Research ArticleLYMPHOCYTE MIGRATION

CD4 T cell sphingosine 1-phosphate receptor (S1PR)1 and S1PR4 and endothelial S1PR2 regulate afferent lymphatic migration

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Science Immunology  15 Mar 2019:
Vol. 4, Issue 33, eaav1263
DOI: 10.1126/sciimmunol.aav1263
  • Fig. 1 LECs specifically promote CD4 T cell TEM to S1P but not to other chemokines.

    Murine CD4 migration: (A) Across plastic or skin or LN-derived iLECs or LECs to S1P or CCL19; (B) across plastic, iSVECs, or SVECs to medium, 100 nM S1P, or 53 nM CCL19; (C) across plastic or iSVECs to varying doses of S1P. (D) Migration of human Teff across plastic, human iLECs or human LECs to CCL19 or S1P. (E) Naïve murine CD4 migration toward S1P or CCL19 across iSVECs, with anti-CCL21 loaded in the upper chamber. (F) Naïve murine CD4 migration across iSVECs toward S1P with various doses of anti-S1P in the upper chamber. (G and H) Individual naïve CD4 migration toward S1P and CCL19, showing velocity (G) and migration time (H) of individual cells. At least three independent experiments, triplicate wells. Data presented as mean ± SEM. One-way ANOVA for multiple comparisons. *P < 0.05; ns, not significant.

  • Fig. 2 S1P induces T cell chemotactic and chemokinetic TEM regulated by fluid flow and inflammation.

    (A) Naïve CD4 migration across iSVECs; S1P loaded in different chambers as indicated. (B to E) Real-time imaging for naïve CD4 chemokinetic or chemotactic migration toward S1P loaded in the lower chamber or in both chambers, respectively. (B) Tracking for individual cells, (C) velocity, (D) migration times, and (E) velocity distribution. (F) Naïve CD4 migration across iSVECs to CCL19 or S1P under no-flow or flow conditions. (G) Migration of naive CD4 pretreated with or without anti–VLA-4 to S1P across iSVECs pretreated with anti–VCAM-1 and/or TNFα 3 hours before anti–VCAM-1. For (A) to (G), three independent experiments with triplicate wells, presented as mean ± SEM; one-way ANOVA for multiple comparisons. (H and I) Footpad migration with or without LPS treatment. One microgram of LPS was injected per footpad; 2 hours later, CD4 T cells without treatment (H) or mixed with control IgG or anti–VCAM-1 (I) were transferred into footpads, and migration to dLN was assessed after 16 hours (n = 6). For (H) and (I), statistical analyses, paired Student’s t test, *P < 0.05.

  • Fig. 3 T cell S1PR1 and S1PR4 regulate TEM.

    (A) iSVECs or CD4 treated with PTX, FTY720. (B) CD4 treated with S1PR1 antagonist. (C) iSVECs or CD4 treated with S1PR4 antagonist. (D) S1PR1−/− or S1PR1+/+ [wild type (WT)]. (E) S1PR4−/− or S1PR4+/+, CD4 treated with S1PR1 or S1PR4 antagonists, migrated to S1P or CCL19. (F) S1PR1-Tg or (G) S1PR1 S5A CD4 treated with S1PR1 antagonist, migrated to S1P or CCL19. (H) CD4 treated with S1PR1, S1PR4, or combined antagonists; iSVEC; and S1P in the lower chamber or in both chambers. (I to M) Naïve CD4 footpad migration: (I) CD4 pretreated with S1PR4 antagonist (n = 12); (J) CD4 pretreated with S1PR1 antagonist (n = 12); (K) S1PR1+/+ or S1PR1−/− CD4 transferred to WT recipients (n = 6); (L) S1PR4+/+ or S1PR4−/− CD4 to WT recipients (n = 12); (M) S1PR1 WT or S5A CD4 to WT recipients (n = 10). (N to S) CD4 Teff or Tmem across iLEC: (N) Teff or (O) Tmem treated with S1PR1, S1PR4, or combined antagonists; S1PR1−/− or S1PR1+/+ (P) Teff or (Q) Tmem; S1PR4−/− or S1PR4+/+ (R) Teff or (S) Tmem. (T and U) Teff footpad migration: (T) Teff pretreated with S1PR4 antagonist (n = 7); or (U) S1PR1 antagonist (n = 6). In paired analysis footpad migration, T cells were transferred to both footpads, with one side treated with inhibitor and the other treated with control. (A) to (H) and (N) to (S), mean ± SEM, one-way ANOVA for multiple comparisons. (I) to (M) and (T) to (U), paired Student’s t test.

  • Fig. 4 S1P gradient and LEC S1PR2 regulate T cell TEM.

    (A) Whole-mount staining, S1P in lymphatics; 100×; scale bars, 8 μm (two ears per treatment per experiment, n = 8). LV, lymphatic vessel. (B) Naïve CD4, footpad migration, anti-S1P treatment (n = 11). (C) Naïve CD4, footpad (treated with LPS) migration, anti-S1P (n = 6). (D) Wild type (WT) into WT, S1PR2−/−, Sphk1−/−, or Sphk2−/− recipients (n = 8 WT, S1PR2−/−; n = 12 Sphk1−/−; n = 6 Sphk2−/−). (E to H) CD4 across iSVECs to S1P: (E) Naïve CD4 (F) Teff and (G) Tmem across iSVECs treated with S1PR2 antagonist. (H) iSVECs infected with shRNA lentivirus. (I to K) CD4 footpad migration: (I) Naive CD4 and (J) Teff footpads pretreated with S1PR2 antagonist (n = 15 naïve and n = 8 Teff); (K) endogenous T cell migration, anti-CD62L mAb intravenously, and S1PR2 antagonist in footpad; total CD4+CD69+ activated and CD4+CD44hi memory in dLN (n = 10). (L and M) CD4 across iLECs to S1P: (L) Naïve CD4 or (M) Teff pretreated with S1PR1, S1PR4, or combined antagonists; iLECs treated with S1PR2 antagonist. (N) Naïve CD4 pretreated with S1PR1 and S1PR4 antagonists into footpad pretreated with S1PR2 antagonist (n = 8). (O) Naïve CD4 treated as indicated; iSVECs treated with S1PR2 antagonist; S1P plus CCL19. (P) Anti-CCR7 or S1PR1 or S1PR4 antagonists added at indicated time points. For (E) to (H), (L) to (M), (O), and (P), mean ± SEM, three independent experiments, one-way ANOVA for multiple comparisons; for (B), (C), (I), (J), and (N), paired Student’s t test; for (K), unpaired Student’s t test; for (D), Mann-Whitney.

  • Fig. 5 S1PRs regulate CD4 T cell afferent lymphatic distribution.

    Naïve CD4, ear pinnae migration. (A) CD4 treated with S1PR4 antagonist, into pinnae. (B) Wild type (WT) and S1PR4−/− CD4, into WT pinnae. After 16 hours, pinnae were stained for Lyve-1. (C) CD4 treated with S1PR1 antagonist, into pinnae. (D) S1PR1-Tg or (E) S1PR1 S5A or littermate CD4, WT pinnae. After 2 hours, pinnae were stained for Lyve-1. (F) CD4, into pinnae treated with anti-S1P. (G) CD4, into pinnae treated with S1PR2 antagonist; and (H) WT CD4, into WT or S1PR2−/− pinnae. After 16 hours, pinnae were stained for Lyve-1. Samples were analyzed by fluorescence microscopy; magnification, ×40; scale bars, 8 μm. Percentage of CD4 in lymphatics out of total CD4, or number of lymphatic vessel lumens with clustered groups of CD4, calculated per high-powered field. Four independent experiments, two ears per treatment per experiment, 6 to 8 mice per group, 10 high-powered fields from each pinna. Statistical analyses, Student’s t test.

  • Fig. 6 S1PR2 regulates LEC layer integrity and junction molecules.

    (A and B) Western blot: SVECs pretreated with S1PR2 or S1PR1 antagonists, stimulated with S1P (A); SVECs pretreated with Sphki, stimulated with S1P (B). (C to F) Permeability assays: Evans Blue, upper chamber; S1P, lower chamber; SVECs treated as indicated; three independent experiments. (G) In vivo permeability, footpad treated with S1PR2 antagonist, Evans Blue to dLN measured at indicated times. (H to J) Junction molecule immunohistochemistry: ears treated with S1PR2 antagonist; Lyve-1, VE-cadherin (H); VE-cadherin, occludin (I); ZO-1 (J). (K) VE-cadherin by WT and S1PR2−/− lymphatics (two ears per treatment per experiment, n = 6); magnification, ×40; scale bars, 21 μm. (L to N) Junction molecule expression, SVECs treated with inhibitors. Mean ± SEM: (C) to (F) and (K), one-way ANOVA for multiple comparisons; (G) to (J), Student’s t test.

  • Fig. 7 S1PR2 regulates junction molecules and S1PRs mediates T cell paracellular and transcellular migrations.

    (A) VE-cadherin; (B) ZO-1; (C) occludin. SVECs pretreated with S1PR2 or S1PR1 antagonists; ERK inhibitor; stimulated with S1P. Magnification, ×100; scale bars, 8 μm; three independent experiments. (D) CD4 migration, iSVECs treated with ERK inhibitor, (E) ROCK or Rho inhibitors; three independent experiments. (F) CFSE-labeled CD4 toward S1P and CCL19 (i); T cells pretreated with S1PR1 or S1PR4 antagonist; eFluor 670–labeled SVECs pretreated with S1PR2 antagonist toward S1P (ii); eFluor 670–labeled SVECs pretreated with anti–VCAM-1 (iii); magnification, ×63; scale bar, 14 μm; arrowheads indicate paracellular (white) and transcellular (yellow) migration. Percentage of T cells in paracellular and transcellular positions (right); >159 cells counted per group; three independent experiments. Mean ± SEM: (L) to (P), one-way ANOVA for multiple comparisons.

  • Fig. 8 LEC S1PR2 regulates VCAM-1 expression.

    (A) VCAM-1 expression, SVECs treated with S1P, S1PR1 antagonist, S1PR2 antagonist, or ERK inhibitor for 3 hours. Flow cytometry analysis, three independent experiments. (B) VCAM-1 expression, SVEC layers pretreated with S1PR2 antagonist, S1PR1 antagonist, or 5 μM ERK inhibitor, and then treated with S1P. Fluorescence microscopy analysis; magnification, ×40; scale bars, 21 μm. (C) VCAM-1 expression, SVECs pretreated with Sphki and then treated with S1P. Fluorescence microscopy analysis; magnification, ×100; scale bars, 8 μm. (D and E) CD4 position, LEC VCAM-1, and actin distribution. CD4 migration to S1P for 1 hour over iSVECs pretreated for 1 hour with (D) S1PR2 antagonist or (E) ERK inhibitor, at least three independent experiments. (F) VCAM-1 expression, afferent lymphatics treated with 0.5 nM S1PR2 antagonist overnight; magnification, ×40; scale bars, 21 μm. (G) Naïve CD4, treated with S1PR1 or S1PR4 antagonists, migrated to S1P, across transwell inserts coated with VCAM-1–Ig, three independent experiments. (H) Naïve CD4, treated with S1PR1 or S1PR4 antagonists, migrated to S1P, across transwell inserts coated with VCAM-1–Ig. Real-time imaging, tracking path (left), velocity (middle), and distances (right) of individual cells. One-way ANOVA for multiple comparisons (B, C, F, and G).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/33/eaav1263/DC1

    Materials and Methods

    Fig. S1. LECs do not promote CD4 T cell migration toward other chemokines and cytokines.

    Fig. S2. LECs and lymphatics express CCL21 but not other chemotactic factors.

    Fig. S3. S1PR antagonists inhibit migration to S1P but not CCL19, and do not induce CD4 T cell apoptosis; Spkh but not S1PR2 deletion decreases S1P expression by afferent lymphatics.

    Fig. S4. S1PRs, CCR7, and receptor inhibitors regulate migration to their cognate ligands.

    Fig. S5. CD4 T cell distribution in LNs after treatment with S1PR antagonists.

    Fig. S6. S1PR2 regulates VCAM-1 expression.

    Fig. S7. Model of T cell lymphatic TEM.

    Table S1. Antibodies for flow cytometry, Western blotting, and immunohistochemistry.

  • Supplementary Materials

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. LECs do not promote CD4 T cell migration toward other chemokines and cytokines.
    • Fig. S2. LECs and lymphatics express CCL21 but not other chemotactic factors.
    • Fig. S3. S1PR antagonists inhibit migration to S1P but not CCL19, and do not induce CD4 T cell apoptosis; Spkh but not S1PR2 deletion decreases S1P expression by afferent lymphatics.
    • Fig. S4. S1PRs, CCR7, and receptor inhibitors regulate migration to their cognate ligands.
    • Fig. S5. CD4 T cell distribution in LNs after treatment with S1PR antagonists.
    • Fig. S6. S1PR2 regulates VCAM-1 expression.
    • Fig. S7. Model of T cell lymphatic TEM.
    • Table S1. Antibodies for flow cytometry, Western blotting, and immunohistochemistry.

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