To better understand vaccine-induced protection and its potential failure in light of recent whooping cough resurgence, we evaluated quantity as well as quality of memory T cell responses in B. pertussis-vaccinated preadolescent children. Using a technique based on flow cytometry to detect proliferation, cytokine production and phenotype of antigen-specific cells, we evaluated residual T cell memory in a cohort of preadolescents who received a whole-cell pertussis (wP; n=11) or an acellular pertussis vaccine (aP; n=13) during infancy, and with a median of 4 years elapsed from the last pertussis booster vaccine, which was aP for all children. We demonstrated that B. pertussis-specific memory T cells are detectable in the majority of preadolescent children several years after vaccination. CD4(+) and CD8(+) T cell proliferation in response to pertussis toxin and/or filamentous hemagglutinin was detected in 79% and 60% of the children respectively, and interferon-γ or tumor necrosis factor-α producing CD4(+) T cells were detected in 65% and 53% of the children respectively. Phenotyping of the responding cells showed that the majority of antigen-specific cells, whether defined by proliferation or cytokine production, were CD45RA(-)CCR7(-) effector memory T cells. Although the time since the last booster vaccine was significantly longer for wP-compared to aP-vaccinated children, their proliferation capacity in response to antigenic stimulation was comparable, and more children had a detectable cytokine response after wP- compared to aP-vaccination. This study supports at the immunological level recent epidemiological studies indicating that infant vaccination with wP induces longer lasting immunity than vaccination with aP-vaccines.

BackgroundType II alveolar epithelial cells (AECII) are well known for their role in the innate immune system. More recently, it was proposed that they could play a role in the antigen presentation to T lymphocytes but contradictory results have been published both concerning their surface expressed molecules and the T lymphocyte responses in mixed lymphocyte cultures. The use of either AECII cell line or fresh cells could explain the observed discrepancies. Thus, this study aimed at defining the most relevant model of accessory antigen presenting cells by carefully comparing the two models for their expression of surface molecules necessary for efficient antigen presentation.MethodsWe have compared by flow cytometry the surface expression of the major markers involved in the immunological synapse on the A549 cell line, the most popular model of type II alveolar epithelial cells, and freshly isolated cells. HLA-DR, CD80, CD86, ICOS-L, CD54, CD58 surface expression were studied in resting conditions as well as after IFN-γ/TNF-α treatment, two inflammatory cytokines, known to modulate some of these markers.ResultsThe major difference found between the two cells types was the very low surface expression of HLA-DR on the A549 cell line compared to its constitutive expression on freshly isolated AECII. The surface expression of co-stimulatory molecules from the B7 family was very low for the CD86 (B7-2) and ICOS-L (B7-H2) and absent for CD80 (B7-1) on both freshly isolated cells and A549 cell line. Neither IFN-γ nor TNF-α could increase the expression of these classical co-stimulatory molecules. However CD54 (ICAM-1) and CD58 (LFA-3) adhesion molecules, known to be implicated in B7 independent co-stimulatory signals, were well expressed on the two cell types.ConclusionsConstitutive expression of MHC class I and II molecules as well as alternative co-stimulatory molecules by freshly isolated AECII render these cells a good model to study antigen presentation.

The treatment of latent tuberculosis infection (LTBI) in target populations is one of the current WHO strategies for preventing active tuberculosis (TB) infection and reducing theT he screening and treatment for LTBI in target populations in order to prevent TB and reduce the Mycobacterium tuberculosis reservoir are some of the main strategies of the WHO's Global Plan to Stop TB (http://www.who.int/tb/publications/global _report/en). However, a major obstacle to the instauration and effectiveness of these preventive measures resides in the lack of a gold standard LTBI screening tool.For several decades, the tuberculin skin test (TST) has been the main screening test for LTBI despite its lack of both sensitivity and specificity (1). Subsequently, T-cell-based gamma interferon release assays (IGRAs) in response to antigens encoded in the M. tuberculosis genomic region of difference 1 (RD-1) and RD-11 were developed and commercialized (QuantiFERON-TB Gold In-Tube [QFT-GIT] and T-SPOT.TB tests), with the objective of offering a more powerful diagnostic tool for LTBI. These tests offer a higher specificity than TST particularly in countries with high Mycobacterium bovis BCG vaccination coverage (2). However, recent studies suggest that these short-incubation RD-1-based IGRAs may have suboptimal sensitivities (3, 4).An alternative IGRA in response to the native mycobacterial antigen heparin-binding hemagglutinin (nHBHA-IGRA) that uses a longer incubation time than the commercialized IGRA has been validated in immunocompetent adults in the screening for LTBI (5). This assay not only demonstrates a high sensitivity and specificity for LTBI diagnosis but also a capacity to detect remote M. tuberculosis infections, a substantial advantage over the commercialized IGRAs (5-7).Remote M. tuberculosis infections are generally believed to be identified through central memory T-cell (Tcm) responses detected with long-incubation IGRAs (3, 4). Here, however, we demonstrate that both recent and remote M. tuberculosis infections can be identified through effector memory T-cell (Tem) responses using a short-incubation nHBHA-IGRA, the upgraded assay presented in this study. Our results suggest that the detection of IFN-␥-producing CD4 ϩ Tem in response to nHBHA reflects the persistence of M. tuberculosis antigens and therefore a true state of latency.(These results were presented in part at the European Congress of Immunology, Glasgow, Scotland, 5 to 8 September 2012, and at the MycoClub,

Preterm differed from term CBMC both by their proportion and phenotype of CD4(+) Treg lymphocytes and by their cytokine secretions. Maturation occurred during infancy with similar IFN-γ secretion but with persistently higher proportion of CD4(+) Treg cells in 1 year preterm infants compared to term neonates.

Based on studies reporting specific antibody titers, it is recommended to vaccinate preterm infants against Bordetella pertussis according to their chronological age. However, as specific T-cell responses also are involved in the protection against B. pertussis, we have determined whether highly preterm infants (<31 weeks) are able to mount these immune responses during vaccination. Forty-eight premature infants were vaccinated at 2, 3, and 4 months of their chronological age with an acellular (Pa; n ‫؍‬ 24) or a whole-cell (Pw; n ‫؍‬ 24) tetravalent diphtheria-tetanus-pertussis-polio vaccine, and blood samples were collected at 2, 3, and 6 months of age. Most of the Pa-and Pw-vaccinated infants developed at 3 or 6 months of age a gamma interferon (IFN-␥) response to the B. pertussis antigens, accompanied by an interleukin-5 (IL-5) and IL-13 secretion for the Pa-vaccinated infants. No association was found between a very low infant birth weight, the occurrence of severe infections, and corticosteroid treatment or the administration of gammaglobulins with a low level of antigen-induced IFN-␥ secretion. We conclude that like full-term infants, most preterm infants are able to mount a specific cellular immune response to the administration of the first doses of an acellular or a whole-cell pertussis vaccine.

Patients with TB display differential effector and regulatory T-cell responses to HBHA in local and circulating lymphocytes with a predominant effector CD4(+) and CD8(+) response locally, compared with a predominant Treg response among circulating lymphocytes. These findings may be helpful for the design of new vaccines against TB, and the detection of HBHA-specific T cells at the site of the infection may be a promising tool for the rapid diagnosis of active TB.

Tuberculosis (TB) in young children differs from adult TB in that the risk of rapid progression to active TB (aTB) is higher in children than in adults. The reasons for this increased risk are not fully understood. Early differentiation remains difficult between children at risk to develop aTB from those who will remain healthy and develop a latent TB infection (LTBI). Biomarkers to differentiate aTB from LTBI in children, especially in very young children, are urgently needed. To identify M. tuberculosis-specific functional T cell subsets related to clinical manifestations in children, we enrolled 87 children exposed to M. tuberculosis. After standard clinical assessment, the children were classified as aTB, LTBI, or uninfected. Their CD4+ T cell cytokine profiles (IFN-γ, TNF-α, IL-2, IL-17) were analyzed at the single-cell level by flow cytometry after stimulation with three mycobacterial antigens, purified protein derivative (PPD), early-secreted-antigenic target-6 (ESAT-6), or heparin-binding hemagglutinin (HBHA). This approach identified age-related discriminative markers between aTB and LTBI. Whereas among the 3- to 15-year-old children, an excellent discrimination between aTB and LTBI was provided by comparing the ratio between the proportions of ESAT-6-induced IFN-γsingle+ and ESAT-6-induced TNF-αsingle+CD4+ T lymphocytes, this was not the case for children younger than 3 years. By contrast, in this group (<3years), the analysis of HBHA-induced IL-17single+CD4+ T lymphocytes allowed us to identify children with LTBI by the high proportion of this cellular lymphocyte subset, whereas this was not the case for children with aTB. The analysis at the single-cell level of T cell immune responses induced by mycobacterial antigens are, thus, different in infected children younger or older than 3 years of age. HBHA-induced IL-17 production by CD4+ T lymphocytes was associated with protection only in children under 3 years who are at high risk for rapid progression to aTB. This suggests that the HBHA-induced IL-17 production by CD4+ T lymphocytes is a potential new correlate of protection against M. tuberculosis in humans, and that the distinction between children with LTBI and those with aTB is possible based on age-related diagnostic markers.

Induction of endoplasmic reticulum stress and activation of the intrinsic apoptotic pathway is widely believed to contribute to β-cell death in type 1 diabetes (T1D). MCL-1 is an antiapoptotic member of the BCL-2 protein family, whose depletion causes apoptosis in rodent β-cells in vitro. Importantly, decreased MCL-1 expression was observed in islets from patients with T1D. We report here that MCL-1 downregulation is associated with cytokine-mediated killing of human β-cells, a process partially prevented by MCL-1 overexpression. By generating a β-cell-specific knockout mouse strain (KO), we observed that, surprisingly, MCL-1 ablation does not affect islet development and function. β-Cells from KO mice were, however, more susceptible to cytokine-induced apoptosis. Moreover,KO mice displayed higher hyperglycemia and lower pancreatic insulin content after multiple low-dose streptozotocin treatment. We found that the kinase GSK3β, the E3 ligases MULE and βTrCP, and the deubiquitinase USP9x regulate cytokine-mediated MCL-1 protein turnover in rodent β-cells. Our results identify MCL-1 as a critical prosurvival protein for preventing β-cell death and clarify the mechanisms behind its downregulation by proinflammatory cytokines. Development of strategies to prevent MCL-1 loss in the early stages of T1D may enhance β-cell survival and thereby delay or prevent disease progression.