Béatrice Corre

T cell receptor for antigen induces linker for activation of T cell–dependent activation of a negative signaling complex involving Dok-2, SHIP-1, and Grb-2

Adaptor proteins positively or negatively regulate the T cell receptor for antigen (TCR) signaling cascade. We report that after TCR stimulation, the inhibitory adaptor downstream of kinase (Dok)-2 and its homologue Dok-1 are involved in a multimolecular complex including the lipid phosphatase Src homology 2 domain–containing inositol polyphosphate 5′-phosphatase (SHIP)-1 and Grb-2 which interacts with the membrane signaling scaffold linker for activation of T cells (LAT). Knockdown of LAT and SHIP-1 expression indicated that SHIP-1 favored recruitment of Dok-2 to LAT. Knockdown of Dok-2 and Dok-1 revealed their negative control on Akt and, unexpectedly, on Zap-70 activation. Our findings support the view that Dok-1 and -2 are critical elements of a LAT-dependent negative feedback loop that attenuates early TCR signal. Dok-1 and -2 may therefore exert a critical role in shaping the immune response and as gatekeepers for T cell tolerance.

Frequencies of Ex Vivo-Activated Human Immunodeficiency Virus Type 1-Specific Gamma-Interferon-Producing CD8+ T Cells in Infected Children Correlate Positively with Plasma Viral Load

ABSTRACT HIV-specific CD8+ T cells are critical in controlling human immunodeficiency virus (HIV) replication. We present the evaluation of a gamma-interferon (IFN-γ)-based enzyme linked immunospot (ELISPOT) assay for the quantification of HIV-specific CD8+ T cells from HIV-infected children. We studied 20 HLA-A∗0201-positive HIV-infected children. The IFN-γ production in response to stimulation with two HLA-A∗0201-restricted immunodominant CD8 epitopes (SLYNTVATL [SL9] in Gag and ILKEPVHGV [IV9] in Pol) was tested using the ELISPOT assay. The results were compared to labeling with the corresponding tetramers. Among the 20 children, 18 had detectable responses against the SL9 and/or the IV9 epitope using the ELISPOT assay (medians, 351 and 134 spot-forming cells/106 peripheral blood mononuclear cells, respectively). Comparison of results from the tetramer and ELISPOT assays suggests that only a fraction of HIV-specific CD8+ T cells were able to produce IFN-γ. Most importantly, we found that the frequencies of IFN-γ-producing CD8+ T cells were positively correlated with the viral load whereas the frequencies of tetramer-binding CD8+ T cells were not. The high sensitivity of the ELISPOT assay and the fact that this functional assay provided information different from that of tetramer labeling support its use for measurement of HIV-specific CD8+ T cells. In conclusion, our results show that the ex vivo-activated IFN-γ-producing HIV-specific CD8+-T-cell subset is dependent upon continuous antigenic stimulation.

Functional characterization of naturally occurring genetic variants in the human TLR1-2-6 gene family†

Toll‐like receptors (TLRs) are considered an essential component of the innate immune system, initiating inflammatory responses following infection of the host. Humans have 10 functional TLRs, differing in their subcellular distributions and the microbial agonists they sense. The phylogenetically conserved TLR1‐2‐6 family is unique in that TLR1 and TLR6 form heterodimers with TLR2 to mediate signalling in response to agonists. Epidemiological genetic studies have identified several TLR variants that appear to influence susceptibility to infectious diseases, but the functional consequences of which remain largely unknown. Here, we assessed the functional impact of the TLR1‐2‐6 variants with altered amino acid sequences segregating naturally in the human population. We used an NF‐κB reporter assay in TLR‐transfected human embryonic kidney 293T cells stimulated with the corresponding TLR agonists. We found that among the 41 naturally occurring variants with amino acid alterations identified in the TLR1‐2‐6 family, 14 of them (five TLR1, four TLR2, and five TLR6 variants) displayed marked impairment of NF‐κB activation. Most of these variants are present at very low population frequencies and are population‐specific. These observations suggest that rare, nonsynonymous TLR mutations are likely to have deleterious effects on immune responses and may therefore contribute to complex susceptibility to infection at the population level. Hum Mutat 32:643–652, 2011.

T cell receptor signal initiation induced by low-grade stimulation requires the cooperation of LAT in human T cells.

Background One of the earliest activation events following stimulation of the T cell receptor (TCR) is the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3-associated complex by the Src family kinase Lck. There is accumulating evidence that a large pool of Lck is constitutively active in T cells but how the TCR is connected to Lck and to the downstream signaling cascade remains elusive. Methodology/Principal Findings We have analyzed the phosphorylation state of Lck and Fyn and TCR signaling in human naïve CD4+ T cells and in the transformed T cell line, Hut-78. The latter has been shown to be similar to primary T cells in TCR-inducible phosphorylations and can be highly knocked down by RNA interference. In both T cell types, basal phosphorylation of Lck and Fyn on their activatory tyrosine was observed, although this was much less pronounced in Hut-78 cells. TCR stimulation led to the co-precipitation of Lck with the transmembrane adaptor protein LAT (linker for activation of T cells), Erk-mediated phosphorylation of Lck and no detectable dephosphorylation of Lck inhibitory tyrosine. Strikingly, upon LAT knockdown in Hut-78 cells, we found that LAT promoted TCR-induced phosphorylation of Lck and Fyn activatory tyrosines, TCRζ chain phosphorylation and Zap-70 activation. Notably, LAT regulated these events at low strength of TCR engagement. Conclusions/Significance Our results indicate for the first time that LAT promotes TCR signal initiation and suggest that this adaptor may contribute to maintain active Lck in proximity of their substrates.

Not all tetramer binding CD8+ T cells can produce cytokines and chemokines involved in the effector functions of virus-specific CD8+ T lymphocytes in HIV-1 infected children.

In the pediatric human immunodeficiency virus type-1 (HIV-1) infection, the presence of cytotoxic T lymphocytes (CTL) is associated with a slow progression to AIDS. The secretion of cytokines by CTLs may be critical in the control of viral infection. We used the combination of cell surface and intracellular staining to study the functionality of tetramer binding CD8+ T cells recognizing two HIV-1 immunodominant epitopes, in peripheral blood mononuclear cells from HIV-1-infected children. A fraction of tetramer positive CD8+ T cells produce cytokines (IFN-γ, TNF-α) or chemokines (CCL4, CCL5) after ex vivo stimulation with the cognate peptide. There was a negative correlation between the plasma viral load and the percentage of CD8+ Tetramer Gag+ T cells secreting IFN-γ. This is the first report in the context of pediatric HIV-1 infection showing that only a fraction of HIV-1-specific CD8+ T cells have the capacity to produce cytokines and chemokines implicated in their antiviral functions.

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4+ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA+CD4+ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10+ Foxp3− IFN‐γ− and favored IL‐10 expression in a fraction of Foxp3+ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.

Expansion of HBV-specific memory CTL primed by dual HIV/HBV genetic immunization during SHIV primary infection in rhesus macaques.

We have previously shown the induction of humoral and cytotoxic responses specific for human immunodeficiency virus (HIV) and hepatitis B virus (HBV) antigens, following genetic immunization of rhesus macaques with a plasmid encoding both the third variable domain of the HIV-1 external envelope glycoprotein and the pseudo-viral particle of hepatitis B surface antigen (HBsAg) as presenting molecules. The DNA-immunized primates and two control animals were then challenged with a chimeric simian/human immunodeficiency virus (SHIV). They were all infected. Significant frequencies of SHIV specific cytotoxic T lymphocyte precursors (CTLp) were detected early in peripheral blood. But, in all DNA-immunized macaques, HBV envelope specific CTLp were detected during the primary infection, and they were correlated with the peak of SHIV viremia. Furthermore, HBV or SHIV specific cytotoxicity corresponded in part to CD8(+) T cells presenting a memory phenotype. Several mechanisms could account for this cellular response. But our results suggest that an expansion of memory cytotoxic CD8(+) cells, not restricted to SHIV specific effectors, could occur in peripheral blood during SHIV primary infection.

Type I interferon-enhanced IL-10 expression in human CD4 T cells is regulated by STAT3, STAT2, and BATF transcription factors

Type I IFN can exert pro‐ and anti‐inflammatory activities in the immune system. Here, we have investigated the mechanism by which IFN‐α enhances early expression of the anti‐inflammatory cytokine IL‐10 in human CD45RA+CD4+ T cells. With the use of transcriptomic and biochemical approaches, we found distinct and combined contributions of the IFN and the TCR signaling pathways to the induction of STAT1/2/3 and the basic leucine zipper activating transcription factor‐like (BATF) family members. Moreover, IFN‐induced STAT3 phosphorylation was prolonged by the TCR response, whereas IFN‐induced STAT2 phosphorylation was of long duration. With the use of RNA interference (RNAi), we identified STAT3 as the major actor and STAT2 as a contributor of the IFN action on IL‐10. Upon TCR/IFN costimulation, STAT3 directly bound at the IL‐10 conserved noncoding sequence (CNS)‐ 9, an enhancer element known to recruit BATF in CD4 T cells. The cosilencing of the 3 BATFs resulted in an overall reduction of IL‐10 expression, but the promoting activity of IFN‐α was retained. These results support the notion that the IFN action is indexed on BATF function and provide evidence for a cooperation between BATFs and STAT3, the latter being activated via early IFN and delayed TCR effects.