PTPN4 and PTPN3 are not required for CD4+ T cell differentiation.

<p>Purified CD4+ T cells were cultured under Th1-, Th2-, or Th17-inducing conditions. T cells were re-stimulated with CD3 antibodies and concentrations of IFN-γ, IL-4, and IL-17, respectively, in culture supernatants was determined by ELISA. A) Shown are results of a representative Th1/Th2 polarization experiment comparing control, PTPN13 ΔPTP/ΔPTP and PTPN4/PTPN3 double-deficient PTPN13 ΔPTP/ΔPTP T cells. Shown is mean cytokine secretion plus 1 standard deviation of triplicate determinations from single littermate mice. Conditions of polarization are indicated on the x-axis. B) Several repeat experiments of the type indicated in A) were performed. In each experiment, a fold increase in IFN-γ, IL-4 or IL-17 expression under conditions of Th1, Th2 or Th17 polarization respectively was calculated for mutant T cells relative to wild-type T cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004014#s2" target="_blank">Materials and Methods</a>). Shown is the mean fold increase+/−one standard error (PTPN4−/−PTPN3−/−PTPN13+/ΔPTP, n = 8; PTPN4+/−PTPN3+/−PTPN13 ΔPTP/ΔPTP, n = 5; PTPN4−/−PTPN3−/−PTPN13 ΔPTP/ΔPTP, n = 4). note that a 0 value fold increase indicates no change in cytokine secretion relative to wild-type. * differences relative to wild-type responses are statistically significant as determined in a paired Student's T-test comparing raw data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004014#s2" target="_blank">Materials and Methods</a>). All other differences relative to wild-type cells are not statistically significant. # p<0.07.</p

PTPN4/PTPN3 double-deficient mice show normal T cell development.

<p>A) Expression of PTPN4 in mice of the indicated genotypes (top) determined by RTPCR. B) Expression of PTPN3 in mice of the indicated genotypes (top) determined by Western blotting. Blots were stripped and reprobed with GAPDH antibodies to confirm equivalent protein loading. C) Flow cytometry plots of thymocytes and peripheral immune cells from PTPN4−/−PTPN3−/−mice and littermate controls showing expression of the indicated markers on live cell populations. In CD44/CD62L plots, the gated population represents recently activated memory T cells. Data are representative of six repeat experiments.</p

Normal T cell development and function in PTPN4-deficient mice.

<p>A) Flow cytometry plots of thymocytes, LN cells, and splenocytes from PTPN4-deficient mice and littermate controls showing expression of the indicated markers on live cell populations. Percentages of cells that fall within the indicated regions are shown. Data are representative of three repeat experiments. B) LN T cells were stimulated with the indicated concentrations of CD3 antibody and 0.5 µg/mL CD28 antibody. Concentrations of cytokines in supernatants were determined by ELISA. Each symbol represents the mean of triplicate determinations from a single mouse. Bars represent the mean cytokine secretion. Differences between PTPN4-deficient and control mice are not statistically significant (Paired Student's T test).</p

Function of PTPN4/PTPN3 double-deficient PTPN13 ΔPTP/ΔPTP T cells.

<p>A) LN T cells were stimulated with the indicated concentrations of CD3 antibody and 0.5 µg/mL CD28 antibody. Concentrations of cytokines in supernatants were determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004014#pone-0004014-g002" target="_blank">Figure 2</a>. Differences between mice are not statistically significant, excepting IFN-γ secretion at low dose anti-CD3 which is not a reproducible finding over four repeat experiments. B) Splenic T cells were labeled with CFSE and stimulated with 0.1 µg/mL CD3 antibody and 0.5 µg/mL CD28 antibody. After 72 h, CFSE dye intensity was measured by flow cytometry. Data are representative of four mice of each genotype.</p

Normal T cell development in PTPN4/PTPN3 double-deficient PTPN13 ΔPTP/ΔPTP mice.

<p>Thymocytes and splenocytes from mice of the indicated genotypes were analyzed by flow cytometry for expression of T cell and B cell markers. Shown is the mean percentage representation plus one standard deviation of the indicated subpopulations among total live cells (n = 4 mice of each genotype). Differences between mice are not statistically significant.</p

LPS induced cell death in differentiated THP-1 cells.

<p>(A) Quantification of percentage of propidium iodide positive THP-1 cells differentiated in the presence of PMA (200 ng/mL) for 2 days (white circles) or 5 days (black circles) following internalization of <i>Salmonella</i> or <i>E</i>. <i>coli</i> DH5α or a 30 min treatment with LPS (10 ng/mL). Data shown as individual experimental means. Statistical significance was determined as compared to uninfected controls using 2-way ANOVA with Tukey’s post hoc test. (B) Representative bright field images of THP-1 cells stained with propidium iodide (red) 120 min after internalization of <i>Salmonella</i> or <i>E</i>. <i>coli</i> or treatment with LPS. Scale bar 15 μm. (C) THP-1 cells were treated with DMSO or staurosporine (1 μM) for 3 h, infected with <i>Salmonella</i> or <i>E</i>.<i>Coli</i> for 30 min, or treated with LPS (10 ng/mL) for 30 min. At 2 h post-treatment cells were incubated with poly-caspase FAM-VAD-FMK before fixation, staining, and analysis. Data are means ± SD from three independent experiments. Statistical significance was determined using 1-way ANOVA with Dunnett post hoc test.</p

Surface receptor expression and cell morphology are dependent on differentiation conditions.

<p>(A) Representative bright field images of THP-1 cells differentiated for 1, 2, or 5 days in the presence of PMA (200 ng/mL). Scale bar is 100 μm. (B) Representative flow cytometric analysis of THP-1 cells stained using anti- CD11b or -CD14. (C) Quantification of mean fluorescence intensity (MFI) for CD11b (black) and CD14 (white). Data are means ± SD from three independent experiments. Statistical significance was determined using 1-way ANOVA with Dunnett post hoc test.</p

Replication of <i>Salmonella</i> in differentiated THP-1 cells.

<p>(A) THP-1 cells differentiated in PMA (200 ng/mL) for 2 days or 5 days and infected with mCherry-<i>Salmonella</i> (red) were stained with DAPI (blue) at 1 and 24 h pi. Scale bars are 25 and 5 μm (inset). (B, C) Intracellular growth of <i>Salmonella</i> in THP-1 cells differentiated 2 days (open circles) or 5 days (closed circles) at 1, 6, 12, and 24 h pi. (C) Growth normalized to 1 h pi. Data are means ± SD from three independent experiments. Statistical significance was determined using 2-way ANOVA with Sidak post hoc test.</p

<i>Salmonella</i> are killed by THP1 cells and primary human macrophage cells.

<p>(A) Intracellular <i>Salmonella</i> in THP-1 cells differentiated for 3 days in the presence of 20 ng/mL PMA (open circles) or primary human monocyte derived macrophages (MDMs, closed circles) differentiated for 7 days in the presence of 100 ng/mL MCSF. Growth normalized to 1 h pi. Data are means ± SD from three independent experiments. (B) Cytokine expression as measured by ELISA for TNF-α, IL-8, and IL-12p40 in THP-1 cells (white bar) and human macrophages (black bars) in uninfected and <i>Salmonella</i>-infected cells at 6 and 24 h pi. Representative plots from three independent experiments. Statistical significance was determined as compared to the uninfected control for each strain using an unpaired <i>t</i>-test. (C) Representative maximum intensity projection images of <i>Salmonella</i> (red) infected MDMs and THP-1 cells differentiated in the presence of either 200 ng/mL PMA (2 day and 5 day) or 20 ng/mL PMA (3 day) at 1 h pi and stained for LAMP1 (green). Scale bars are 15μm and 5uM (insert). (D) Quantification of LAMP1 positive <i>Salmonella</i> in MDMs (red circles) and THP1 cells differentiated for 2 days (open circles), 3 days (grey squares), and 5 days (closed circles). Data are means ± SD from three independent experiments.</p

THP-1 surface receptor expression in cells differentiated with 20 ng/mL PMA.

<p>(A) Representative flow cytometric analysis of THP-1 cells stained using anti- CD11b or -CD14 antibodies. THP-1 cells were differentiated for 2 days (red), 3 days (blue), and 4 days (green) in the presence of PMA (20 ng/mL). (B) Quantification of mean fluorescence intensity (MFI) for CD11b (black) and CD14 (white). Data are means ± SD from three independent experiments. Statistical significance was determined using 1-way ANOVA with Dunnett post hoc test.</p