Dengue is a member of the Flaviviridae, a large group of related viruses some of which co-circulate in certain regions (e.g. dengue and Yellow fever in South America). Immune responses cross-reactive between different dengue serotypes are important in the pathogenesis of dengue disease but it is not known whether previous infection with one flavivirus might affect the clinical course of subsequent infections with other members of the family. CD4+ T cells have been shown to be important in the production of cytokines in response to dengue infection and can demonstrate significant epitope cross-reactivity. Here, we describe the generation and characterisation of CD4+ T cell clones from a patient experiencing acute dengue infection. These clones were DRB1 *15+ and recognised epitope variants not only within other dengue viruses but certain other flaviviruses. This cross-reactivity was dependent upon the presence of a five-amino acid core region, consistent with structural observations of class II MHC binding to TCR demonstrating that only a subset of residues within an epitope bound to a class II molecule are “read out” by the TCR. This capacity of CD4+ T cell clones to recognise a given epitope despite considerable variation between viruses may be of pathological significance, particularly in regions where related viruses co-circulate.
Dengue; Epitope; T cell
The 2009/10 pandemic (pH1N1) highlighted the need for vaccines conferring heterosubtypic immunity against antigenically shifted influenza strains. Although cross-reactive T cells are strong candidates for mediating heterosubtypic immunity, little is known about the population-level prevalence, frequency, and cytokine-secretion profile of heterosubtypic T cells to pH1N1. To assess this, pH1N1 sero-negative adults were recruited. Single-cell IFN-γ and IL-2 cytokine-secretion profiles to internal proteins of pH1N1 or live virus were enumerated and characterised. Heterosubtypic T cells recognising pH1N1 core proteins were widely prevalent, being detected in 90% (30 of 33) of pH1N1-näive individuals. Although the last exposure to influenza was greater than 6 months ago, the frequency and proportion of the IFN-γ-only-secreting T-cell subset was significantly higher than the IL-2-only-secreting subset. CD8+ IFN-γ-only-secreting heterosubtypic T cells were predominantly CCR7−CD45RA− effector-memory phenotype, expressing the tissue-homing receptor CXCR3 and degranulation marker CD107. Receipt of the 2008–09 influenza vaccine did not alter the frequency of these heterosubtypic T cells, highlighting the inability of current vaccines to maintain this heterosubtypic T-cell pool. The surprisingly high prevalence of pre-existing circulating pH1N1-specific CD8+ IFN-γ-only-secreting effector memory T cells with cytotoxic and lung-homing potential in pH1N1-seronegative adults may partly explain the low case fatality rate despite high rates of infection of the pandemic in young adults.
Heterosubtypic immunity; Influenza vaccines; Pandemic influenza; T-cell memory
Macrophages exhibit remarkable plasticity and can change their phenotype in response to different environmental cues. They can become activated to kill intracellular microbes or they can assume regulatory properties to modulate immune responses. Regulatory macrophages are fundamentally different from classically, and we propose from non-classically activated macrophages; they arise in response to different stimuli and perform different physiological functions. They are likely to express unique biochemical markers that could be exploited to identify and potentially target these macrophage subsets in tissue. Furthermore, inducers of regulatory macrophages may have the potential to be used as anti-inflammatory therapeutics. Therefore, a better understanding of the various macrophage phenotypes may pave the way for new therapies that are directed at modulating macrophage functions or manipulating individual macrophage subsets.
Langerhans cells (LCs) are a distinct subset of DCs that resides in the epidermis and other epithelia. They are potent antigen-presenting cells and strong inducers of T-cell responses. Like other DC types, LCs express C-type lectins that serve as antigen/pathogen uptake receptors, with Langerin/CD207 being the characteristic LC C-type lectin. In this issue of the European Journal of Immunology, Geijtenbeek and colleagues [Eur. J. Immunol. 2011. 41: 2619–2631] assign a role to Langerin on human LCs for binding and capturing measles virus. Interestingly, however, this function does not correlate with productive infection or with cross-presentation of measles virus. These authors show that measles virus does not infect the LCs via Langerin, and that LCs cannot cross-present the virus to CD8+ T cells; however, presentation of this virus to CD4+ T cells occurs and is dependent on virus capture by Langerin. Thus, cross-presentation of measles virus may be left to skin DCs other than LCs. This highlights the complexity of anti-viral T-cell responses that originate in the skin and also emphasizes the need for intensified investigations into human skin DCs in order to be able to ultimately harness their potential for immunotherapy.
DCs; Dermatology; Host/pathogens interactions; Innate immunity; Langerhans cells
Mutations in TNFRSF1A encoding TNF receptor 1 (TNFR1) cause the autosomal dominant TNF receptor-associated periodic syndrome (TRAPS): a systemic autoinflammatory disorder. Misfolding, intracellular aggregation, and ligand-independent signaling by mutant TNFR1 are central to disease pathophysiology. Our aim was to understand the extent of signaling pathway perturbation in TRAPS. A prototypic mutant TNFR1 (C33Y), and wild-type TNFR1 (WT), were expressed at near physiological levels in an SK-Hep-1 cell model. TNFR1-associated signaling pathway intermediates were examined in this model, and in PBMCs from C33Y TRAPS patients and healthy controls. In C33Y-TNFR1-expressing SK-Hep-1 cells and TRAPS patients’ PBMCs, a subtle, constitutive upregulation of a wide spectrum of signaling intermediates and their phosphorylated forms was observed; these were associated with a proinflammatory/antiapoptotic phenotype. In TRAPS patients’ PBMCs, this upregulation of proinflammatory signaling pathways was observed irrespective of concurrent treatment with glucocorticoids, anakinra or etanercept, and the absence of overt clinical symptoms at the time that the blood samples were taken. This study reveals the pleiotropic effect of a TRAPS-associated mutant form of TNFR1 on inflammatory signaling pathways (a proinflammatory signalome), which is consistent with the variable and limited efficacy of cytokine-blocking therapies in TRAPS. It highlights new potential target pathways for therapeutic intervention.
Autoinflammation; Protein microarray; Signalome; TNF receptor 1; TRAPS
Immunotherapies that augment anti-tumor T cells have had recent success for treating patients with cancer. Here we examined whether tumor-specific CD4+ T cells enhance CD8+ T-cell adoptive immunotherapy in a lymphopenic environment. Our model employed physiological doses of TRP-1-CD4+ T cells and pmel-CD8+ T cells that when transferred individually were subtherapeutic; however, when transferred together provided significant (p≤0.001) therapeutic efficacy. Therapeutic efficacy correlated with increased numbers of effector and memory CD8+ T cells with tumor-specific cytokine expression. When combined with CD4+ T cells, transfer of total (naïve and effector) or effector CD8+ T cells were highly effective, suggesting CD4+ T cells can help mediate therapeutic effects by maintaining function of activated CD8+ T cells. In addition,, CD4+ T cells had a pronounced effect in the early post-transfer period, as their elimination within the first 3-days significantly (p<0.001) reduced therapeutic efficacy. The CD8+ T cells recovered from mice treated with both CD8+ and CD4+ T cells had decreased expression of PD-1 and PD-1-blockade enhanced the therapeutic efficacy of pmel-CD8 alone, suggesting that CD4+ T cells help reduce CD8+ T cell exhaustion. These data support combining immunotherapies that elicit both tumor-specific CD4+ and CD8+ T cells for treatment of patients with cancer.
T cell; cancer immunotherapy; CD4 T-cell help; metastatic melanoma
Vaccines are very effective at preventing infectious disease but not all recipients mount a protective immune response to vaccination. Recently, gene expression profiles of peripheral blood mononuclear cell samples in vaccinated individuals have been used to predict the development of protective immunity. However, the magnitude of change in gene expression that separates vaccine responders and non-responders is likely to be small and distributed across networks of genes, making the selection of predictive and biologically relevant genes difficult. Here we apply a new approach to predicting vaccine response based on coordinate up-regulation of sets of biologically informative genes in post vaccination gene expression profiles. We found that enrichment of gene sets related to proliferation and immunoglobulin genes accurately segregated high responders to influenza vaccination from low responders (AUC 0.94) and achieved a prediction accuracy of 88% in an independent clinical trial. Many of the genes in these gene sets would not have been identified using conventional, single-gene level approaches because of their subtle up-regulation in vaccine responders. Our results demonstrate that gene set enrichment method can capture subtle transcriptional changes and may be a generally useful approach for developing and interpreting predictive models of the human immune response.
Systems biology; Gene Expression; Vaccine Efficacy; Immune Response; B Cell Proliferation
Two-dimensional (2D) kinetic analysis directly measures molecular interactions at cell-cell junctions, thereby incorporating inherent cellular effects. By comparison, three-dimensional (3D) analysis probes the intrinsic physical chemistry of interacting molecules isolated from the cell. To understand how T-cell tumor reactivity relates to 2D and 3D binding parameters and to directly compare them, we performed kinetic analyses of a panel of human T-cell receptors (TCR) interacting with a melanoma self-antigen peptide (gp100209–217) bound to major histocompatibility complex (pMHC) in the absence and presence of coreceptor CD8. We found that while 3D parameters are inadequate to predict T-cell function, 2D parameters (which do not correlate with their 3D counterparts) show a far broader dynamic range and significantly improved correlation with T-cell function. Thus, our data support the general notion that 2D parameters of TCR–pMHC–CD8 interactions determine T-cell responsiveness and suggest a potential 2D-based strategy to screen TCRs for tumor immunotherapy.
2D kinetics; T cell activation; micropipette adhesion frequency assay; thermal fluctuation assay
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of anti-nucleic acid autoantibodies, high levels of circulating type I interferon (IFN-I), and an IFN-I-dependent elevated expression of activating FcγR. Increases in neutrophils and monocytes are often observed in clinical SLE, but how these contribute to autoantibody and IFN-I production is poorly understood. We are analyzing SLE pathogenesis in 564Igi mice, an SLE-model strain carrying gene-targeted heavy and light chain antibody genes encoding an anti-RNA autoantibody in a C57BL/6 background. Similar to human SLE patients, 564Igi mice produce anti-RNA autoantibodies and expanded neutrophil and monocyte populations. These myeloid cells produce IFN-I and exhibit increased FcγRIV expression induced via an IFN-I autocrine loop. A direct effect of IFN-I on 564Igi bone marrow B cells and neutrophils is supported by their up-regulation of “IFN-I signature genes”. In addition, 564Igi developing B cells show up-regulated TLR7 resulting in IgG2a/2b class switch recombination and autoantibody production. Our results indicate that the production of anti-RNA autoantibody is sufficient to induce an increase of bone marrow, blood and spleen IFN-I-producing neutrophils, and suggest a mechanism by which autoantibody and IFN-I contribute to SLE by activating B lymphocytes, neutrophils and monocyte effector cells in vivo.
Type I interferon; autoantibody; Fcγ receptors; myeloid cells; interferon signature genes; systemic lupus erythematosus
CD161++CD8+T cells represent a novel subset that is dominated in adult peripheral blood by mucosal-associated invariant T (MAIT) cells, as defined by the expression of a Vα7.2-Jα33 TCR, and IL-18Rα. Stimulation with IL-18+IL-12 is known to induce IFN-γ by both NK cells and, to a more limited extent, T cells. Here, we show the CD161++ CD8+T cell population is the primary T cell population triggered by this mechanism. Both CD161++Vα7.2+ and CD161++Vα7.2− T cell subsets responded to IL-12+IL-18 stimulation, demonstrating this response was not restricted to the MAIT cells, but to the CD161++ phenotype. Bacteria and TLR-agonists also indirectly triggered IFN-γ expression via IL-12 and IL-18. These data show that CD161++T cells are the predominant T cell population that responds directly to IL-12+IL-18 stimulation. Furthermore, our findings broaden the potential role of MAIT cells beyond bacterial responsiveness to potentially include viral infections and other inflammatory stimuli.
CD161++T cells; IL-12; IL-18; MAIT cells; T cells
Natural killer (NK) cells are cytotoxic cells that are able to rapidly kill viruses, tumor cells,
parasites, bacteria, and even cells considered “self”. The activity of NK cells is
controlled by a fine balance of inhibitory and activating signals mediated by a complex set of
different receptors. However, the function of NK cells is not restricted only to the killing of
target cells, NK cells also possess other properties such as the secretion of proangiogenic factors
during pregnancy. Here, we demonstrate another unique NK-cell activity, namely the regulation of
T-cell mediated allergic responses, which is dependent on the NK-cell specific receptor NKp46 (Ncr1
in mice). Using mice in which the Ncr1 gene has been replaced with a green fluorescent protein, we
demonstrate reduced delayed-type hypersensitivity and airway hypersensitivity. Interestingly, we
show that this reduction in airway hypersensitivity is due to differences in the stimulation of T
cells resulting in an altered cytokine profile.
Allergy; Dendritic cells; NK cells; NKp46; Ncr1
Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca2+]i) elevation. TCR activation triggers increased [Ca2+]i and can arrest T-cell motility in vitro. However the requirement for [Ca2+]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca2+ release-activated Ca2+ (CRAC) channel pathway required for [Ca2+]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4+ T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca2+]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen specific ORAI1-DN T cells had a two-fold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest.
antigen recognition; calcium signaling; intravital microscopy and T-cell activation
CD4 T-follicular helper cells (TFH) are central for generation of long-term B cell immunity. A defining phenotypic attribute of TFH cells is expression of the chemokine receptor CXCR5, and TFH cells are typically identified by co-expression of CXCR5 together with other markers such as programmed death (PD)-1. Herein we report high-level expression of the nutrient transporter, folate receptor (FR)4 on TFH cells in acute viral infection. Distinct from the expression profile of conventional TFH markers, FR4 was highly expressed by naive CD4 T cells, was down regulated after activation and subsequently re-expressed on TFH cells. Furthermore, FR4 was maintained, albeit at lower levels, on memory TFH cells. Comparative gene expression profiling of FR4hi, versus FR4lo antigen-specific CD4 effector T cells revealed a molecular signature consistent with TFH and TH1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto-enzyme CD73, were enriched in TFH cells compared to TH1 cells, and phenotypic analysis confirmed expression of CD73 on TFH cells. As there is now considerable interest in developing vaccines that will induce optimal TFH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of TFH cells following vaccination and infection.
CD4 T cells; folate receptor 4; CD73; viral infection; B cells
Th17 cytokines can play both protective and pathologic roles in the airway. An emerging theme in Th17 cytokine biology is that these responses can mediate tissue pathology when downstream effector cells are dysfunctional such as neutrophils lacking functional NADPH oxidase in the case of chronic granulomatous disease or epithelial cells lacking appropriate ion transport, as in the case of cystic fibrosis. In this mini-review we highlight recent advances in the protective and pathologic roles of Th17 cytokines in the context of pulmonary infection.
Approximately 2 billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), and an estimated 1.5 million individuals die annually from TB. Presently, Mycobacterium bovis BCG remains the only licensed TB vaccine; however, previous studies suggest its protective efficacy wanes over time and fails in preventing pulmonary TB. Therefore, a safe and effective vaccine is urgently required to replace BCG or boost BCG immunizations. Our previous studies revealed that mycobacterial proteins are released via exosomes from macrophages infected with M. tuberculosis or pulsed with M. tuberculosis culture filtrate proteins (CFP). In the present study, exosomes purified from macrophages treated with M. tuberculosis CFP were found to induce antigen-specific IFN-γ and IL-2-expressing CD4+ and CD8+ T cells. In exosome-vaccinated mice there was a similar TH1 immune response but a more limited TH2 response compared to BCG vaccinated mice. Using a low-dose M. tuberculosis mouse aerosol infection model, exosomes from CFP-treated macrophages were found to both prime a protective immune response as well as boost prior BCG immunization. The protection was equal to or superior to BCG. In conclusion, our findings suggest that exosomes might serve as a novel cell-free vaccine against an M. tuberculosis infection.
Mycobacterium tuberculosis; Exosomes; Vaccine
The initial interaction between HIV-1 and the host occurs at the mucosa during sexual intercourse. In cervical mucosa, HIV-1 exists both as free and opsonized virions and this might influence initial infection. We used cervical explants to study HIV-1 transmission, the effects of opsonization on infectivity, and how infection can be prevented. Complement opsonization enhanced HIV-1 infection of dendritic cells (DCs) compared with that by free HIV-1, but this increased infection was not observed with CD4+ T cells. Blockage of the α4-, β7-, and β1-integrins significantly inhibited HIV-1 infection of both DCs and CD4+ T cells. We found a greater impairment of HIV-1 infection in DCs for complement-opsonized virions compared with that of free virions when αM/β2- and α4-integrins were blocked. Blocking the C-type lectin receptor macrophage mannose receptor (MMR) inhibited infection of emigrating DCs but had no effect on CD4+ T-cell infection. We show that blocking of integrins decreases the HIV-1 infection of both mucosal DCs and CD4+ T cells emigrating from the cervical tissues. These findings may provide the basis of novel microbicidal strategies that may help limit or prevent initial infection of the cervical mucosa, thereby reducing or averting systemic HIV-1 infection.
CD4+ T cells; Complement system; DCs; HIV; Integrins