Mechanisms of memory CD8 T reactivation and orchestration of protective immune responses.

<p>Upon contact with microbial pathogen, different myeloid subpopulations may rapidly activate memory CD8 T cells through cytokinic and antigenic signals (Phase 1). In turn, memory CD8 T cells produce various cytokines and chemokines (IFNγ, CCL3) that allow initial recruitment and licencing of innate immune cells (Phase 2). Myeloid cells further amplify recruitment (CXCL9/10) of more memory T and innate effectors cells leading to pathogen containement and protective immunity (Phase 3).</p

Pathway of conventional and unconventional CD8 T cell memory differentiation.

<p>Naïve CD8 T cells undergoing cognate antigen recognition in the context of an infection or an immunization differentiate into effector cells and form “true” antigen-experienced memory cells or "conventional memory." Under physiological conditions, naïve CD8 T cells may also acquire a memory phenotype in the absence of non-self cognate antigenic stimulation. This may occur in the thymus or in the periphery under the control of cytokines such as IL-4, IL-15, and type I IFN and give rise to “virtual memory” or "innate/memory-like" CD8 T cells.</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

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 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

Age-dependent upregulation of CpG-induced production of IP-10 and MIG.

<p>Whole blood samples were stimulated with CpG A+B combination and culture supernatants were collected after 16–18 h. Production in the different infant groups was compared to adult values. Cord blood (CB, n = 13), 3-month (3 m, n = 10), 6&9-month (6 m, n = 3, 9 m, n = 5), 12-month (12 m, n = 11) old infants and healthy adults (n = 10). Data are represented as median+interquartile range. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 as compared to stimulated adult samples.</p

Decreased production of IL-12p70 and IL-23 in response to LPS+R848 stimulation in frail old individuals.

<p>Whole blood cells from frail (n = 10) and non frail (n = 9) old individuals were stimulated with LPS+R848 during 18 h. IL-12p70 and IL-23 were measured in cell-free supernatants by ELISA. Each dot represents a single donor and the bars represent median values ± interquartile range. ***p<0.001.</p

HLA DR expression on the surface of circulating monocytes, myeloid and plasmacytoid DCs.

<p>Blood samples were incubated with PBS or the indicated stimulant. The different subpopulations were gated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010407#s2" target="_blank">Material and Methods</a> section. Expression was compared in samples from the different age groups: Cord blood (CB, n = 10), 3-month (3 m, n = 10), 6&9-month (6–9 m pooled data from n = 4 and 8, respectively), 12-month (12 m, n = 9) old infants and healthy adults (n = 16). Data are represented as median+interquartile range. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 as compared to stimulated adult samples.</p

CD80 expression on the surface of circulating monocytes, myeloid and plasmacytoid DCs.

<p>The different subpopulations were gated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010407#s2" target="_blank">Material and Methods</a> section. Expression was compared in samples from the different age groups: Cord blood (CB, n = 10), 3-month (3 m, n = 10), 6 and 9-month (6–9 m, pooled data from n = 4 and 8, respectively), 12-month (12 m, n = 9) old infants and healthy adults (n = 16). Data are represented as median+interquartile range. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p