Tfh-like cells express MyoR mRNA.

<p>Naive CD62L⁺CD4⁺ T cells purified from the spleen of C57BL/6 mice were stimulated with plastic-coated anti-CD3 and anti-CD28 mAbs under neutral conditions (medium) or in the presence of IL-6 (Tfh-like condition). Expression level of the indicated genes was assessed by quantitative RT-PCR and expressed as relative expression to RPL32 mRNA. (A) Kinetic expression of MyoR under Th0 and Tfh culture conditions; (B) Compilation of individual experiments showing increased MyoR expression in 72 h cultured-Tfh-like cells; (C) Expression of a set of Tfh-associated genes in 72 h-cultured cells in the presence of IL-6; (D) MyoR expression in resting versus TcR activated, IL-6-treated T lymphocytes; (E) Expression of MyoR in 48 h-Tfh-like activated wild type and STAT3-deficient T cells; (F) MyoR, T-bet, GATA-3 and RORγT mRNA expression in 72 h-polarized Th0, Th1, Th2, Th17 and Tfh-like cells. The 72 h activated-Th0 condition (48 h in panel E) was set to 1. Histograms represent the mean ± SD of duplicates and are representative of three independent experiments (A, C-E and F-panels T-bet, GATA-3, RORγT) or the mean ± SD of three independent experiments (F-panel MyoR). Dots in panel B represent individual paired Th0 and Tfh cultures data sets. Differences between groups in B were analyzed with the Mann-Whitney test for 2-tailed data. * p<0.05; n.d. =  not detectable</p

MyoR mRNA is expressed in Tfh cells induced <i>in vivo</i>.

<p>Tfh and non Tfh cells from NP-KLH/Alum immunized mice were tested for relative MyoR mRNA expression. (A) Representative flow cytometric plot showing the percentage of Tfh cells in non-immune and NP-KLH/Alum treated mice and the gating strategy for sorted Tfh and non-Tfh cells from immune mice on day 7. (B) Relative expression of MyoR and selected genes in sorted Tfh and non Tfh cells. Histograms represent the mean ± SD of duplicates and are representative of three independent experiments.</p

Myor/ABF-1 Mrna Expression Marks Follicular Helper T Cells but Is Dispensable for Tfh Cell Differentiation and Function <i>In Vivo</i>

<div><p>Follicular T helper cells (Tfh) are crucial for effective antibody responses and long term T cell-dependent humoral immunity. Although many studies are devoted to this novel T helper cell population, the molecular mechanisms governing Tfh cell differentiation have yet to be characterized. MyoR/ABF-1 is a basic helix-loop-helix transcription factor that plays a role in the differentiation of the skeletal muscle and Hodgkin lymphoma. Here we show that MyoR mRNA is progressively induced during the course of Tfh-like cell differentiation <i>in vitro</i> and is expressed in Tfh responding to Alum-precipitated antigens <i>in vivo</i>. This expression pattern suggests that MyoR could play a role in the differentiation and/or function of Tfh cells. We tested this hypothesis using MyoR-deficient mice and found this deficiency had no impact on Tfh differentiation. Hence, MyoR-deficient mice developed optimal T-dependent humoral responses to Alum-precipitated antigens. In conclusion, MyoR is a transcription factor selectively up-regulated in CD4 T cells during Tfh cell differentiation <i>in vitro</i> and upon response to alum-protein vaccines <i>in vivo</i>, but the functional significance of this up-regulation remains uncertain.</p></div

MyoR is dispensable for T-dependent humoral responses.

<p>(A–F) WT and MyoR^−/− mice were immunized against NP-KLH/Alum (A–D) or KLH/Alum (E, F) and tested for Tfh cell marker expression in the draining lymph nodes. Representative contour plots showed the expression of CXCR5⁺PD-1⁺ (A), CXCR5⁺ICOS⁺ (C) or CXCR5⁺ BCL-6 ⁺ (E) among CD4⁺ T cells. Histograms in (B, D, F) represent the mean ± SD of marker-positive cells from 4 individual mice and are representative of 3 independent experiments. (G) Aliquots of cells were stimulated for 4 h with PMA and ionomycin and tested for IL-21 production by intracellular staining. Histograms represent the mean ± SD of IL-21⁺ cells in the Tfh (CXCR5⁺PD-1⁺) and non-Tfh (CXCR5⁻PD-1⁻) subsets of WT and MyoR^−/− mice (4 mice/group). (H) NP-specific serum IgG1 titers of WT (open symbols) and MyoR^−/− (closed symbols) mice were determined at different timings after i.p. immunization with NP-KLH/Alum. (I) sera from panel H were tested for NP-affinity. Relative affinities are expressed as ratio of 50% binding on NP₂-BSA and NP₃₀-BSA- coated plates. Each dot represents a mouse. Data are representative of two independent experiments. *: p<0.05; **: p<0.01; ns, not significant; n.i., non immune mouse.</p

STAT3 promotes <i>ICOS</i> transcription through direct interaction with the STAT#1 binding site.

<p>A) Naive cord blood CD4 T cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence or absence of rIL-6 before measuring ICOS mRNA expression by qRT-PCR. Data are one representative out of 3 experiments on different donors. B) EL4 cells were co-transfected with the (−684/+20) ICOS reporter construct containing a luciferase element and STAT3C or control plasmids. Data are mean ± SEM of triplicates of one experiment out of 5 independent experiments. C) EL4 cells were co-transfected with the indicated reporter plasmid and STAT3C. Twenty-four hours after transfection, cells were incubated with rIL-6 or medium alone for an additional 24 hours before measuring luciferase activity. Data were normalized against unstimulated conditions for each construct and are mean ± SEM of triplicates of 4 independent experiments. D) EL4 cells were co-transfected with the (−174/+20) WT or mutated ICOS reporter construct and STAT3C. Cells were then incubated with rIL-6 or medium alone for an additional 24 hours before measuring luciferase activity. Data are mean ± SEM of triplicates of 4 independent experiments. The sequences of the STAT#1 binding site (nt −57/−43) and the mutation introduced in (−174/+20) MUT constructs are depicted. E and F) ChIP experiments. Naive cord blood CD4 T cells were stimulated with anti-CD3 mAb in the presence of rIL-6 or rIL-21. Chromatin samples were immunoprecipitated with anti-STAT3 or control antibodies. Purified DNA samples were subjected to qPCR amplification using primers encompassing the STAT#1 site from the ICOS promoter or specific for the proximal GAPDH promoter region. Data are mean ± SEM of triplicates of one representative out of two experiments on different donors. R.U.: relative units.</p

Induction of IL-21 and ICOS expression by IL-6 is STAT3-dependent.

<p>Naive cord blood CD4 T cells were transfected with STAT3 specific or control siRNAs in the presence of IL-2. After 48 h, cells were stimulated for 72 hours with plate bound anti-CD3 (5 µg/ml) and soluble anti-CD28 (1 µg/ml) mAbs in the presence of rIL-6 or medium alone before measuring IL-21 production by qRT-PCR (A), membrane expression of ICOS by flow cytometry (A and B), membrane expression of CD69 by flow cytometry and IFN-γ production by ELISA (D). Data are individual results or mean ± SEM of 3 independent experiments on different donors. R.U.: relative units. *p<0.05 as determined by Student's t-test.</p

STAT3 is critical for the differentiation of CD4 T cells helping B cells induced by IL-6.

<p>A) Naive cord blood CD4 T cells were stimulated with the indicated cytokines before measuring phospho (p)STAT3 expression by flow cytometry. B and C) Naive cord blood CD4 T cells were incubated with STAT3 specific or control siRNAs in the presence of IL-2 for 48 hours. Transfected cells were stimulated for an additional 48 hours with plate-bound anti-CD3 (5 µg/ml) and soluble anti-CD28 (1 µg/ml) mAbs in the presence of rIL-6 or medium alone. We then assessed the expression of STAT3 mRNA by qRT-PCR and of pSTAT3 by flow cytometry. D) CD4 T cells were incubated in the presence of heterologous B cells before measuring the production of Ig as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071029#pone-0071029-g001" target="_blank">Fig. 1</a>. Data are individual results or mean ± SEM of one representative of two experiments on different donors.</p

IL-6, IL-12 and IL-27 promote the differentiation of CD4 T cells helping B cells.

<p>A) Naive cord blood CD4 T cells were primed with plate bound anti-CD3 (5 µg/ml) and soluble anti-CD28 (1 µg/ml) mAbs during 72 hours in the presence of supernatant from immature moDCs activated with LPS, Poly(I∶C) or incubated with medium alone. T cells were then thoroughly washed before T/B co-culture to avoid potential carry-over effect of the DC supernatants and were incubated with anti-CD3 mAb and heterologous B cells before measuring Ig production. B) Experiments were performed as in (A) except that anti-IL-6, anti-IL-12 or control mAbs (10 µg/ml) were added to DC culture supernatants before CD4 T cell priming. C) Experiments were performed as in (A) except that recombinant cytokines were used instead of DC culture supernatants. D) Experiments were performed as in (C) except that TSST was used in the T/B co-culture instead of anti-CD3 mAb. Data are mean ± SEM of triplicates from one representation of 3 (A), of 4 (B) or 6 (C and D) independent experiments on different donors. FI/None: fold increase as compared to no cytokine. *p<0.05, **p<0.01 and ***p<0.001 as determined by paired Wilcoxon signed-rank test (A, C and D) or paired Student's t-test (B).</p

IL-17A deficiency does not prevent obliterative airway disease.

<p>Fully allogeneic BALB/C tracheas were heterotopically grafted into WT or IL-17A^-/- C57BL/6 recipients and harvested after 28 days. <b>A</b>, pathologic scores. <b>B</b> & <b>C</b>, histologic analysis of allografts harvested from one representative WT and IL-17A^-/- recipient, respectively. Masson’s trichrome staining of a whole section (magnification x40). <b>D</b>, percentages of lumenal fibrosis. The bars represent the mean ± SEM of 12-15 organs in each group. Data are pooled from three independent experiments.</p

APO866 reduces intracellular NAD in PEC <i>in vivo</i> and inhibits TNFα production after LPS challenge.

<p>(a) Mice were treated with thioglycollate to elicit PEC, and then received 10 mg/kg APO866 by ip injection. PEC were obtained by lavage after different time points and intracellular NAD was determined. Data are mean+sem of 3 mice per group. (b) Mice were treated with thioglycollate to elicit PEC, and then received 10 mg/kg APO866 or placebo by ip injection 7 h before ip challenge with LPS. Serum TNFα at 90 min (mean+sem of 3 mice per group is shown. PEC were obtained by lavage and intracellular NAD was determined. Data are mean+sem of 3 mice per group. This panel is representative of at least 4 experiments performed. <i>P</i><0.05 9 h versus 0 h in panel a, or APO866 versus placebo in panel b.</p