Bystander T cells in human immune responses to dengue antigens

<p>Abstract</p> <p>Background</p> <p>Previous studies of T cell activation in dengue infection have focused on restriction of specific T cell receptors (TCRs) and classical MHC molecules. However, bystander T cell activation, which is TCR independent, occurs via cytokines in other viral infections, both in vitro and in vivo, and enables T cells to bypass certain control checkpoints. Moreover, clinical and pathological evidence has pointed to cytokines as the mediators of dengue disease severity. Therefore, we investigated bystander T cell induction by dengue viral antigen.</p> <p>Results</p> <p>Whole blood samples from 55 Thai schoolchildren aged 13-14 years were assayed for in vitro interferon-gamma (IFN-γ) induction in response to inactivated dengue serotype 2 antigen (Den2). The contribution of TCR-dependent and independent pathways was tested by treatment with cyclosporin A (CsA), which inhibits TCR-dependent activation of T cells. ELISA results revealed that approximately 72% of IFN-γ production occurred via the TCR-dependent pathway. The major IFN-γ sources were natural killer (NK) (mean ± SE = 55.2 ± 3.3), CD4⁺T (24.5 ± 3.3) and CD8⁺T cells (17.9 ± 1.5), respectively, as demonstrated by four-color flow cytometry. Interestingly, in addition to these cells, we found CsA-resistant IFN-γ producing T cells (CD4⁺T = 26.9 ± 3.6% and CD8⁺T = 20.3 ± 2.1%) implying the existence of activated bystander T cells in response to dengue antigen in vitro. These bystander CD4⁺and CD8⁺T cells had similar kinetics to NK cells, appeared after 12 h and were inhibited by anti-IL-12 neutralization indicating cytokine involvement.</p> <p>Conclusions</p> <p>This study described immune cell profiles and highlighted bystander T cell activation in response to dengue viral antigens of healthy people in an endemic area. Further studies on bystander T cell activation in dengue viral infection may reveal the immune mechanisms that protect or enhance pathogenesis of secondary dengue infection.</p

Interleukin 10 inhibits pro-inflammatory cytokine responses and killing of Burkholderia pseudomallei.

Melioidosis, caused by Burkholderia pseudomallei, is endemic in northeastern Thailand and Northern Australia. Severe septicemic melioidosis is associated with high levels of pro-inflammatory cytokines and is correlated with poor clinical outcomes. IL-10 is an immunoregulatory cytokine, which in other infections can control the expression of pro-inflammatory cytokines, but its role in melioidosis has not been addressed. Here, whole blood of healthy seropositive individuals (n = 75), living in N. E. Thailand was co-cultured with B. pseudomallei and production of IL-10 and IFN-γ detected and the cellular sources identified. CD3- CD14+ monocytes were the main source of IL-10. Neutralization of IL-10 increased IFN-γ, IL-6 and TNF-α production and improved bacteria killing. IFN-γ production and microbicidal activity were impaired in individuals with diabetes mellitus (DM). In contrast, IL-10 production was unimpaired in individuals with DM, resulting in an IL-10 dominant cytokine balance. Neutralization of IL-10 restored the IFN-γ response of individuals with DM to similar levels observed in healthy individuals and improved killing of B. pseudomallei in vitro. These results demonstrate that monocyte derived IL-10 acts to inhibit potentially protective cell mediated immune responses against B. pseudomallei, but may also moderate the pathological effects of excessive cytokine production during sepsis

Phenotypic and Functional Characterization of Human Memory T Cell Responses to Burkholderia pseudomallei

The Gram-negative bacterium, Burkholderia pseudomallei, is a public health problem in southeast Asia and northern Australia and a Centers for Disease Control and Prevention listed Category B potential bioterrorism agent. It is the causative agent of melioidosis, and clinical manifestations vary from acute sepsis to chronic localized and latent infection, which can reactivate decades later. B. pseudomallei is the major cause of community-acquired pneumonia and septicemia in northeast Thailand. In spite of the medical importance of B. pseudomallei, little is known about the mechanisms of pathogenicity and the immunological pathways of host defense. There is no available vaccine, and the mortality rate in acute cases can exceed 40% with 10–15% of survivors relapsing or being reinfected despite prolonged and complete treatments. In this article, we describe cell-mediated immune responses to B. pseudomallei in humans living in northeast Thailand and demonstrate clear evidence of T cell priming in healthy seropositive individuals and patients who recovered from melioidosis. This is the most detailed study yet performed on the cell types that produce interferon-gamma to B. pseudomallei in humans and the antigens that they recognize and the first to study large sample numbers in the primary endemic focus of melioidosis in the world

Quantitative Proteomic Analysis of Burkholderia pseudomallei Bsa Type III Secretion System Effectors Using Hypersecreting Mutants

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. We constructed a mutant lacking bsaP, a homolog of the T3SS “gatekeeper” family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hypersecrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion. Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei

1α,25‐dihydroxyvitamin D3 in combination with transforming growth factor‐β increases the frequency of Foxp3+ regulatory T cells through preferential expansion and usage of interleukin‐2

A high prevalence of vitamin D insufficiency and deficiency exists worldwide, which is associated with an increased incidence and severity of a range of immune‐mediated diseases. This has resulted in considerable interest in the immunodulatory functions of vitamin D. The active form of vitamin D, 1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to increase the frequency of Foxp3+ CD4+ T regulatory (Treg) cells when present at high concentrations or under strong T‐cell stimulation in culture. Supporting evidence exists in vivo for a positive association between serum 25(OH)D and Foxp3+ Treg cell numbers in humans. The aim of this work was to identify the cytokine milieu required in vitro to promote Foxp3+ Treg cells in cultures containing 1,25(OH)2D3 at more moderate concentrations (10−7 m). Stimulation of human CD4+ T cells with a combination of 1,25(OH)2D3 and transforming growth factor‐β (TGF‐β) greatly increased the frequency of Foxp3+ Treg cells, which is proposed to result from the preferential expansion of Foxp3+ Treg cells, as compared with the Foxp3− effector T cells, in culture. The differential effect on proliferation may result from enhanced availability and usage of interleukin‐2 by the Foxp3+ Treg cells compared with Foxp3− effector T cells. In summary, modulation of the cytokine environment to one high in TGF‐β in the presence of 1,25(OH)2D3 (10−7 m) significantly increased Foxp3+ Treg cell frequency. These data provide additional evidence for the important immunomodulatory properties of 1,25(OH)2D3 that exist and may help to control inflammatory diseases

Human Immune Responses to Burkholderia pseudomallei Characterized by Protein Microarray Analysis

Background. We aimed to determine the antibody and T cell responses to Burkholderia pseudomallei of humans to select candidate vaccine antigens