Functional and pathogenic differences of Th1 and Th17 cells in experimental autoimmune encephalomyelitis

Background

There is consensus that experimental autoimmune encephalomyelitis (EAE) can be mediated by myelin specific T cells of Th1 as well as of Th17 phenotype, but the contribution of either subset to the pathogenic process has remained controversial. In this report, we compare functional differences and pathogenic potential of “monoclonal” T cell lines that recognize myelin oligodendrocyte glycoprotein (MOG) with the same transgenic TCR but are distinguished by an IFN-γ producing Th1-like and IL-17 producing Th17-like cytokine signature.

Methods and Findings

CD4⁺ T cell lines were derived from the transgenic mouse strain 2D2, which expresses a TCR recognizing MOG peptide 35–55 in the context of I-A^b. Adoptive transfer of Th1 cells into lymphopenic (Rag2^−/−) recipients, predominantly induced “classic” paralytic EAE, whereas Th17 cells mediated “atypical” ataxic EAE in approximately 50% of the recipient animals. Combination of Th1 and Th17 cells potentiated the encephalitogenicity inducing classical EAE exclusively. Th1 and Th17 mediated EAE lesions differed in their composition but not in their localization within the CNS. While Th1 lesions contained IFN-γ, but no IL-17 producing T cells, the T cells in Th17 lesions showed plasticity, substantially converting to IFN-γ producing Th1-like cells. Th1 and Th17 cells differed drastically by their lytic potential. Th1 but not Th17 cells lysed autoantigen presenting astrocytes and fibroblasts in vitro in a contact-dependent manner. In contrast, Th17 cells acquired cytotoxic potential only after antigenic stimulation and conversion to IFN-γ producing Th1 phenotype.

Conclusions

Our data demonstrate that both Th1 and Th17 lineages possess the ability to induce CNS autoimmunity but can function with complementary as well as differential pathogenic mechanisms. We propose that Th17-like cells producing IL-17 are required for the generation of atypical EAE whereas IFN-γ producing Th1 cells induce classical EAE.     

Therapeutic effect of the potent IL-12/IL-23 inhibitor STA-5326 on experimental autoimmune uveoretinitis

Introduction

The purpose of this study was to determine if oral administration of the interleukin (IL) 12/IL-23 inhibitor, STA-5326, is effective in experimental autoimmune uveoretinitis (EAU).

Methods

C57BL/6J mice were immunised with human interphotoreceptor retinoid binding protein peptide (IRBP_1–20). STA-5326 at a dose of either 5 mg/kg or 20 mg/kg, or vehicle alone, was orally administered once a day for six days a week from day 0 to day 14. Fundus examination was performed on day 14 and day 18 after immunisation. Mice were euthanased on day 18 and the eyes were enucleated for histopathological examination. In vivo-primed draining lymph node cells were stimulated with IRBP_1–20 and culture supernatant was harvested for assay of interferon (IFN)-γ and IL-17 by ELISA. Intracellular expression of IFN-γ and IL-17 in CD4⁺ T cells of cultured draining lymph node cells was assessed by flow cytometry. The level of IL-12 p40 in serum was examined in STA-5326-treated or vehicle-treated mice receiving immunisation.

Results

The level of IL-12 p40 in serum was decreased in mice treated with STA-5326. Oral administration of either 5 mg/kg or 20 mg/kg STA-5326 reduced the severity of EAU on day 14 and 18. In addition, mice treated with 20 mg/kg STA-5326 showed significantly decreased severity of EAU by histopathological analysis. Although IFN-γ production of draining lymph node cells was increased in STA-5326-treated mice by ELISA analysis, the proportion of IFN-γ-producing cells was not significantly altered. However, IL-17 production and the proportion of IL-17-producing cells were significantly reduced in STA-5326-treated mice. Furthermore, oral administration of STA-5326 during the effector phase reduced the severity of EAU.

Conclusions

These results indicate that oral administration of the IL-12/IL-23 inhibitor STA-5326 is effective in suppressing inflammation in the EAU model, and reduces the expansion of IL-17-producing cells. STA-5326 may represent a new therapeutic modality for human refractory uveitis.     

Use of praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice

Background

H5N1 is a highly pathogenic influenza A virus, which can cause severe illness or even death in humans. Although the widely used killed vaccines are able to provide some protection against infection via neutralizing antibodies, cytotoxic T-lymphocyte responses that are thought to eradicate viral infections are lacking.

Methodology/Principal Findings

Aiming to promote cytotoxic responses against H5N1 infection, we extended our previous finding that praziquantel (PZQ) can act as an adjuvant to induce IL-17-producing CD8⁺ T cells (Tc17). We found that a single immunization of 57BL/6 mice with killed viral vaccine plus PZQ induced antigen-specific Tc17 cells, some of which also secreted IFN-γ. The induced Tc17 had cytolytic activities. Induction of these cells was impaired in CD8 knockout (KO) or IFN-γ KO mice, and was even lower in IL-17 KO mice. Importantly, the inoculation of killed vaccine with PZQ significantly reduced virus loads in the lung tissues and prolonged survival. Protection against H5N1 virus infection was obtained by adoptively transferring PZQ-primed wild type CD8⁺ T cells and this was more effective than transfer of activated IFN-γ KO or IL-17 KO CD8⁺ T cells.

Conclusions/Significance

Our results demonstrated that adding PZQ to killed H5N1 vaccine could promote broad Tc17-mediated cytotoxic T lymphocyte activity, resulting in improved control of highly pathogenic avian influenza virus infection.     

Praziquantel facilitates IFN-γ-producing CD8+ T cells (Tc1) and IL-17-producing CD8+ T cells (Tc17) responses to DNA vaccination in mice

Background

CD8⁺ cytotoxic T lymphocytes (CTLs) are crucial for eliminating hepatitis B virus (HBV) infected cells. DNA vaccination, a novel therapeutic strategy for chronic virus infection, has been shown to induce CTL responses. However, accumulated data have shown that CTLs could not be effectively induced by HBV DNA vaccination.

Methodology/Principal Findings

Here, we report that praziquantel (PZQ), an anti-schistoma drug, could act as an adjuvant to overcome the lack of potent CTL responses by HBV DNA vaccination in mice. PZQ in combination with HBV DNA vaccination augmented the induction of CD8⁺ T cell-dependent and HBV-specific delayed hypersensitivity responses (DTH) in C57BL/6 mice. Furthermore, the induced CD8⁺ T cells consisted of both Tc1 and Tc17 subtypes. By using IFN-γ knockout (KO) mice and IL-17 KO mice, both cytokines were found to be involved in the DTH. The relevance of these findings to HBV immunization was established in HBsAg transgenic mice, in which PZQ also augmented the induction of HBV-specific Tc1 and Tc17 cells and resulted in reduction of HBsAg positive hepatocytes. Adoptive transfer experiments further showed that PZQ-primed CD8⁺ T cells from wild type mice, but not the counterpart from IFN-γ KO or IL-17 KO mice, resulted in elimination of HBsAg positive hepatocytes.

Conclusions/Significance

Our results suggest that PZQ is an effective adjuvant to facilitate Tc1 and Tc17 responses to HBV DNA vaccination, inducing broad CD8⁺ T cell-based immunotherapy that breaks tolerance to HBsAg.     

Effect of pregnancy on serum cytokines in SLE patients

Introduction

The aim of this study was to evaluate an extensive panel of cytokines involved in immune regulation during pregnancy in patients with systemic lupus erythematosus (SLE) and in healthy women.

Methods

A total of 47 consecutive successful pregnancies in 46 SLE patients and 56 pregnancies in 56 matched healthy subjects, as controls, were prospectively studied. Serum interleukin (IL)-1-α, IL-1-β, IL-2, IL-6, IL-8, IL-10, IL-12p70, interferon (INF)-γ and tumor necrosis factor (TNF)-α were detected in sera obtained at the first and third trimester of pregnancy by a highly sensitive, multiplexed sandwich ELISA.

Results

Medians (pg/ml) of serum levels of most helper T (Th)1-type cytokines were significantly lower in the third trimester compared with those observed in the first trimester of pregnancy in healthy women: INF-γ 2.0 vs 3.4, TNF-α 10.2 vs 11.5, IL-1-α 0.9 vs 1.1, IL-1-β 0.6 vs 1.0, IL-2 3.0 vs 3.5, and IL-12p70 4.9 vs 5.6 (P-values < 0.02 for all). By contrast, only the IL-1-α serum levels were lower in the third trimester compared with the first trimester in SLE patients (P = 0.006). IFN-γ/IL-6 and IFN-γ/IL-10 ratios were higher in controls than in SLE (P = 0.002, and P = 0.001, respectively); moreover, they were significantly reduced in the third compared to the first trimester of pregnancy in healthy women, but not in SLE.

Conclusions

In SLE patients, Th1/Th2 cytokine serum level ratio does not decrease during pregnancy progression as much as in healthy pregnant women. This could account for the observation of a low frequency of disease flares in the third trimester of gestation.     

Variation of Mycobacterium tuberculosis Antigen-Specific IFN-γ and IL-17 Responses in Healthy Tuberculin Skin Test (TST)-Positive Human Subjects

Objective

To determine the variation of IFN-γ and IL-17 responses to M. tuberculosis antigens in healthy TST+ humans.

Methods

We isolated peripheral blood mononuclear cells from 21 TST+ healthy adults, stimulated them with phytohemagglutinin (PHA), PPD, Ag85B, ESAT-6, and live M. bovis BCG, and assayed IFN-γ and IL-17 secretion by ELISA in supernatants after 24 or 72 hours of incubation respectively.

Results

As in other studies, we found a wide range of IFN-γ responses to M. tuberculosis antigens; the variation significantly exceeded that observed in the same donors to the polyclonal T cell stimulus, phytohemagglutinin (PHA). In addition, we assayed IL-17 secretion in response to the same stimuli, and found less subject-to-subject variation. Analysis of the ratio of IFN-γ to IL-17 secretion on a subject-to-subject basis also revealed a wide range, with the majority of results distributed in a narrow range, and a minority with extreme results all of which were greater than that in the majority of subjects. The data suggest that study of exceptional responses to M. tuberculosis antigens may reveal immunologic correlates with specific outcomes of M. tuberculosis infection.

Conclusion

Variation of IFNγ and IFN-γ/IL-17 responses to mycobacterial antigens exceeds that of responses to the polyclonal stimulus, PHA, in TST positive healthy humans. This indicates a quantitative spectrum of human immune responses to infection with M. tuberculosis. Since the outcome of human infection with M. tuberculosis varies greatly, systematic study of multiple immune responses to multiple antigens is likely to reveal correlations between selected immune responses and the outcomes of infection.     

Enumeration of functional T-cell subsets by fluorescence-immunospot defines signatures of pathogen burden in tuberculosis

Background

IFN-γ and IL-2 cytokine-profiles define three functional T-cell subsets which may correlate with pathogen load in chronic intracellular infections. We therefore investigated the feasibility of the immunospot platform to rapidly enumerate T-cell subsets by single-cell IFN-γ/IL-2 cytokine-profiling and establish whether immunospot-based T-cell signatures distinguish different clinical stages of human tuberculosis infection.

Methods

We used fluorophore-labelled anti-IFN-γ and anti-IL-2 antibodies with digital overlay of spatially-mapped colour-filtered images to enumerate dual and single cytokine-secreting M. tuberculosis antigen-specific T-cells in tuberculosis patients and in latent tuberculosis infection (LTBI). We validated results against established measures of cytokine-secreting T-cells.

Results

Fluorescence-immunospot correlated closely with single-cytokine enzyme-linked-immunospot for IFN-γ-secreting T-cells and IL-2-secreting T-cells and flow-cytometry-based detection of dual IFN-γ/IL-2-secreting T-cells. The untreated tuberculosis signature was dominated by IFN-γ-only-secreting T-cells which shifted consistently in longitudinally-followed patients during treatment to a signature dominated by dual IFN-γ/IL-2-secreting T-cells in treated patients. The LTBI signature differed from active tuberculosis, with higher proportions of IL-2-only and IFN-γ/IL-2-secreting T-cells and lower proportions of IFN-γ-only-secreting T-cells.

Conclusions

Fluorescence-immunospot is a quantitative, accurate measure of functional T-cell subsets; identification of cytokine-signatures of pathogen burden, distinct clinical stages of M. tuberculosis infection and long-term immune containment suggests application for treatment monitoring and vaccine evaluation.     

Natural variation in immune responses to neonatal Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Vaccination in a Cohort of Gambian infants

Background

There is a need for new vaccines for tuberculosis (TB) that protect against adult pulmonary disease in regions where BCG is not effective. However, BCG could remain integral to TB control programmes because neonatal BCG protects against disseminated forms of childhood TB and many new vaccines rely on BCG to prime immunity or are recombinant strains of BCG. Interferon-gamma (IFN-γ) is required for immunity to mycobacteria and used as a marker of immunity when new vaccines are tested. Although BCG is widely given to neonates IFN-γ responses to BCG in this age group are poorly described. Characterisation of IFN-γ responses to BCG is required for interpretation of vaccine immunogenicity study data where BCG is part of the vaccination strategy.

Methodology/Principal Findings

236 healthy Gambian babies were vaccinated with M. bovis BCG at birth. IFN-γ, interleukin (IL)-5 and IL-13 responses to purified protein derivative (PPD), killed Mycobacterium tuberculosis (KMTB), M. tuberculosis short term culture filtrate (STCF) and M. bovis BCG antigen 85 complex (Ag85) were measured in a whole blood assay two months after vaccination. Cytokine responses varied up to 10 log-fold within this population. The majority of infants (89–98% depending on the antigen) made IFN-γ responses and there was significant correlation between IFN-γ responses to the different mycobacterial antigens (Spearman''s coefficient ranged from 0.340 to 0.675, p = 10⁻⁶–10⁻²²). IL-13 and IL-5 responses were generally low and there were more non-responders (33–75%) for these cytokines. Nonetheless, significant correlations were observed for IL-13 and IL-5 responses to different mycobacterial antigens

Conclusions/Significance

Cytokine responses to mycobacterial antigens in BCG-vaccinated infants are heterogeneous and there is significant inter-individual variation. Further studies in large populations of infants are required to identify the factors that determine variation in IFN-γ responses.     

Regulatory effects of IFN-β on the development of experimental autoimmune uveoretinitis in B10RIII mice

Background

Experimental autoimmune uveoretinitis (EAU) serves as a model for human intraocular inflammation. IFN-β has been used in the treatment of certain autoimmune diseases. Earlier studies showed that it ameliorated EAU; however, the mechanisms involved in this inhibition are still largely unknown.

Methodology/Principal Findings

B10RIII mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 161–180 in Complete Freund''s adjuvant. Splenocytes from different time points after immunization were used to evaluate the expression of IFN-β. An increased expression of IFN-β was observed during EAU and its highest expression was observed on day 16, 3 days after the peak of intraocular inflammation. Splenocytes and draining lymph node cells from mice immunized with IRBP_161-180 on day 13 and control mice were activated with anti-CD3/anti-CD28 antibodies or IRBP_161-180 to evaluate the production of IFN-γ and IL-17. The results showed that IFN-γ and IL-17 were significantly higher in immunized mice as compared to the control mice when exposed to anti-CD3/anti-CD28 antibodies. However, the production of IFN-γ and IL-17 was detected only in immunized mice, but not in the control mice when stimulated with IRBP_161-180. Multiple subcutaneous injections of IFN-β significantly inhibited EAU activity in association with a down-regulated expression of IFN-γ, IL-17 and an enhanced IL-10 production. In an in vitro system using cells from mice, IFN-β suppressed IFN-γ production by CD4⁺CD62L⁻ T cells, IL-17 production by CD4⁺CD62L^+/- T cells and proliferation of CD4⁺CD62L^+/- T cells. IFN-β inhibited the secretion of IL-6, but promoted the secretion of IL-10 by monocytes. IFN-β-treated monocytes inhibited IL-17 secretion by CD4⁺CD62L^+/- T cells, but did not influence IFN-γ expression and T cell proliferation.

Conclusions/Significance

IFN-β may exert its inhibitory effect on EAU by inhibiting Th1, Th17 cells and modulating relevant cytokines. IFN-β may provide a potential treatment for diseases mediated by Th1 and Th17 cells.     

Immune events associated with high level protection against Schistosoma japonicum infection in pigs immunized with UV-attenuated cercariae

Background

The vaccination of radiation-attenuated Schistosoma japonicum cercariae can induce effective protection in artiodactyl, but the immune events related to protective immunity are not fully understood. To provide a paradigm for a human recombinant antigen vaccine, we have undertaken a vaccination and challenge experiment in pigs, which was recognized as an appropriate animal model in this type of study because of their similarity to human in immunology, and investigated the relative immune events induced by the radiation-attenuated S. japonicum cercariae.

Methods and Findings

We found that pigs immunized once with 400 µw UV-irradiated cercariae exhibited 63.84% and 71.82% reductions in worm burden and hepatic eggs respectively. Protective immunity in vaccinated pigs was associated with high level productions of IgM, total IgG, IgG1 and IgG2; IgG2 was significantly increased in the acute infection. IFN-γ levels could be elicited by immunization. At week 6 post-infection, IFN-γ, IL-4 and IL-10 levels also showed a dramatic rise synchronously in vaccinated pigs. Moreover, the granzyme b, nk-lysin, ifnγ, il4 and il10 mRNA levels in early skin-draining lymph nodes of immunized pigs were higher than those in pigs with non-irradiated cercariae infection. In addition, cytotoxicity-related genes in the mesenteric lymph nodes were significantly upregulated in vaccinated pigs in the acute infection.

Conclusion/Significance

Our results demonstrated that IFN-γ and IgG2 antibody production, as well as genes related to cytotoxicity are associated with the high level protection induced by UV-irradiated Schistosoma japonicum vaccine. These findings indicated that optimal vaccination against S. japonicum required the induction of IFN-γ, IgG2 antibody related to Th1 responses and cytotoxicity effect.     

Co-administration of a plasmid DNA encoding IL-15 improves long-term protection of a genetic vaccine against Trypanosoma cruzi

Background

Immunization of mice with the Trypanosoma cruzi trans-sialidase (TS) gene using plasmid DNA, adenoviral vector, and CpG-adjuvanted protein delivery has proven highly immunogenic and provides protection against acute lethal challenge. However, long-term protection induced by TS DNA vaccines has not been reported. The goal of the present work was to test whether the co-administration of a plasmid encoding IL-15 (pIL-15) could improve the duration of protection achieved through genetic vaccination with plasmid encoding TS (pTS) alone.

Methodology

We immunized BALB/c mice with pTS in the presence or absence of pIL-15 and studied immune responses [with TS-specific IFN-γ ELISPOT, serum IgG ELISAs, intracellular cytokine staining (IFN-γ, TNF-α, and IL-2), tetramer staining, and CFSE dilution assays] and protection against lethal systemic challenge at 1 to 6 months post vaccination. Mice receiving pTS alone developed robust TS-specific IFN-γ responses and survived a lethal challenge given within the first 3 months following immunization. The addition of pIL-15 to pTS vaccination did not significantly alter T cell responses or protection during this early post-vaccination period. However, mice vaccinated with both pTS and pIL-15 challenged 6 months post-vaccination were significantly more protected against lethal T. cruzi challenges than mice vaccinated with pTS alone (P<0.05). Improved protection correlated with significantly higher numbers of TS-specific IFN-γ producing total and CD8⁺ T cells detected>6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro re-stimulation.

Conclusion

Addition of pIL-15 during genetic vaccination greatly improved long-term T cell survival, memory T cell expansion, and long-term protection against the important human parasite, T. cruzi.

Author Summary

Over 11 million individuals are infected with Trypanosoma cruzi, the causative agent of Chagas disease, which kills >50,000 people annually. Although recent vector control efforts and increased use and effectiveness of chemotherapeutic drugs including benznidazole have reduced infection rates and mortality, a safe, effective vaccine is needed. Vaccination with the T. cruzi trans-sialidase (TS) has been used effectively in mice to reduce mortality and chronic disease, however, the establishment of vaccine-induced long-term protective immunity remains elusive. Co-immunization strategies utilizing immune regulators such as interleukin-12 (IL-12) and interleukin-15 (IL-15) can be used to enhance antigen-specific T cell responses and prolong protective immunity. In the present report, we show that genetic vaccination of BALB/c mice with plasmid DNA encoding both TS and IL-15 compared with plasmid DNA encoding TS alone significantly enhanced CD4⁺ and CD8⁺ T cell responses including increased TNF-α, IFN-γ, and IL-2 production, and long-term protection against lethal systemic parasite challenge.     

Baseline levels of influenza-specific CD4 memory T-cells affect T-cell responses to influenza vaccines

Background

Factors affecting immune responses to influenza vaccines have not been studied systematically. We hypothesized that T-cell and antibody responses to the vaccines are functions of pre-existing host immunity against influenza antigens.

Methodology/Principal Findings

During the 2004 and 2005 influenza seasons, we have collected data on cellular and humoral immune reactivity to influenza virus in blood samples collected before and after immunization with inactivated or live attenuated influenza vaccines in healthy children and adults. We first used cross-validated lasso regression on the 2004 dataset to identify a group of candidate baseline correlates with T-cell and antibody responses to vaccines, defined as fold-increase in influenza-specific T-cells and serum HAI titer after vaccination. The following baseline parameters were examined: percentages of influenza-reactive IFN-γ⁺ cells in T and NK cell subsets, percentages of influenza-specific memory B-cells, HAI titer, age, and type of vaccine. The candidate baseline correlates were then tested with the independent 2005 dataset. Baseline percentage of influenza-specific IFN-γ⁺ CD4 T-cells was identified as a significant correlate of CD4 and CD8 T-cell responses, with lower baseline levels associated with larger T-cell responses. Baseline HAI titer and vaccine type were identified as significant correlates for HAI response, with lower baseline levels and the inactivated vaccine associated with larger HAI responses. Previously we reported that baseline levels of CD56^dim NK reactivity against influenza virus inversely correlated with the immediate T-cell response to vaccination, and that NK reactivity induced by influenza virus depended on IL-2 produced by influenza-specific memory T-cells. Taken together these results suggest a novel mechanism for the homeostasis of virus-specific T-cells, which involves interaction between memory helper T-cells, CD56^dim NK and DC.

Significance

These results demonstrate that assessment of baseline biomarkers may predict immunologic outcome of influenza vaccination and may reveal some of the mechanisms responsible for variable immune responses following vaccination and natural infection.     

Regulatory T cell induction during Plasmodium chabaudi infection modifies the clinical course of experimental autoimmune encephalomyelitis

Background

Experimental autoimmune encephalomyelitis (EAE) is used as an animal model for human multiple sclerosis (MS), which is an inflammatory demyelinating autoimmune disease of the central nervous system characterized by activation of Th1 and/or Th17 cells. Human autoimmune diseases can be either exacerbated or suppressed by infectious agents. Recent studies have shown that regulatory T cells play a crucial role in the escape mechanism of Plasmodium spp. both in humans and in experimental models. These cells suppress the Th1 response against the parasite and prevent its elimination. Regulatory T cells have been largely associated with protection or amelioration in several autoimmune diseases, mainly by their capacity to suppress proinflammatory response.

Methodology/Principal Findings

In this study, we verified that CD4⁺CD25⁺ regulatory T cells (T regs) generated during malaria infection (6 days after EAE induction) interfere with the evolution of EAE. We observed a positive correlation between the reduction of EAE clinical symptoms and an increase of parasitemia levels. Suppression of the disease was also accompanied by a decrease in the expression of IL-17 and IFN-γ and increases in the expression of IL-10 and TGF-β1 relative to EAE control mice. The adoptive transfer of CD4⁺CD25⁺ cells from P. chabaudi-infected mice reduced the clinical evolution of EAE, confirming the role of these T regs.

Conclusions/Significance

These data corroborate previous findings showing that infections interfere with the prevalence and evolution of autoimmune diseases by inducing regulatory T cells, which regulate EAE in an apparently non-specific manner.     

Multiple cytokines are released when blood from patients with tuberculosis is stimulated with Mycobacterium tuberculosis antigens

Background

Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection.

Methods

Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens.

Results

Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines.

Conclusions

Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone.     

Cellular immunity confers transient protection in experimental Buruli ulcer following BCG or mycolactone-negative Mycobacterium ulcerans vaccination

Background

Buruli ulcer (BU) is an emerging infectious disease caused by Mycobacterium ulcerans that can result in extensive necrotizing cutaneous lesions due to the cytotoxic exotoxin mycolactone. There is no specific vaccine against BU but reports show some degree of cross-reactive protection conferred by M. bovis BCG immunization. Alternatively, an M. ulcerans-specific immunization could be a better preventive strategy.

Methodology/Principal Findings

In this study, we used the mouse model to characterize the histological and cytokine profiles triggered by vaccination with either BCG or mycolactone-negative M. ulcerans, followed by footpad infection with virulent M. ulcerans. We observed that BCG vaccination significantly delayed the onset of M. ulcerans growth and footpad swelling through the induction of an earlier and sustained IFN-γ T cell response in the draining lymph node (DLN). BCG vaccination also resulted in cell-mediated immunity (CMI) in M. ulcerans-infected footpads, given the predominance of a chronic mononuclear infiltrate positive for iNOS, as well as increased and sustained levels of IFN-γ and TNF. No significant IL-4, IL-17 or IL-10 responses were detected in the footpad or the DLN, in either infected or vaccinated mice. Despite this protective Th1 response, BCG vaccination did not avoid the later progression of M. ulcerans infection, regardless of challenge dose. Immunization with mycolactone-deficient M. ulcerans also significantly delayed the progression of footpad infection, swelling and ulceration, but ultimately M. ulcerans pathogenic mechanisms prevailed.

Conclusions/Significance

The delay in the emergence of pathology observed in vaccinated mice emphasizes the relevance of protective Th1 recall responses against M. ulcerans. In future studies it will be important to determine how the transient CMI induced by vaccination is compromised.     

Regulatory T Cells Suppress Natural Killer Cells during Plasmid DNA Vaccination in Mice,Blunting the CD8+ T Cell Immune Response by the Cytokine TGFβ

Background

CD4⁺CD25⁺ regulatory T cells (Tregs) suppress adaptive T cell-mediated immune responses to self- and foreign-antigens. Tregs may also suppress early innate immune responses to vaccine antigens and might decrease vaccine efficacy. NK and NKT cells are the first responders after plasmid DNA vaccination and are found at the site of inoculation. Earlier reports demonstrated that NKT cells could improve plasmid DNA efficacy, a phenomenon not found for NK cells. In fact, it has been shown that under certain disease conditions, NK cells are suppressed by Tregs via their release of IL-10 and/or TGFβ. Therefore, we tested the hypothesis that NK cell function is suppressed by Tregs in the setting of plasmid DNA vaccination.

Methodology/Principal Findings

In this study we show that Tregs directly inhibit NK cell function during plasmid DNA vaccination by suppressing the potentially 10-fold, NK cell-mediated, augmentation of plasmid DNA antigen-specific CD8⁺ T cells. We found that this phenomenon is dependent on the secretion of cytokine TGFβ by Tregs, and independent of IL-10.

Conclusions

Our data indicate a crucial function for Tregs in blocking plasmid DNA vaccine-elicited immune responses, revealing potentially novel strategies for improving the efficiency of plasmid DNA vaccines including chemical- or antibody-induced localized blockage of Treg-mediated suppression of NK cells at the site of plasmid DNA vaccine inoculation.     

T cell responses to the RTS,S/AS01(E) and RTS,S/AS02(D) malaria candidate vaccines administered according to different schedules to Ghanaian children

Background

The Plasmodium falciparum pre-erythrocytic stage candidate vaccine RTS,S is being developed for protection of young children against malaria in sub-Saharan Africa. RTS,S formulated with the liposome based adjuvant AS01_E or the oil-in-water based adjuvant AS02_D induces P. falciparum circumsporozoite (CSP) antigen-specific antibody and T cell responses which have been associated with protection in the experimental malaria challenge model in adults.

Methods

This study was designed to evaluate the safety and immunogenicity induced over a 19 month period by three vaccination schedules (0,1-, 0,1,2- and 0,1,7-month) of RTS,S/AS01_E and RTS,S/AS02_D in children aged 5–17 months in two research centers in Ghana. Control Rabies vaccine using the 0,1,2-month schedule was used in one of two study sites.

Results

Whole blood antigen stimulation followed by intra-cellular cytokine staining showed RTS,S/AS01_E induced CSP specific CD4 T cells producing IL-2, TNF-α, and IFN-γ. Higher T cell responses were induced by a 0,1,7-month immunization schedule as compared with a 0,1- or 0,1,2-month schedule. RTS,S/AS01_E induced higher CD4 T cell responses as compared to RTS,S/AS02_D when given on a 0,1,7-month schedule.

Conclusions

These findings support further Phase III evaluation of RTS,S/AS01_E. The role of immune effectors and immunization schedules on vaccine protection are currently under evaluation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00360230","term_id":"NCT00360230"}}NCT00360230     

Interferon-α abrogates tolerance induction by human tolerogenic dendritic cells

Background

Administration of interferon-α (IFN-α) represents an approved adjuvant therapy as reported for malignancies like melanoma and several viral infections. In malignant diseases, tolerance processes are critically involved in tumor progression. In this study, the effect of IFN-α on tolerance induction by human tolerogenic dendritic cells (DC) was analyzed. We focussed on tolerogenic IL-10-modulated DC (IL-10 DC) that are known to induce anergic regulatory T cells (iTregs).

Methodology/Principal Findings

IFN-α promoted an enhanced maturation of IL-10 DC as demonstrated by upregulation of the differentiation marker CD83 as well as costimulatory molecules. IFN-α treatment resulted in an increased capacity of DC to stimulate T cell activation compared to control tolerogenic DC. We observed a strengthened T cell proliferation and increased IFN-γ production of CD4⁺ and CD8⁺ T cells stimulated by IFN-α-DC, demonstrating a restoration of the immunogenic capacity of tolerogenic DC in the presence of IFN-α. Notably, restimulation experiments revealed that IFN-α treatment of tolerogenic DC abolished the induction of T cell anergy and suppressor function of iTregs. In contrast, IFN-α neither affected the priming of iTregs nor converted iTregs into effector T cells.

Conclusions/Significance

IFN-α inhibits the induction of T cell tolerance by reversing the tolerogenic function of human DC.