Figure 1.
LPA induces the migration, but not chemotaxis of naïve CD4+ T cells.
(A.) Naïve mouse CD4+ T cells were added to the upper chamber of a Transwell and LPA (1∶1 mixture of 18∶1 and 16∶0 LPA at 1 µM final concentration) was added to the bottom chamber or top chamber as indicated or (B.) top chamber at various concentrations. Cells were allowed to migrate for 2 hours at 37°C and the number of cells that migrated to the bottom chamber was quantified by hemocytometry. CCL21 (100 ng/ml) was added to the bottom chamber to induce chemotaxis, as a positive control. Data are mean +/− SEM of 3–5 experiments. *p<0.05; **p<0.01; ****p<0.0001.
Figure 2.
LPA induces enhanced migration of naïve CD4+ T cells.
Naïve mouse CD4+ T cells from wild-type C57BL/6 mice were added to microchambers coated with ICAM-1 and CCL21 in the absence of LPA or with 1 µM or 10 µM LPA. T cell migration was imaged every 15 s for 15 min and tracked by Volocity software. (A). Spider-plots of individual cell tracks over 15 min without LPA or with 1 µM LPA. (B). Mean track length, (C). Mean displacement, (D). Mean velocity with (1 µM and 10 µM) LPA or without LPA. Data are mean +/− SEM and representative of three independent experiments. **p<0.01, ****p<0.0001.
Figure 3.
Mouse CD4+ T cells differentially express LPA1–6 over the course of T cell activation and polarization.
(A). Naïve mouse CD4+ T cells were collected and activated by plate-bound anti-CD3 and anti-CD28 antibodies for 24, 48, or 72 hours. At each time point, cells were harvested and the mRNA expression of LPA1,2,3,4,5,6 was determined by semi-quantitative real-time PCR, performed in triplicate. Expression levels were normalized to mouse GAPDH using the 2∧-deltaCt method. Relative Value Units (RVU) = 2∧-deltaCt×1000. Data are mean of three independent experiments. (B). Protein lysates were collected at 0, 24, and 72 hours post-activation and protein expression of LPA1, LPA2, and LPA3 was measured by Western blot. GAPDH was used as a lane loading control. Data are representative of one-two experiments. (C). Naïve mouse CD4+ T cells were collected and activated by plate-bound anti-CD3 and anti-CD28 antibodies and cultured under Th1 (IFN-γ, anti-IL-4), Th2 (IL-4, anti-IFN-γ), or Th17 (TGF-β, IL-6, anti-IL-4, anti-IFN-γ) polarizing conditions for 72 hours. Cells were harvested and the mRNA expression of LPA2 and LPA6 was determined by semi-quantitative real-time PCR, performed in triplicate. Expression levels were normalized to mouse GAPDH and compared to naïve CD4+ T cells using the 2∧-deltadeltaCt method. Data are mean +/− SEM of three independent experiments.
Figure 4.
Naïve CD4+ T cells from lpa2−/− mice still migrate in response to LPA.
Naive mouse CD4+ T cells from wild-type C57BL/6 mice (open bars) or lpa2−/− mice (dark bars) were added to the upper chamber of a Transwell (5 µm pore size) in the presence or absence of LPA (1 µM; 18∶1 and 16∶0). Cells were allowed to migrate for 2 hours at 37°C and cell migration analyzed as in Figure 2. Data are expressed as Migration Index, or the number of cells that migrated in response to LPA relative to the number of cells that migrated in the presence of serum-free media only. Data are mean +/− SEM of four independent experiments. *p<0.05. n.s. = not significant.
Figure 5.
Lpa2-deficiency results in early defects in intranodal T cell migration.
Naïve mouse CD4+ T cells from wild-type C57BL/6 mice and lpa2−/− mice were labeled with CFSE and CMTMR, respectively. (5–10)×106 cells mixed at a 1∶1 ratio together with Texas Red Dextran were given to anesthetized wild-type recipients by injection into the orbital sinus immediately before imaging the microsurgically exposed popliteal lymph node. MP-IVM was performed to visualize intranodal T cell motility, and an HEV-specific mask was created. Images were acquired with a pixel dwell time of 2 µs, using step sizes of 2 µm to a depth of 50 µm every 45 seconds, and analyzed using Volocity software (see Methods). Data are the averaged±SEM of two independent experiments, represented separately for early (0–15 mins) and later (15–30 mins) phases of imaging of both intravascular (A–D) and intranodal (E–H) cells. *p<0.05, **p<0.005, ***p<0.0001.
Figure 6.
LPA2 does not play a crucial role in steady-state T cell homing to lymph nodes.
Wild-type (CD90.2+, CD45.1+) and lpa2−/− (CD90.2+, CD45.1−) CD4+ T cells were adoptively transferred through tail vein injection into wild-type recipient mice. Forty-two hours post-transfer, the inguinal, brachial, cervical lymph nodes and spleen were harvested and the number of donor CD4+ T cells were enumerated by flow cytometry. Data are mean +/− SEM of two independent experiments, n = 5 recipient mice/experiment.