments. PBS (n = 9), pIC (n = 9). k, RT-qPCR for Sphk1 in bone marrow of pIC-treated Sphk1f/fSphk2−/− (n = 5 animals, bone marrow cells split into 2 samples before RNA purification), pIC-treated Sphk1f/fSphk2−/−Mx1-Cre+ (n = 7 animals, bone marrow cells split into 2 samples before RNA purification), and Sphk1−/− (n = 2 animals). Compilation of 2 experiments. l, RT-qPCR for Sphk1 in blood of pIC-treated Sphk1f/fSphk2−/− (n = 4) and pIC-treated Sphk1f/fSphk2−/−Mx1-Cre+(n = 4) animals. One experiment. mr, UBC-GFP+ mice were lethally irradiated, reconstituted with pIC-treated Sphk1f/f Sphk2−/− Mx1-Cre+ CD45.2+ (SPHK-KO) or littermate control (LitCtl) BM, and left to recover for 12-14 weeks. Chimaeras received CD45.1+ CD69-KO T cells i.v. 24h later, chimaeras were injected s.c. with pIC or PBS. 14h later, dLN were analysed. m, S1PR1 on CD69-KO CD8+ T cells in LitCtl chimaeras (left) or SPHK-KO chimaeras (right). FT720-treated mouse served as a negative control. n, Compilation of 3 experiments. The S1PR1 MFI on the CD69-KO CD8+ T cells in each mouse was divided by the mean S1PR1 MFI on the CD69-KO CD8+ T cells in the PBS-treated group. LitCtl (PBS n = 7, pIC n = 9); SPHK-KO (PBS n = 7, pIC n = 7). o, Percent CD69+ among CD4+ T cells. Compilation of 3 experiments. LitCtl (PBS n = 7, pIC n = 9); SPHK-KO (PBS n = 7, pIC n = 8). p, Percent CD69+ among CD8+ T cells. Compilation of 3 experiments. LitCtl (PBS n = 7, pIC n = 9); SPHK-KO (PBS n = 7, pIC n = 7). q, r, CD69-KO CD45.1+ lymphocytes were transferred into LitCtl or SPHK-KO BM chimaeras. 24h later, the chimaeras were injected s.c. with pIC. 14h later, half of the mice in each group were euthanized and the cells in the dLN were counted. The remaining mice were injected i.v. with anti-αL and anti-α4 neutralizing antibodies. These antibodies blocked any further lymphocyte entry into the LN. 4h later, these mice were euthanized and the cells in the dLN were counted. The decline in cell numbers in the LN over 4 h, with no further cell entry, indicated the exit rate. Compilation of 3 experiments. SPHK-KO (t = 0 n = 8; t = 4h n = 8), LitCtl (t = 0 n = 6, t = 4h n = 8). q, Percent exit of CD69-KO CD8+ T cells. r, Percent exit of endogenous CD69low CD8+ T cells. Each point represents one mouse at t = 4h relative to the average at t = 0. Data presented as mean values +/− SEM. Mann–Whitney two-tailed t-test.

Source data

Extended Data Fig. 3 CD11b+LY6ChiCCR2+ 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 (Ly6ChiCD11bhi). 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. ch, 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). im, 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); CCR2DTR:LitCtl (PBS n = 6; pIC n = 6); LitCtl:SPHK-KO (PBS n = 4; pIC n = 4); CCR2DTR: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); CCR2DTR:LitCtl (PBS n = 6, pIC n = 4); LitCtl:SPHK-KO (PBS n = 4, pIC n = 4); CCR2DTR: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); CCR2DTR:LitCtl (PBS n = 4, pIC n = 3); LitCtl:SPHK-KO (PBS n = 4, pIC n = 3); CCR2DTR: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 CD69low CD4+ T cells in each mouse in the PBS-treated group was divided by the mean S1PR1 MFI of the CD69low CD4+ T cells in the PBS-treated group; the S1PR1 MFI of the CD69high CD4+ T cells in each mouse of the pIC-treated group was similarly divided by the mean MFI of the CD69low CD4+ T cells in the PBS-treated group. LitCtl:LitCtl (PBS n = 7, pIC n = 6); CCR2DTR:LitCtl (PBS n = 6, pIC n = 6); LitCtl:SPHK-KO (PBS n = 4, pIC n = 4); CCR2DTR:SPHK-KO (PBS n = 8, pIC n = 12). Compilation of 5 experiments. Bars represent mean +/− s.e.m. Mann–Whitney two-tailed t-test.

Source data

Extended Data Fig. 4 iMo are sufficient to supply LN S1P.

af, C57BL/6 mice received CD69-KO CD45.1+ lymphocytes i.v. 1d later, the mice received an intra-LN injection of Ly6ChiCD11b+ iMo sorted from LN of mixed BM chimaeras (WT+CD69-KO or WT+SPHK-KO). Alternately, mice were injected intra-LN with PBS (sham), injected s.c. with pIC, or left untreated. 14h later, the injected LN were analysed. Compilation of 3 experiments, untreated (n = 6); sham (n = 6); pIC (n = 8); iMo WT (n = 6); iMo SPHK-KO (n = 9); iMo CD69-KO (n = 7). a, Representative dot plots (gated on live single cells) showing CD11b+Ly6Chi iMo in the injected LN. b, Number of iMo in the injected LN. c, Percent CD69+ among CD4+ T cells in the injected LN. d, S1PR1 on CD69-KO CD4+ T cells in the indicated mice. e, S1PR1 on CD69low endogenous CD4+ T cells in the indicated mice. f, Compilation of S1PR1 on CD69low endogenous CD4+ T cells. g, CD69-KO S1P-sensor+ T cells were cultured for 8h across a transwell from media or the indicated Ly6ChiCD11b+ iMo sorted from LN of pIC-treated mixed BM chimaeras (either WT:CD69-KO or WT:SPHK-KO). Sensor+ cells were analysed by confocal microscopy. Images representative of cells quantified in Fig. 2g, scale bar 5μm. h, i, CD69-KO Sensor+ T cells were cultured for 8h across a transwell from media alone or Ly6ChiCD11b+ iMo sorted from bone marrow of PBS-treated mice, spleen of PBS-treated mice, or LN of pIC-treated mice. Sensor cells were analysed by confocal microscopy. h, Representative images of the cells quantified in (i). Scale bar, 5μm. i, Quantification of S1P reporting, as in Fig. 1f. Compilation of 3 experiments. Each point is the ratio of surface GFP:RFP on one cell (media n = 26, iMo BM n = 60, iMo spleen n = 99, iMo LN n = 75). j, k, CD69-KO T cells were cultured across a transwell from media or sorted iMo from LN of pIC-treated WT, SPHK-KO, or CD69-KO mice. After 12h, surface S1PR1 on the CD69-KO CD4+ T cells was analysed by flow cytometry. j, Experiment diagram and representative histograms. k, Compilation of 5 experiments. Each symbol represents one well relative to the average of the media control wells. Media n = 7, iMo WT n = 11, iMo SPHK-KO n = 4, iMo CD69-KO n = 7. Data presented as mean values +/− SEM. Mann–Whitney two-tailed t-test.

Source data

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 CD69low 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 CD69low 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 CD69low 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). in, 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); CCR2DTR:LitCtl (PBS n = 6, pIC n = 8); LitCtl:CD69-KO (PBS n = 5, pIC n = 5); CCR2DTR: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); CCR2DTR:LitCtl (PBS n = 7, pIC n = 7); LitCtl:CD69-KO (PBS n = 5, pIC n = 5); CCR2DTR: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 CD69low CD4+ T cells in each mouse in the PBS-treated group was divided by the mean S1PR1 MFI of the CD69low CD4+ T cells in the PBS-treated group; the S1PR1 MFI of the CD69high CD4+ T cells in each mouse of the pIC-treated group was similarly divided by the mean MFI of the CD69low CD4+ T cells in the PBS-treated group. Compilation of 5 experiments. LitCtl:LitCtl (PBS n = 7, pIC n = 7); CCR2DTR:LitCtl (PBS n = 10, pIC n = 12); LitCtl:CD69-KO (PBS n = 7, pIC n = 9); CCR2DTR: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); CCR2DTR: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); CCR2DTR:CD69-KO (PBS n = 4; pIC n = 6). Data presented as mean values +/− SEM. Mann–Whitney two-tailed t-test.

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Extended Data Fig. 6 Characterization of CD69-KO iMo.

a, Volcano plot showing transcripts from congenically marked WT and CD69-KO iMo, sorted from dLN of mixed BM chimaeras (1:1 WT CD45.2:CD69-KO CD45.1 BM) 14h after pIC injection. b, c, CD69-KO mice were injected s.c. with PBS or pIC, and dLN were analysed 14h later. b, Representative t-SNE plots (12-colour flow cytometry). NK cells (CD3-NKp46+), B cells (CD19+), CD3+CD4+ T cells, CD3+CD8+ T cells, plasmacytoid dendritic cells (pDC) (CD11c+B220+SiglecH+), classical dendritic cells (DC) (CD11c+SiglecH-B220-), neutrophils (CD11b+Ly6G+ Ly6C-CCR2-), iMo (CD11b+ Ly6Chi CCR2+Ly6G-), and other CD11b+ cells (CD11b+Ly6G-Ly6Clow) are shown. Representative of 2 experiments. c, Number of the indicated cells. Compilation of 5 experiments (some experiments did not include all 12 antibodies). iMo (PBS n = 11, pIC n = 11); NK cells (PBS n = 4, pIC n = 4); neutrophils (PBS n = 5, pIC n = 5); other CD11b+ cells (PBS n = 9, pIC n = 8); pDC (PBS n = 7, pIC n = 6); DC (PBS n = 7 pIC n = 6). d, LN section from a pIC-treated CCR2-RFP mouse, stained with antibodies to Ly6C (green) and CD11b (blue). Left image shows Ly6C and CD11b, right image shows CCR2-RFP (red) and CD11b, arrows indicate double-positive cells (Ly6C+CD11b+ or CCR2-RFP CD11b+). Scale bar 50 μm. Image representative of 2 experiments. e, Littermate control (or WT), CD69-KO and CD69-KO S1PR5-KO mice were injected with PBS or PIC s.c. 14h later, dLN were analysed by confocal microscopy. Inflammatory monocytes (arrows) were identified as CD11b+Ly6C+. T zone, B follicles, and medulla (M) were distinguished by CD4 and Lyve1 staining. Scale bar, 50μm. LN section representative of data compiled in Fig. 3e. f, CD69-KO S1P-sensor+ T cells were cultured for 12h across a transwell from media alone or the indicated Ly6ChiCD11b+ inflammatory monocytes sorted from LN of pIC-treated mixed BM chimaeras (WT with CD69-KO, or WT with CD69-KO S1PR5-KO). Reporter cells were analysed by confocal microscopy. Images representative of cells quantified in Fig. 3f. Scale bar, 10 μm. g, h, The indicated sorted iMo were cultured for 5h with 50 nM S1P. Then CD69-KO S1P-sensor+ T cells were added across a transwell from the iMo for 2h. As controls, the T cells were cultured for 2h across a transwell from media alone or media + 50 nM S1P. g, Experiment design and representative images of the cells quantified in (h). Scale bar, 5 μm. h, Quantification of S1P reporting, as in Fig. 2g. Compilation of 4 experiments. Media (n = 65), S1P (n = 89), iMo CD69-KO+S1P (n = 21), iMo CD69-KO S1PR5-KO+S1P (n = 114). i, j, WEHI-231 cells were transduced with S1pr5 or vector control. The S1pr5+ or control lines were cultured for 5 h with 50 nM S1P. Then CD69-KO S1P-sensor+ T cells were added across a transwell from the WEHI-231 cultures for 2 h. As controls, the T cells were cultured for 2 h across a transwell from media with 50 nM S1P or media alone. i, Experiment design and images representative of cells quantified in (j). Scale bar, 5 μm. j, Quantification of S1P reporting, as in Fig. 2g. Compilation of 3 experiments. Empty vector (media n = 39, S1P n = 61); S1PR5 (media n = 36, S1P n = 49). k, RT-qPCR analysis of sorted iMo from LN of pIC-treated WT, CD69-KO, and SPHK-KO mice. Sorted CD4+ T cells from WT mice served as a negative control for Spns2. Compilation of 3 experiments for Sphk1 (iMo WT n = 4, iMo CD69-KO n = 7, iMo SPHK-KO n = 3 mice). Compilation of 4 experiments for Sphk2 (iMo WT n = 6, iMo CD69-KO n = 8, iMo Sphk-KO n = 3 mice). Compilation of 5 experiments for Spns2 (iMo WT n = 7, iMo CD69-KO n = 11, WT T cells n = 3 mice). For some mice, technical duplicates are included in the compilation (sorted cells were divided before RNA purification). Data presented as mean values +/− SEM. Mann–Whitney two-tailed t-test.

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Extended Data Fig. 7 S1P increases in the dLN in EAE.

a, (left) Percent CD69+ among total CD4 T cells in the cervical LN of healthy WT mice or WT mice with EAE (d9). Compilation of 4 experiments (no EAE n = 6, EAE n = 11). (right) MOG/Ab-specific 2D2 TCR-transgenic T cells were transferred i.v. to WT recipients. After EAE induction, CD69 expression on the transferred 2D2 T cells, endogenous MOG/Ab tetramer+ T cells, and endogenous MOG/Ab tetramer- T cells in the cervical LN was followed over time. Compilation of 2 experiments (3-5 mice per time point, 33 mice total) for endogenous cells, and 1 experiment (2-3 mice per time point, 18 mice total) for 2D2 T cells. b, Percent CD69+ among total CD4+ (n = 11), Tet+ (CD4+ MOG/Ab-tetramer+) (n = 10), Th17 (CD4+Foxp3-Rorγt+) (n = 10), Tfh (CD4+CXCR5+PD-1high) (n = 9), Th1 (CD4+Foxp3-Tbet+) (n = 9), Treg (CD4+Foxp3+) (n = 10), CD4+ Ki67+ (n = 9), CD4+CD44low (n = 9), CD4+CD44high (n = 9), CD8+ (n = 12), γδ T (n = 9), NK (NK1.1+CD3-) (n = 7), and NKT (NK1.1+CD3+) (n = 9) cells in the cervical LN of WT mice with EAE (d9). Compilation of 4 experiments. c, EAE was induced in WT mice. Left axis: Kinetics of surface S1PR1 expression on CD4+CD69low T cells in the cervical LN, relative to CD4+CD69low T cells in the cervical LN of healthy controls. 1-4 mice per time point, no EAE n = 11 mice total, EAE n = 14 mice total. Right axis: iMo recruitment to the cervical LN, as a percent of total CD45+ cells. 1-4 mice per time point, no EAE n = 8 mice total, EAE n = 14 mice total. Compilation of 2 experiments. d, e, Representative histograms (d) and compilation (e) of S1PR1 on transferred CD69-KO, endogenous CD69low, and endogenous CD69high CD4 T cells in the cervical LN of WT mice with EAE (d9-d12) or healthy controls. For the compilation, the S1PR1 MFI of endogenous CD69low or endogenous CD69high CD4+ cells in each mouse was divided by the mean S1PR1 MFI of endogenous CD69low CD4+ cells in healthy controls. Compilation of 6 experiments (no EAE n = 8, EAE CD69low n = 15, EAE CD69high n = 15). f, g, CD69-KO CD45.1 lymphocytes were transferred i.v. into C57BL/6 mice. The following day, EAE was induced (controls were treated with PBS). 12d-14d after EAE induction, the dLN (cervical) was analysed. f, Representative S1PR1 on endogenous (WT) CD69low CD8+ T cells (left) and CD69-KO CD8+ T cells (right). g, Compilation of 2 experiments. (WT) CD69low CD8+ T cells (no EAE n = 3, EAE n = 4); CD69-KO CD8+ T cells (no EAE n = 3, EAE n = 4). hm, Lethally irradiated C57BL/6 mice were reconstituted with a 1:1 mix of WT and CD45.1+ CD69-KO BM, and allowed to reconstitute for 12-14 weeks. EAE was induced, or chimaeras were treated with PBS. 10d-12d later, the cervical LN was analysed. h, Experiment diagram. i, Number CD11b+Ly6Chi cells in the dLN. Compilation of 5 experiments. No EAE n = 6, EAE n = 6. j, S1PR1 on WT CD69low (left) and CD69-KO (right) CD4+ T cells. k, Compilation of 3 experiments. No EAE n = 7, EAE n = 7. l, S1PR1 on WT CD69low (left) and CD69-KO (right) CD8+ T cells. m, Compilation of 2 experiments. No EAE n = 5, EAE n = 5. Data presented as mean values +/− SEM. Mann–Whitney two-tailed t-test.

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Extended Data Fig. 8 IMo supply S1P in the dLN during EAE.

a, Left: Experiment design to test the effect of haematopoietic S1P on T cell exit from the cervical LN in EAE. Right: Percent cells exiting the LN in 6h. Each point represents one mouse at t = 6h relative to the average at t = 0. Compilation of 3 experiments. LitCtl (t = 0h n = 5; t = 6h n = 7), SPHK-KO (t = 0 n = 6; t = 6h n = 7). Note that the control and SPHK-KO chimaeras are at different stages of disease, so the change in T cell residence time may in part reflect differences in LN architecture. b, c, Total CD45+ cells and CD11b+Ly6Chi iMo were sorted from the cervical LN of littermate control or SPHK-KO BM chimaeras with EAE (d9). CD69-KO S1P-sensor+ T cells were cultured for 8h across a transwell from media alone or the indicated cells, and analysed by confocal microscopy. b, Representative images of the cells quantified in (c). Scale bar, 5μm. c, Quantification of S1P reporting, as in Fig. 1f. Each symbol represents the ratio of surface GFP:RFP on one cell. Compilation of 2 experiments. Media (n = 63), CD45+ (n = 101), iMo LitCtl (n = 147), iMo SPHK-KO (n = 83). Data presented as mean values +/− SEM. Mann–Whitney two-tailed t-test.

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Extended Data Fig. 9 IMo supply S1P in the dLN during EAE.

a, b, The indicated chimaeras (as in Fig. 4c) received CD69-KO CD45.1+ lymphocytes i.v. on d0. On d0 and every 3d after, they were injected i.p. with DT. On d1 EAE was induced (or mice were treated with PBS). 10d-12d later, S1PR1 levels on CD69-KO CD8+ (a) and endogenous (WT) CD4+ (b) T cells in dLN were analysed. Top, representative histograms. Bottom, compilation of 4 experiments. LitCtl:LitCtl (PBS n = 9, EAE n = 8); CCR2DTR:LitCtl (PBS n = 9, EAE n = 8); LitCtl:SPHK-KO (PBS n = 7, EAE n = 9); CCR2DTR:SPHK-KO (PBS n = 9, EAE n = 9). For the compilation of endogenous CD4 T cells, the S1PR1 MFI of the CD69low CD4+ T cells in each mouse in the PBS-treated group was divided by the mean S1PR1 MFI of the CD69low CD4+ T cells in the PBS-treated group; the S1PR1 MFI of the CD69high CD4+ T cells in each mouse of the pIC-treated group was similarly divided by the mean MFI of the CD69low CD4+ T cells in the PBS-treated group. cf, The indicated chimaeras (as in Fig. 4k) received CFSE-labelled CD69-KO lymphocytes i.v. on d0. On d0 and every 3d after, they were injected i.p. with DT. On d1 EAE was induced (or mice were treated with PBS). 10d-12d later, S1PR1 levels in the cervical LN were analysed. c, Representative histograms (top) and compilation of 4 experiments (bottom) for endogenous CD69-KO CD4+ T cells LitCtl:CD69-KO (PBS n = 7, EAE n = 6); CCR2DTR:CD69-KO (PBS n = 7, EAE n = 7). d, Representative histograms (top) and compilation of 3 experiments (bottom) for transferred CD69-KO CD8+ T cells. LitCtl:LitCtl (PBS n = 4, EAE n = 5); CCR2DTR:LitCtl (PBS n = 5, EAE n = 4); LitCtl:CD69-KO (PBS n = 5, EAE n = 5); CCR2DTR:CD69-KO (PBS n = 6, EAE n = 6). e, Representative histograms (top) and compilation of 3 experiments (bottom) for endogenous CD69-KO CD8+ T cells. LitCtl:CD69-KO (PBS n = 5, EAE n = 5); CCR2DTR:CD69-KO (PBS n = 6, EAE n = 6). f, Representative histograms (top) and compilation of 4 experiments (bottom) for endogenous WT CD4+ T cells. For the compilation, the S1PR1 MFI of the CD69low CD4+ T cells in each mouse in the PBS-treated group was divided by the mean S1PR1 MFI of the CD69low CD4+ T cells in the PBS-treated group; the S1PR1 MFI of the CD69high CD4+ T cells in each mouse of the pIC-treated group was similarly divided by the mean MFI of the CD69low CD4+ T cells in the PBS-treated group. LitCtl:LitCtl (PBS n = 5, EAE n = 7); CCR2DTR:LitCtl (PBS n = 6, EAE n = 6); LitCtl:CD69-KO (PBS n = 6, EAE n = 7); CCR2DTR:CD69-KO (PBS n = 8, EAE n = 8). g, Representative staining of CD4+PD1hiCXCR5hi Tfh cells in the dLN. h, Representative staining of CD4+Rorγt+Foxp3- Th17 cells in the dLN (top) and in the CNS (bottom). For each graph bars represent mean +/− s.e.m. Mann–Whitney two-tailed t-test.

Source data

Extended Data Fig. 10 S1P regulates Tfh and Th17 numbers in EAE.

We sought to address how haematopoietic S1P regulates Tfh and Th17 accumulation in EAE. Possibilities include that this S1P may promote priming by delaying naïve T cell exit from LN; promote proliferation or differentiation by delaying activated T cell exit from LN; promote survival, proliferation, or differentiation due to residence-time independent effects of S1P signalling in T cells in LN18,36; or act indirectly through iMo S1P secretion in the CNS. ag, The results of experiments similar to those in Fig. 4 but using BM chimaeras in which WT mice were reconstituted with SPHK-KO or littermate control BM, to avoid repeated DT injections. hk, The results of experiments similar to those in Fig. 4 but using mice treated with an S1P lyase inhibitor, to test the effect of increased LN S1P. lq, The results of experiments designed to distinguish effects of S1P signalling on T cell residence time from other effects. We generated T cells that overexpressed S1PR1. We expected these cells to exit tissues more quickly than control T cells9, similar to T cells that could not ‘see’ iMo-derived S1P, while experiencing enhanced S1PR1 signalling compared to control T cells due to their higher S1PR1 levels, unlike T cells that could not ‘see’ iMo-derived S1P. We observed that overexpression of S1PR1 reduced the frequency of Th17 and Tfh cells, consistent with a cell-intrinsic effect of trafficking on T cell numbers and with previous findings17. We also found that the influx of iMo into the cervical LN and the increased S1P peaked just before the onset of EAE symptoms, which is more consistent with an effect on differentiation than priming (Extended Data Fig. 7c). Although not definitive, these experiments provide impetus for future research on the effect of LN residence time on T cell differentiation. ag, Lethally irradiated WT mice were reconstituted with BM from pIC-treated Sphk1f/f Sphk2−/− Mx1-Cre+ (SPHK-KO) or littermate control (LitCtl) animals. 14-25 weeks after reconstitution, EAE was induced in the chimaeras. T cells in the cervical LN were analysed 9d after EAE induction. a, Symptoms over time. LitCtl (n = 9), SPHK-KO (n = 9). 1 experiment. b, Representative tetramer staining of CD4+ T cells. Number indicates mean percent tetramer+ +/− SEM. Compilation of 5 experiments. LitCtl (n = 11), SPHK-KO (n = 11). c, Number tetramer+ CD4 T cells. Compilation of 5 experiments. LitCtl (n = 11), SPHK-KO (n = 11). d, Representative contour plots identifying Tfh among CD4+ cells, top gated on MOG/Ab tetramer+ cells and bottom gated on MOG/Ab tetramer- cells. Number indicates mean percent Tfh +/− SEM. Compilation of 4 experiments. LitCtl (n = 11), SPHK-KO (n = 9). e, Compilation of the experiments in (d), showing ratio of the number of MOG/Ab tetramer+ Tfh cells (left) and MOG/Ab tetramer- Tfh cells (right) in SPHK-KO versus littermate chimaeras (each point represents the ratio of one SPHK-KO animal to the average of the LitCtl animals in the experiment). f, Representative contour plots identifying Th17 among CD4+FoxP3- cells, top gated on MOG/Ab tetramer+ cells and bottom gated on MOG/Ab tetramer- cells. Number indicates mean percent Th17 +/− SEM from 5 experiments. LitCtl (n = 13), SPHK-KO (n = 13). g, Compilation of the experiments in (f), showing ratio of the number of MOG/Ab tetramer+ Th17 cells (left) and MOG/Ab tetramer- Th17 cells (right) in SPHK-KO versus littermate chimaeras (each point represents the ratio of one SPHK-KO animal to the average of the LitCtl animals in the experiment). hk, WT mice were treated to induce EAE. 4d after EAE induction, the S1P lyase inhibitor 4-deoxypyridoxine (DOP) was added to the drinking water. After 5d of DOP treatment, T cells in the cervical LN were analysed. h, Representative gating for Tfh among CD4+ T cells. Number indicates mean percent Tfh +/− SEM from 2 experiments, Ctl (n = 6), DOP (n = 7). i, Compilation of 2 experiments. Ctl (n = 5), DOP (n = 7). j, Representative gating for Th17 among CD4+FoxP3- T cells. Number indicates mean percent Th17 +/− SEM. Compilation of 2 experiments, Ctl (n = 6), DOP (n = 7). k, Compilation of 2 experiments. Ctl (n = 6); DOP (n = 7) lq, WT BM progenitors were transduced with a vector encoding S1PR1_IRES_GFP or a control vector encoding IRES_GFP. Lethally irradiated WT hosts were reconstituted with S1PR1-overexpressing BM or control BM. Because transduction was inefficient, each mouse harboured a mix of GFP+ transduced and GFP- untransduced BM. After reconstitution, EAE was induced in the chimaeras. T cells in the cervical LN were analysed 9d after EAE induction. l, Experiment diagram. m, Representative histograms of S1PR1 expression by GFP+ and GFP- CD4+ cells in a mouse that received S1PR1_IRES_GFP+ BM (left) and a mouse that received IRES_GFP+ BM (right). n, Representative contour plots showing gating for Tfh cells among GFP+ CD4 T cells in a mouse that received vector-transduced BM (left) or S1PR1-transduced BM (right). Number indicates mean percent Tfh +/− SEM, compiling 4 experiments, empty vector (n = 7), S1PR1 (n = 8). o, Each point represents, for a single mouse, the ratio of the % Tfh among GFP+ CD4 T cells to the % Tfh among GFP- CD4 T cells. Compilation of 4 experiments, empty vector (n = 7), S1PR1 (n = 8). p, Representative contour plots showing gating for Th17 cells among GFP+ FoxP3- CD4 T cells in a mouse that received vector-transduced BM (left) or S1PR1-transduced BM (right). Number indicates mean percent Th17 +/− SEM. Compilation of 4 experiments, empty vector (n = 8), S1PR1 (n = 7). q, Each point represents, for a single mouse, the ratio of the % Th17 among GFP+ CD4 T cells to the % Th17 among GFP- CD4 T cells. Compilation of 4 experiments, empty vector (n = 8), S1PR1 (n = 7). Mann–Whitney two-tailed t-test. For EAE curve, two-way ANOVA with Geisser-Greenhouse correction.

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This file contains the ImageJ macro for monocyte localization.

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Supplementary Table 1

A list of antibodies used for flow cytometry.

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Baeyens, A., Bracero, S., Chaluvadi, V.S. et al. Monocyte-derived S1P in the lymph node regulates immune responses. Nature 592, 290–295 (2021). https://doi.org/10.1038/s41586-021-03227-6

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