Journal: Arthritis

Article Title: Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes

doi: 10.1155/2017/6365857

Figure Lengend Snippet: EL4 T cells. T cells were grown in the absence (a–c) or presence (d–f) of PMA/Ionomycin stimulus. Cells were cotransfected with miR control or experimental (miRlet7f, miR-10a, miR-20b, or miR-21) and luciferase reporter vectors for detecting IL17a, TNF, and TLR10 expression or STAT3 and STAT1 activity. (a) Effect of IL-27 (50 ng/ml) on luciferase expression. (b) Effect of experimental miRNA on luciferase expression. (c) Effect of miRNA+IL27 combination on luciferase expression. Black rectangle frames (□) highlight the data with significant expression changes ( P < 0.05) relative to negative control (miR ctrl); EL4 T cells cultured in the absence (a–c) or presence of PMA/I (d–f) as per Materials and Methods. Yellow arrowheads, proposed best combinations of miR and IL-27. Color bar, range of fold change in expression, from green (−2.4- or −2.6-fold change or decrease) to red (12.0- or 16.7-fold change or increase).

Article Snippet: EL4 murine T cells were obtained from ATCC and grown on DMEM (Mediatech) with 10% horse serum and 1x antibiotic-antimycotic and Jurkat human T cells were obtained from ATCC and grown on RPMI-1640 (Mediatech) and 10% FBS with 1x antibiotic-antimycotic.

Techniques: Control, Luciferase, Expressing, Activity Assay, Negative Control, Cell Culture

Journal:

Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity

doi:

Figure Lengend Snippet: Chemotaxis of human and murine T lymphocytes to conditioned media of genetically modified DCs. (a) CEM cells. Human T lymphocyte CEM cells placed in the upper chamber of a transwell chamber were assayed for chemotaxis in response to serial dilution of supernatant from DCs transduced with AdMDC, AdNull, or PBS alone (naive control) in the lower chamber. (b) EL4 cells. The study was similar to that in a, but the murine T lymphocyte EL4 cell line was used. (c) Suppression of AdMDC-mediated chemotaxis. CEM and EL4 cells were assayed for chemotaxis in response to 1:2 dilution of supernatant from AdMDC-modified DCs. Where indicated, anti–MDC neutralizing Ab or mouse IgG control Ab was added to the supernatant in the lower chambers at 10 μg/ml at the initiation of assay or cells were placed to the upper chamber in the presence of recombinant (r) MDC or TARC at 100 ng/ml. For all parts, the number of cells migrating to the lower chamber at 37°C for 4 hours was counted by FACS analysis. Migration index was calculated as the number of cells migrating to the conditioned media over the number of cells migrating to control medium. Results represent the mean ± SE (n = 3 per data point).

Article Snippet: To demonstrate the function of the MDC protein expressed by the AdMDC-modified DCs, human T lymphocyte CEM cells or murine T lymphocyte EL4 cells (no. CCL-119 and TIB-39, respectively; American Type Culture Collection, Manassas, Virginia, USA) were suspended in 100 μl of RPMI-1640 media containing 1 mg/ml BSA at 10 7 cells/ml and loaded to the upper wells of 5-μm pore, polycarbonate, transwell chambers in 24-well plates (Corning Costar Inc., Corning, New York, USA).

Techniques: Chemotaxis Assay, Genetically Modified, Serial Dilution, Transduction, Control, Modification, Recombinant, Migration

Journal: Nature Communications

Article Title: PD-L1- and IL-4-expressing basophils promote pathogenic accumulation of T follicular helper cells in lupus

doi: 10.1038/s41467-024-47691-w

Figure Lengend Snippet: a Flow cytometry gating strategy used to identify human circulating T follicular helper T cells (cTFH) defined as CD3 + CD8α – CD4 + CXCR5 + ICOS + PD-1 + . Among cTFH cells, cTFH1 cells were defined as CXCR3 + CCR6 – , cTFH2 as CXCR3 – CCR6 – , and cTFH17 as CXCR3 – CCR6 + . b Proportions (%) among CD4 + T cells of cTFH as defined in ( a ) in blood from healthy controls (CT) and inactive (inact.), mild, or active SLE patients ( n = 16/11/12/21) as determined by flow cytometry. Proportions (%) among CD4 + T cells of cTFH2 ( c ), cTFH1 ( d ) and cTFH17 ( e ) as defined in ( a ) in blood from healthy controls and inactive, mild, or active SLE patients ( n = 16/10/12/20) as determined by flow cytometry. f Correlation (and linear regression with 95% confidence intervals) between Log (cTFH2 cell numbers per mL of blood) and SLEDAI (Spearman r = 0.3682, p = 0.02, n = 37). g Left, Histogram plot representing the CD203c expression levels on blood basophils from a healthy control (CT, black), a patient with active SLE (red), and isotype control staining (gray filled). Right, CD203c expression levels on blood basophils from CT and inact, mild or active SLE patients ( n = 43/61/46/98) as determined by flow cytometry. h Left, Representative histogram plot of PD-L1 expression levels on blood basophils as in ( g ). Right, PD-L1 expression levels on blood basophils from CT and inact, mild or active SLE patients ( n = 39/60/45/96) as determined by flow cytometry. ( b – e , g , h ) Data are presented as violin plots with median (solid line) and quartiles (dotted lines). Statistical analyses were Kruskal–Wallis tests followed by Dunn’s multiple comparisons tests and p values are shown above each bracket. NS not significant. A.U. arbitrary units. Source data are provided in the file.

Article Snippet: For basophil/CD4 + T cell mouse co-culture experiments, F(ab’)2 anti-CD3 (clone145-2C11 at 0,5 µg/mL) and anti-CD28 (clone PV-1 at 0,5 µg/mL) antibodies (BioXcell) were coated overnight at 4 °C in culture 96 well plates (Costar) in sterile-filtered coating buffer (20 mM carbonate buffer (pH 9.6) containing 2 mM MgCl 2 and 0.01% NaN 3 ).

Techniques: Flow Cytometry, Expressing, Staining

Journal: Nature Communications

Article Title: PD-L1- and IL-4-expressing basophils promote pathogenic accumulation of T follicular helper cells in lupus

doi: 10.1038/s41467-024-47691-w

Figure Lengend Snippet: a – g CD3/CD28-stimulated wild-type (WT) naïve CD4 + T cells cultured for three days without (–, gray) or with basophils from Mcpt8 CT/+ (WT) (blue), Mcpt8 CT/+ Il4 fl/fl (red), Mcpt8 CT/+ Il6 fl/fl (orange), or Mcpt8 CT/+ Pdl1 fl/fl (green) mice. a Proportions (%) of TFH cells among CD4 + T cells ( n = 17/31/12/12/15). Proportions (%) of IL-21- ( b ), IL-6- ( c ), IL-4- ( d ), and IL-13- ( e ) producing cells among TFH cells non-restimulated ( n = 11/16/5/10/8). f ( Top ) Proportions (%) of TFH cells among CD4 + T cells ( n = 10/9/20/9). Bottom RT-qPCR done for the indicated targets on resorted CD4 + T cells. Relative results are presented with the color scale indicated from 0% (blue, the least abundant) to 100% (red, the most abundant). g Ratio of Bcl6 on Bach2 mRNA ( Top ) ( n = 10/5/17/8) and of Batf on Bach2 mRNA ( Bottom ) ( n = 13/5/17/8) as described in ( f ). h Top Proportions (%) of TFH cells among CD4 + T cells cultured without basophils for three days either alone (–, gray) or with coated anti-PD-1 antibody (red) or soluble IL-4 (green) or both (blue) ( n = 10/12/12/8). Bottom. Relative mRNA expression of the indicated targets as in ( f ). i Ratio of Bcl6 on Bach2 mRNA ( Top ) ( n = 10/5/4/6) and of Batf on Bach2 mRNA ( Bottom ) ( n = 13/5/4/6) on samples described in ( h ). b – e Results for PMA/Ionomycin restimulated cells are shown in Supplementary Fig. . a – i Results are from at least three independent experiments and presented as individual values in bars representing the mean ± s.e.m. Statistical analyses were done by Kruskal–Wallis test followed by Dunn’s multiple comparisons tests ( a ) or by one way ANOVA followed by Tukey’s multiple comparisons tests ( b – i ) between the indicated groups. P values are shown above each bracket. NS not significant. A.U. arbitrary units. Source data are provided in the file.

Article Snippet: For basophil/CD4 + T cell mouse co-culture experiments, F(ab’)2 anti-CD3 (clone145-2C11 at 0,5 µg/mL) and anti-CD28 (clone PV-1 at 0,5 µg/mL) antibodies (BioXcell) were coated overnight at 4 °C in culture 96 well plates (Costar) in sterile-filtered coating buffer (20 mM carbonate buffer (pH 9.6) containing 2 mM MgCl 2 and 0.01% NaN 3 ).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: PD-L1- and IL-4-expressing basophils promote pathogenic accumulation of T follicular helper cells in lupus

doi: 10.1038/s41467-024-47691-w

Figure Lengend Snippet: a Histograms of PD-L1 expression on basophils cultured without (dotted) or with (solid) activated naïve CD4 + T cells as in Fig. (gray filled: isotype control staining). b PD-L1 expression levels on basophils cultured without (–, lighter colors) or with (+, darker colors) activated naïve CD4 + T cells ( n = 14/31/5/12/6/12/5/12). c Left. Intracellular IL-3 staining in WT CD3/CD28-activated naïve CD4 + T cells after 3 days of culture (red line) (gray filled: isotype control staining). Right. Proportions (%) of IL-3 + cells among CD4 + T cells non-restimulated in the same samples as in Fig. ( n = 6/9/16/9). d PD-L1 expression induction on WT basophils after stimulation of splenocytes with 1 ng/mL of IL-4 (red), of IL-3 (blue), both (purple), 100 ng/mL of anti-IgE without (light green) or with (green) IL-3 for 20 h ( n = 8/8/8/8/4/4) (normalized to unstimulated conditions mean value). e Contour plots of IL-6 and IL-4 spontaneous production by basophils of the indicated genotype after co-culture without (–) or with activated CD4 + T cells. Proportions (%) of spontaneous IL-4 + ( f ) and IL-6 + ( g ) basophils of the indicated genotypes co-cultured (+) or not (–) with WT CD3/CD28-activated naïve CD4 + T cells ( n = 11/16/3/5/6/8/4/8). f , g Results for IL-13 and PMA/Ionomycin restimulated cells are shown in Supplementary Fig. . a – g Results are from at least three independent experiments and presented as individual values in bars representing the mean ± s.e.m. b – d , f , g Statistical analyses were by one way ANOVA followed by Tukey’s multiple comparisons tests between the indicated groups. P values are shown above each bracket. NS not significant. A.U. arbitrary units. Source data are provided in the file.

Article Snippet: For basophil/CD4 + T cell mouse co-culture experiments, F(ab’)2 anti-CD3 (clone145-2C11 at 0,5 µg/mL) and anti-CD28 (clone PV-1 at 0,5 µg/mL) antibodies (BioXcell) were coated overnight at 4 °C in culture 96 well plates (Costar) in sterile-filtered coating buffer (20 mM carbonate buffer (pH 9.6) containing 2 mM MgCl 2 and 0.01% NaN 3 ).

Techniques: Expressing, Cell Culture, Staining, Co-Culture Assay

Journal: Nature Communications

Article Title: PD-L1- and IL-4-expressing basophils promote pathogenic accumulation of T follicular helper cells in lupus

doi: 10.1038/s41467-024-47691-w

Figure Lengend Snippet: Contour plots showing CD3/CD28-activated human CD4 + T cells cultured for three days without ( a ) or with ( b ) purified human basophils at a 5:1 ratio (left) and the condition-induced TFH differentiation of the CD4 + T cells (right). Basophils were defined as FcεRIα + CRTH2 + CCR3 + cells and TFH cells were defined as CD4 + PD-1 + CXCR5 + ICOS + cells. c Proportions (%) of TFH cells among CD3/CD28-activated CD4 + T cells cultured without (0:1) or with the indicated ratio of purified human basophils ( n = 4 per group). d Proportions (%) of TFH cells among CD3/CD28-activated CD4 + T cells cultured without or with purified human basophils at a ratio of 1:5 in the absence (–) (blue) or presence of antibodies blocking IL-4 (αIL-4) (red), IL-6 (αIL-6) (orange) or PD-1 (αPD-1) (green) or the corresponding isotype controls (Iso) (blue) ( n = 4/3/6/6/6/6/5/5/8/8/8/8). e Contour plots showing subsets of TFH cells as defined in Fig. on cells as in ( a , b ). f Proportions (%) of TFH2 cells among TFH cells after culture as described in ( d ) ( n = 5/5/8/8/8/8). TFH2 cells were defined as CD4 + PD-1 + CXCR5 + ICOS + CCR6 – CXCR3 – cells. c , d , f Data are presented as individual values in bars representing the mean values ± s.e.m. c One representative experiment out of two with cells from 4 different donors is shown. d , f Results are from three independent experiments. c , d , f Statistical analyses were done by one-way ANOVA test followed by Tukey’s multiple comparisons tests between the indicated groups. P values are shown above each bracket. NS not significant. Source data are provided in the file.

Article Snippet: For basophil/CD4 + T cell mouse co-culture experiments, F(ab’)2 anti-CD3 (clone145-2C11 at 0,5 µg/mL) and anti-CD28 (clone PV-1 at 0,5 µg/mL) antibodies (BioXcell) were coated overnight at 4 °C in culture 96 well plates (Costar) in sterile-filtered coating buffer (20 mM carbonate buffer (pH 9.6) containing 2 mM MgCl 2 and 0.01% NaN 3 ).

Techniques: Cell Culture, Purification, Blocking Assay