Caractéristation des lymphocytes T auxiliaires impliqués dans la régulation de la réponse humorale

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Interleukin-6/STAT3 signaling regulates the ability of naive T cells to acquire B-cell help capacities

The conditions leading to the activation/differentiation of T-helper (Th) cells dedicated for B-cell antibody production are still poorly characterized. We now demonstrate that interleukin-6 (IL-6) promotes the differentiation of naive T lymphocytes into helper cells able to promote B-cell activation and antibody secretion. IL-6–driven acquisition of B-cell help capacity requires expression of the signal transducer and activator of transcription 3 (STAT3), but not STAT4 or STAT6 transcription factors, suggesting that the ability to provide help to B cells is not restricted to a well-defined Th1 or Th2 effector population. T cell–specific STAT3-deficient mice displayed reduced humoral responses in vivo that could not be related to an altered expansion of CXCR5-expressing helper T cells. IL-6 was shown to promote IL-21 secretion, a cytokine that was similarly found to promote the differentiation of naive T cells into potent B-cell helper cells. Collectively, these data indicate that the ability to provide B-cell help is regulated by IL-6/IL-21 through STAT3 activation, independently of Th1, Th2, Th17, or follicular helper T cell (TFH) differentiation

STAT3 signaling induces the differentiation of human ICOS(+) CD4 T cells helping B lymphocytes.

The generation of high-affinity antibodies and the development of B cell memory are dependent on the help provided by CD4 T cells. Mouse studies indicate that STAT3 signaling in CD4 T cells promotes the acquisition of the B cell help function. However, the role of STAT3 in humans has been controversial. In this study, we show that IL-6 and other STAT3 activating cytokines (IL-21 and IL-27) induce the differentiation of CD4 T cells promoting antibody production by B cells. The acquisition of B cell stimulating properties by naive cord blood CD4 T cells required the STAT3-dependent expression of ICOS and IL-21. Gene reporter and ChIP experiments unambiguously demonstrated that upon IL-6 stimulation, STAT3 induces the transcription of the ICOS gene through direct recruitment to the proximal promoter region indicating that STAT3 acts in part through the direct activation of the ICOS gene.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

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

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

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