Several human CD4 T cell subsets can produce AR.

<p>(A) Allogeneic Th1 and Th2 cell lines from three subjects were stimulated with PMA + ionomycin for 6 hours. The percentage of cells expressing IFNγ, IL-4, and AR was analyzed by ICS. (B) The expression of AR and other cytokines was measured in SEB-stimulated PBMC from four subjects by ICS, calculating the frequencies of single cytokine producers, and all possible combinations of double-producers, among the CD154+ CD4+ T cells. The figure shows the ratio between the <u>observed</u> frequencies of double-producing T cells for each cytokine pair, and the <u>expected</u> frequencies (calculated as the product of the individual frequencies for each cytokine). Values represent the ratios for the double-producer combination defined by the row and column labels. Ratios above or below 1 are indicated by solid or open symbols, respectively. (C) IL-4, IFNγ and IL-2 mRNA levels were measured by RT-PCR in the sorted populations described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039072#pone-0039072-g004" target="_blank">Figure 4C</a>. (D) PBMC were treated with influenza H1N1 peptides or tetanus (five subjects each), or the allergens Fel d1 (solid symbols) or Der p1 (open symbols)(three subjects each). The numbers of memory CD4 T cells expressing AR and other cytokines were measured by ICS. The backgrounds (no antigen) have been subtracted. Each symbol represents one individual and the filled bar is the mean of all tested subjects. (E) CD69+ CD4+ T cells (Control_CD69+) were sorted from PBMC incubated in medium alone. CD69+IFNγ+ and CD69+IFNγ- CD4 T cells were sorted from influenza peptide-treated PBMC using the cytokine secretion assay. The mRNA levels of IFNγ and AR were measured by RT-PCR. Results in (A-C) are representative of at least three experiments, (D) represents two experiments using a total of 5 independent subjects, and (E) represents two experiments.</p

Both naïve and memory human CD4 T cells expressed AR during TCR activation.

<p>(A) PBMC were treated with medium alone or allogeneic EBV-transformed B cells for 10 hours and analyzed by ICS. The gating strategy to identify activated CD4+ and CD8+ T cells is shown. (B) AR, IL-2, IFNγ or IL-4 expression was measured in four subjects in CD45RA+ (open) and CD45RA- (solid) CD4+ and CD8+ T cells after allogeneic EBV-transformed B cell stimulation. Background values have been subtracted. (C) PBMC were treated with medium alone or SEB in the presence of TAPI-1 for 8 hours. Then six populations were sorted based on surface AR, CD69 and CD45RA expression (left). AR mRNA in each population was measured by RT-PCR (right). Results in (A) and (B) represent at least three experiments, (C) represents two experiments.</p

AR is produced by T cell subsets expressing different chemokine receptors and surface markers.

<p>PBMC were treated with medium alone, anti-CD3+ anti-CD28 antibodies, or SEB in the presence of TAPI-1 for 8 hours. Cells were stained for AR and cell-surface markers and analyzed by flow cytometry. Representative of two experiments.</p

TCR and cAMP synergize to induce AR production in human CD4 T cells.

<p>Purified CD4 T cells were incubated with or without TCR stimulation (anti-CD3/CD28 beads) and the cAMP agonist. (A) AR and HB-EGF mRNA expression was measured by RT-PCR. (B) The concentrations of AR in the supernatant and cell lysates were measured by ELISA. (C) Enriched CD45RA+CD45RO- (naïve) and CD45RA-CD45RO+ (memory) CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of cAMP agonist (1 ∼ 1000 µM). The concentration of AR in the supernatant at 24 hours was measured by ELISA. (D) Purified CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of the cAMP-modifying agents shown. RNA was extracted at 4 hours, and AR mRNA was measured by RT-PCR. The concentration of AR in the 24-hour supernatant was measured by ELISA. (E) PBMC were treated with anti-CD3+ anti-CD28 antibodies in the presence or absence of cAMP agonist or antagonist for 8 hours. CD4 T cells were purified by cell sorting and RNA was extracted. The mRNA levels of AR and other cytokines were measured by RT-PCR. All results are representative of at least three experiments.</p

TCR activation induced AR expression in human PBMC T cells.

<p>(A) PBMC were treated as indicated and analyzed by ICS. The upper panels show the gating strategy to identify activated (CD69+) CD4 or CD8 T cells expressing AR. The lower panels show the induction of AR by different stimuli in CD4 or CD8 T cells. (B) AR and IL-2 mRNA were measured by RT-PCR in purified CD4 and CD8 T cells after activation by anti-CD3+anti-CD28 beads. Results in (A) and (B) are representative of at least three experiments.</p

Mouse CD4+ T cells differentially express LPA1–6 over the course of T cell activation and polarization.

<p>(<b>A</b>). 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>B</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. (<b>C</b>). 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.</p

Regulation of T Cell Motility <i>In Vitro</i> and <i>In Vivo</i> by LPA and LPA2

<div><p>Lysophosphatidic acid (LPA) and the LPA-generating enzyme autotaxin (ATX) have been implicated in lymphocyte trafficking and the regulation of lymphocyte entry into lymph nodes. High local concentrations of LPA are thought to be present in lymph node high endothelial venules, suggesting a direct influence of LPA on cell migration. However, little is known about the mechanism of action of LPA, and more work is needed to define the expression and function of the six known G protein-coupled receptors (LPA 1–6) in T cells. We studied the effects of 18∶1 and 16∶0 LPA on naïve CD4+ T cell migration and show that LPA induces CD4+ T cell chemorepulsion in a Transwell system, and also improves the quality of non-directed migration on ICAM-1 and CCL21 coated plates. Using intravital two-photon microscopy, <i>lpa2−/−</i> CD4+ T cells display a striking defect in early migratory behavior at HEVs and in lymph nodes. However, later homeostatic recirculation and LPA-directed migration <i>in vitro</i> were unaffected by loss of <i>lpa2</i>. Taken together, these data highlight a previously unsuspected and non-redundant role for LPA2 in intranodal T cell motility, and suggest that specific functions of LPA may be manipulated by targeting T cell LPA receptors.</p></div

LPA induces enhanced migration of naïve CD4+ T cells.

<p> 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. (<b>A</b>). Spider-plots of individual cell tracks over 15 min without LPA or with 1 µM LPA. (<b>B</b>). Mean track length, (<b>C</b>). Mean displacement, (<b>D</b>). 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.</p

LPA2 does not play a crucial role in steady-state T cell homing to lymph nodes.

<p>Wild-type (CD90.2+, CD45.1+) and <i>lpa2</i>−/− (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.</p

Naïve CD4+ T cells from <i>lpa2</i>−/− mice still migrate in response to LPA.

<p>Naive mouse CD4+ T cells from wild-type C57BL/6 mice (open bars) or <i>lpa2</i>−/− 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101655#pone-0101655-g002" target="_blank">Figure 2</a>. 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.</p