The S1P-analog FTY720 differentially modulates T-cell homing via HEV: T-cell-expressed S1P1 amplifies integrin activation in peripheral lymph nodes but not in Peyer patches - PubMed (original) (raw)

The S1P-analog FTY720 differentially modulates T-cell homing via HEV: T-cell-expressed S1P1 amplifies integrin activation in peripheral lymph nodes but not in Peyer patches

Cornelia Halin et al. Blood. 2005.

Abstract

Sphingosine-1-phosphate (S1P) and its receptor S1P1 control T-cell egress from thymus and secondary lymphoid organs (SLOs). To further define the role of S1P1 in lymphocyte trafficking, we performed adoptive transfer experiments and intravital microscopy (IVM) using both S1P1-/- lymphocytes and recipient wild-type (WT) mice treated with FTY720, an immunosuppressant that downmodulates S1P receptors. S1P1 deficiency and FTY720 caused rapid disappearance of T cells from blood, prolonged retention in SLOs, and accumulation in bone marrow, but did not alter interstitial T-cell motility in peripheral lymph nodes (PLNs) as assessed by multiphoton IVM. However, S1P1-/- lymphocytes displayed reduced short-term homing to PLNs due to attenuated integrin-mediated firm arrest in high endothelial venules (HEVs). By contrast, S1P1-/- T cells homed normally to Peyer patches (PPs), whereas S1P1-/- B cells had a marked defect in homing to PPs and arrested poorly in PP HEVs. Therefore, S1P1 not only controls lymphocyte egress from SLOs, but also facilitates in a tissue- and subset-specific fashion integrin activation during homing. Interestingly, FTY720 treatment enhanced accumulation of both S1P1 sufficient and S1P1-/- T cells in PPs by enhancing integrin-mediated arrest in HEVs. Thus, FTY720 exerts unique effects on T-cell traffic in PPs that are independent of T-cell-expressed S1P1.

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Figures

Figure 1.

Figure 1.

Effect of FTY720 and FTY-P on homing and tissue distribution of adoptively transferred T cells. (A-B) TRITC-labeled splenocytes and PLN cells from T-GFP mice were injected intravenously into WT mice (n = 6 mice/group), which had been pretreated 2.5 hours earlier with FTY720, FTY-P, or saline. At different time-points, PBL (1 mL), SLOs, and BM were harvested and single-cell suspensions were analyzed by FACS for the presence of TRITC+GFP+ T cells. At 2.5 hours after adoptive transfer (A), homed T cells were reduced in PLNs by 44% and 30% in FTY-P– and FTY720-treated mice, respectively, and were increased in PPs by 46% and in BM by 48% after FTY720 treatment. At 16 hours after transfer (B), homed T cells in PLNs remained reduced by approximately 46%. T-cell homing to PPs increased over control: 2.8-fold for FTY-P; 3.9-fold for FTY720; and in BM 3.7-fold for FTY-P; 4.3-fold for FTY720. Data are shown as total number of homed cells per million injected. Error bars indicate SEM. *P < .05; **P < .01 (versus control).

Figure 2.

Figure 2.

Role of S1P1 in T- and B-cell trafficking to SLOs. (A-B) Thymocytes from S1P1+/ or S1P1–/– FL chimeras were differentially labeled with TRITC or CFSE, mixed, and adoptively transferred into WT mice (n = 6 mice/time-point). After 2.5 hours (A) or 20 hours (B), PBL, SLOs, and BM were harvested and single-cell suspensions were stained for CD4 and CD8α and analyzed by FACS for the presence of TRITC+ and CFSE+ SP T cells. (C-D) Differentially labeled splenocytes from S1P1+/ and S1P1–/– FL chimeras were adoptively transferred into WT mice (n = 4 mice/time-point).After 2.5 hours (C) and 20 hours (D), PBL, single-cell suspensions from SLOs, and BM were stained for B220 and analyzed by FACS for the presence of TRITC+ and CFSE+ B cells. Data are shown as total number of homed cells per million injected. Error bars indicate SEM. *P < .05; **P < .01.

Figure 3.

Figure 3.

S1P1 deficiency does not affect T-cell motility within the popliteal LN. (A-D) Differentially fluorescently labeled S1P1+/ and S1P1–/– CD4+ SP cells were adoptively transferred into WT mice and their migratory behavior was analyzed in the same LN after 24 hours and 48 hours using MP-IVM (pooled data, n = 2 mice/time-point). Parameters analyzed are (A) the median instantaneous 3D velocity (S1P1+/: 12.0 ± 0.7 μm minute–1 versus S1P1–/–: 12.4 ± 0.4 μm minute–1), (B) the cumulative instantaneous 3D velocity, (C) motility coefficients (S1P1+/: 73.5 ± 5.1 μm2 minute–1 versus S1P1–/–: 74.8 ± 4.0 μm2 minute–1), and (D) turning angles. (E-G) Differentially fluorescently labeled untreated (control) or CD4+ T cells from FTY720-treated WT donors were adoptively transferred into WT recipients (n = 2 mice, 2-3 recordings/mouse). After 3 hours, cell motility was analyzed in the popliteal LN. (E) Median instantaneous 3D velocity (control: 11.3 ± 0.5 μm minute–1 versus FTY720: 13.2 ± 0.9μm minute–1), (F) cumulative instantaneous 3D velocity, (G) motility coefficients (control: 82.3 ± 8.2 μm2 minute–1 versus FTY720: 95.6 ± 8.5 μm2 minute–1). Horizontal bars in panels A, C, E, G represent the mean of the measurements. Dashed horizontal lines in panels B and F indicate a frequency of 0.5, corresponding to the median. Vertical dashed lines connect these data points to the x-axis, indicating the median values for instantaneous 3D velocity.

Figure 4.

Figure 4.

S1P1–/– T cells display a defect in integrin-mediated firm arrest in PLN HEVs. The intravascular behavior of calcein-labeled CD4+ SP cells from S1P1+/ or S1P1–/– FL chimeras was analyzed by IVM in inguinal LN of WT mice (n = 2 mice). (A) Rolling and (B) sticking fractions (44.1% ± 5.6% of S1P1+/ versus 26.5% ± 6.2% of S1P1–/–; *P < .05) of cells were determined in venular orders III, IV, and V. Error bars indicate SEM.

Figure 5.

Figure 5.

S1P1–/– B cells but not S1P1–/– T cells display a defect in integrin-mediated firm arrest in PP HEVs. (A-B) The intravascular behavior of calcein-labeled B cells from S1P1+/ or S1P1–/– FL chimeras was analyzed by IVM in PP HEVs (n = 3 mice). (A) Rolling (32.1% ± 4.1% of S1P1+/ versus 24.2% ± 3.6% of S1P1–/–; **P < .01) and (B) sticking fractions (11.3% ± 1.8% of S1P1+/ versus 5.9% ± 1.3% of S1P1–/–;* P < .05). (C-D) Calcein-labeled CD4+ SP cells from S1P1+/ or S1P1–/– FL chimeras were analyzed by IVM in PP HEVs (n = 3 mice). No difference was observed in (C) rolling (37.4% ± 4.4% of S1P1+/ versus 28.4% ± 3.5% of S1P1–/–) and (D) sticking fractions (12.5% ± 2.5% of S1P1+/ versus 11.5% ± 2.8% of S1P1–/–). Error bars indicate SEM.

Figure 6.

Figure 6.

FTY720 promotes accumulation of S1P1–/– T cells in PPs. TRITC- or CFSE-labeled thymocytes from S1P1+/ (A) or S1P1–/– (B) FL chimeras were adoptively transferred into control or FTY720-treated WT recipients (n = 4 mice/group). After 20 hours, mice were killed and PBL and SLOs were harvested. Single-cell suspensions were stained for CD4 and CD8α and analyzed by FACS for the presence of TRITC+ and CFSE+ donor SP cells. (C) TRITC- or CFSE-labeled thymocytes from S1P1+/ or S1P1–/– FL chimeras were adoptively transferred into control or FTY720-treated WT recipients (n = 5). After 2.5 hours mice were killed and PPs were harvested and single-cell suspensions were stained for CD4 and CD8α and analyzed by FACS. Error bars indicate SEM. *P < .05.

Figure 7.

Figure 7.

Treatment of mice with FTY720 induces T-cell sticking in PP HEVs. (A) Splenocytes and PLN cells from T-GFP mice were injected intravenously into WT mice (n = 6 mice/group), which had been pretreated 2.5 hours earlier with FTY720 or saline. After 30 minutes, PPs were harvested and single-cell suspensions were analyzed by FACS for the presence of GFP+ T cells. Homed T cells were increased in PPs of FTY720-treated mice by 62%; *P < .05. (B-C) The intravascular behavior of calcein-labeled WT and S1P1–/– T cells in PP HEVs of untreated or FTY720-treated recipients was analyzed by IVM (n = 4 mice/group). (B) WT T-cell rolling and sticking fractions (9.4% ± 2.2% in control mice versus 14.4% ± 2.0% in FTY720-treated mice; P = .05) and (C) S1P1–/– T-cell rolling and sticking fractions (11.5% ± 2.8% in control mice versus 26.0% ± 5.0% in FTY720-treated mice. Error bars indicate SEM. *P < .05).

Figure 8.

Figure 8.

Summary of the effects of S1P1 deficiency or FTY720 treatment on the trafficking of adoptively transferred lymphocytes. The effects of S1P1 deficiency and FTY720 treatment on cell entry or cell exit to various lymphoid organs are presented for T cells (top) and B cells (bottom). Conclusions on “cell entry” are derived from our short-term homing and IVM experiments, whereas “cell exit” summarizes our findings in emigration experiments, as well as observations made by others. “Cell content in organ” refers to our results in long-term (16 to 20 hours) homing experiments and provides a measure of the cell numbers of adoptively transferred cells found in a particular organ. Open arrows within the organ drawings symbolize the entry and exit routes of naive lymphocytes for each organ. Other symbols and abbreviations are explained as follows: upward arrows indicate enhancement; downward arrows, reduction; lateral arrows, indicate no effect; and ND, not determined. Arrows in parentheses indicate a pronounced, although not statistically significant, tendency. Footnotes: a, based on the fact that cell exit from PP is reduced; b, occurs independently of T cell–expressed S1P1; c, the profound lymphopenia in blood suggests a reduction in lymphocyte exit from spleen; d, although not addressed experimentally, it remains possible that T-cell accumulation in BM is increased not only due to enhanced cell entry but also due to decreased cell exit; e, based on the analysis of endogenous single-positive CD4+ or CD8+ T cells in thymus of S1P1–/– chimeras and FTV720-treated mice,,; f, suggested by thoracic duct lymph canulation experiments, which have revealed a substantial reduction of S1P1-deficient or FTY720-treated T and B cells in lymph; g, as shown by Cinamon et al; h, the fact that B-cell entry is unaffected, but S1P1 deficiency or FTY720 treatment leads to accumulation of cells in BM after long-term adoptive transfer, suggests that B-cell exit from BM is reduced.

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