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 -- Halin et al. 106 (4): 1314 Data Supplement - Supplemental materials for: Halin et al, Vol. 106, Issue 4, 1314-1322 -- Blood

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
Blood Halin et al. 106: 1314

Supplemental materials for: Halin et al, Vol. 106, Issue 4, 1314-1322

Files in this Data Supplement:

Document S1. Supplemental materials and methods (PDF, 74.3 KB)
Table S1. Total number of lymphocytes in PBL (per 1 mL) and total number of leukocytes in SLOs in control mice and in mice treated for 2.5 hours or 16 hours with FTY-P or FTY720 (n = 6 mice/group) (PDF, 52 KB) - Averages of cell numbers are expressed as 1 × 10⁶ ± SEM; *P < .05; **P < .01; ***P < .001 (versus control).
Figure S1. Accumulation of lymphocytes in thoracic duct lymph after short- and long-term adoptive transfer (PDF, 40 KB) - Similar numbers of differentially labeled T or B cells were injected intravenously into recipient mice (n > 4) 18 hours before (–18 h) and 2.5 hours before (–2.5 h) lymph collection. At 0 hours lymph was collected from thoracic duct of mice. Experimental setup (A), representative FACS plot of lymph for T cells (B) and for B cells (C).
Figure S2. FTY720 and FTY-P have little influence on B-cell homing (PDF, 80 KB) - TRITC-labeled pooled splenocytes and PLN cells from T-GFP mice were injected intravenously into WT mice (n = 6 mice/group). At 2.5 hours (A) and 16 hours (B) after injection, PBLs and SLOs were harvested and single-cell suspensions were analyzed by FACS for the presence of TRITC+GFPneg B cells. *P < .05; **P < .01 (versus control).
Figure S3. SP cells are the predominant subset among the thymocytes that home to PLNs (PDF, 85 KB) - TRITC- or CFSE-labeled thymocytes from S1P₁^+/ and S1P₁^-/- FL chimeras were stained with CD4 and CD8α and analyzed by FACS (A). Both populations were then mixed at a 1:1 ratio and injected intravenously into WT mice (n = 6 mice). Twenty hours later, PBLs (B) and PLNs (C) were harvested, and single-cell suspensions were stained for CD4 and CD8α and analyzed by FACS for the presence of homed donor cells. Data are representative of 3 individual experiments.
Figure S4. Effect of S1P₁ on retention of SP T cells in PLNs (PDF, 36 KB) - TRITC- or CFSE-labeled thymocytes from either S1P₁^+/ or S1P₁^-/- FL chimeras were adoptively transferred into WT mice (n = 6 mice/group). Four hours later, some mice were treated with L-selectin–blocking mAb Mel-14. Mice were sacrificed 24 hours later, PBLs and SLOs were harvested, and single-cell suspensions were stained for CD4 and CD8α and analyzed by FACS. S1P₁^+/ SP cells were reduced in PBLs and PLNs of Mel-14–treated mice by 87% and 90%, respectively. Data are shown as total number of homed cells per million injected. *P < .05.
Figure S5. Effect of FTY-P on lymphocyte retention in PLNs. (A) Schematic diagram of the experimental protocol (PDF, 75 KB) - Four hours after adoptive transfer of T-GFP cells into WT recipients, some mice were treated with anti–L-selectin mAb Mel-14. TRITC-labeled splenocytes were injected 30 minutes later. Some mice were injected intraperitoneally with FTY-P 5 and 20 hours after T-GFP cell transfer, and all animals were sacrificed after 24 hours (n > 4 mice/group). PBLs and SLOs were harvested and single-cell suspensions were analyzed by FACS. (B) Frequency of homed T-GFP cells in PBLs and SLOs. *P < .05; **P < .01 (versus control); ##P < .01; ###P < .001 (Mel-14 versus Mel-14+FTY-P-treated). (C) Frequency of TRITC-labeled cells in PBLs and SLOs. Data are shown as total number of homed cells per million injected. *P < .05; **P < .01 (versus control). Of note, although Mel-14 reduced the number of cells recovered from MLNs, FTY-P had no significant effect on T cell accumulation in this organ. This could possibly reflect the unique anatomic connectivity of MLNs, which receive afferent lymph from the entire intestine, including PPs. FTY-P is likely blocking lymphocyte egress via lymphatics both within and upstream of MLNs, resulting in no significant net effect on T cell numbers in MLNs compared to control mice.
Video S1. S1P₁ does not affect cell motility within the popliteal LNs (MOV, 3.31 MB) - CMFDA-labeled S1P₁^+/ (green) and CMTMR-labeled S1P₁^-/- CD4⁺ SP T cells (red) were adoptively transferred into WT mice and their migratory behavior analyzed after 24 hours.