Multifunctional CD4+ T cells correlate with active Mycobacterium tuberculosis infection

Th1 CD4⁺ T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN‐γ/IL‐2/TNF‐α triple expressors, IFN‐γ/IL‐2, IFN‐γ/TNF‐α or TNF‐α/IL‐2 double expressors or IFN‐γ, IL‐2 or TNF‐α single expressors) of CD4⁺ T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85–90%TB patients, were only present in 10–15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12‐ to 15‐fold) proportions of IL‐2/IFN‐γ double and IFN‐γ single expressors as compared with the other CD4⁺ T‐cell subsets. Proportions of the other double or single CD4⁺ T‐cell expressors did not differ between TB and LTBI subjects. These distinct IFN‐γ, IL‐2 and TNF‐α profiles of M. tuberculosis‐specific CD4⁺ T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB‐infected patients after completion of anti‐mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4⁺ T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti‐mycobacterial therapy.     

Multi‐functional flow cytometry analysis of CD4+ T cells as an immune biomarker for latent tuberculosis status in patients treated with tumour necrosis factor (TNF) antagonists

Although monitoring tuberculosis (TB) infection during long‐term treatment with tumour necrosis factor (TNF) antagonists is of great importance, no monitoring strategy has yet proved successful. Indeed, even the newly proposed interferon‐gamma release assays (IGRAs) are known to produce dynamic changes in IFN‐γ plasma levels, making them unreliable indicators of patients' pathological/clinical status. We used intracellular cytokine flow cytometry (ICCFC) to investigate the performance of multi‐functional CD4⁺ T cells producing IFN‐γ, interleukin (IL)‐2 and/or TNF in response to Mycobacterium tuberculosis‐specific antigens in subjects treated with TNF antagonists. Patients were classified into three groups based on their TB status before commencement of treatment and on IFN‐γ level fluctuations evaluated by IGRA during a 36‐month follow‐up period. The cytokine profile of M. tuberculosis‐specific CD4⁺ T cells showed that latent tuberculosis infection (LTBI) subjects had a higher frequency of double‐positive IFN‐γ⁺ IL‐2⁺ CD4⁺ T cells and triple‐positive IFN‐γ⁺ IL‐2⁺ TNF⁺ CD4⁺ T cells compared to those without LTBI, who showed IFN‐γ‐level fluctuations over time. In contrast, this latter group of patients showed similar proportions of cells producing IFN‐γ alone, IL‐2 alone and IL‐2 in combination with TNF in response to M. tuberculosis‐specific antigens. It therefore appears that patients with and without LTBI infection are characterized by different intracellular cytokine profiles. This is the first study evaluating ICCFC in patients treated with TNF antagonists, and suggests that multi‐functional analysis of CD4⁺ T cells could be useful for ruling out TB infection in patients classified at screening as LTBI‐negative but who show IGRA fluctuations under long‐term TNF antagonist treatment.     

Differences in T‐cell responses between Mycobacterium tuberculosis and Mycobacterium africanum‐infected patients

In The Gambia, Mycobacterium tuberculosis (Mtb) and Mycobacterium africanum (Maf) are major causes of tuberculosis (TB). Maf is more likely to cause TB in immune suppressed individuals, implying differences in virulence. Despite this, few studies have assessed the underlying immunity to the two pathogens in human. In this study, we analyzed T‐cell responses from 19 Maf‐ and 29 Mtb‐infected HIV‐negative patients before and after TB chemotherapy following overnight stimulation of whole blood with TB‐specific antigens. Before treatment, percentages of early secreted antigenic target‐6(ESAT‐6)/culture filtrate protein‐10(CFP‐10) and purified protein derivative‐specific single‐TNF‐α‐producing CD4⁺ and CD8⁺ T cells were significantly higher while single‐IL‐2‐producing T cells were significantly lower in Maf‐ compared with Mtb‐infected patients. Purified protein derivative‐specific polyfunctional CD4⁺ T cells frequencies were significantly higher before than after treatment, but there was no difference between the groups at both time points. Furthermore, the proportion of CD3⁺CD11b⁺ T cells was similar in both groups pretreatment, but was significantly lower with higher TNF‐α, IL‐2, and IFN‐γ production in Mtb‐ compared with that of Maf‐infected patients posttreatment. Our data provide evidence of differences in T‐cell responses to two mycobacterial strains with differing virulence, providing some insight into TB pathogenesis with different Mtb strains that could be prospectively explored as biomarkers for TB protection or susceptibility.     

Correlation Between Natural Cytotoxicity Receptors and Intracellular Cytokine Expression of Peripheral Blood NK Cells in Women with Recurrent Pregnancy Losses and Implantation Failures

Problem Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. We investigated whether women with recurrent pregnancy losses (RPLs) and implantation failures have aberrant correlation between NCRs and intracellular cytokine expression of NK cells. Method of study Peripheral blood NK cells (CD56^dim and CD56^bright) were analyzed for NCRs (NKp46, NKp44 and NKp30) and cytokine expression (TNF‐α, IFN‐γ, IL‐4, IL‐10) using flow cytometry in RPL (n = 22), implantation failures (n = 23) or controls (n = 15). Results In type 1 cytokine studies, CD56^bright/NKp30⁺ cells in controls (r = 0.696, P < 0.05) were positively correlated with CD56^bright/IFN‐γ⁺/TNF‐α⁺ cells. CD56^bright/NKp46⁺ cells in implantation failures (r = ?0.76, P < 0.01) were negatively correlated with CD56^bright/IFN‐γ⁺/TNF‐α^? cells. RPL did not have any correlation. In type 2 cytokine studies, CD56⁺/NKp46⁺ cells (r = 0.758, P < 0.01) and CD56⁺/NKp30⁺ cells (r = 0.637, P < 0.05) were positively correlated with CD56^bright/IL‐4⁺/IL‐10⁺ cells in controls. CD56⁺/NKp30⁺ cells in implantation failures (r = ?0.778, P < 0.05) were negatively correlated with CD56^bright/IL‐10⁺/IL‐4⁺ cells. There were no correlations in RPL. Conclusion Recurrent pregnancy losses and implantation failures have lack of, or negative correlation between NCRs and intracellular cytokines expression. This observation suggests that excessive pro‐inflammatory cytokine expression in NK cells in RPL and implantation failures may be exerted through the NCRs or interruption of signal transduction processes.     

Intracellular cytokine production in peripheral blood lymphocytes: a comparison of values in infertile and fertile women

Citation
Horká P, Jaro?ová R, Malí?ková K, Janatková I, Mare?ková H, Zima T, Kalousová M. Intracellular cytokine production in peripheral blood lymphocytes: a comparison of values in infertile and fertile women. Am J Reprod Immunol 2011; 65: 466–469 Problem To analyze the relation of the fertility and pregnancy of women of childbearing age to the intracellular (IC) production of tumor necrosis factor alpha (TNF‐α), interferon gamma (IFN‐γ), and interleukins 2 and 4 (IL‐2 and IL‐4). Method of study Intracellular cytokine production in peripheral blood CD3⁺ CD4⁺ lymphocytes was analyzed by flow cytometry in 185 women being treated for infertility and 50 fertile women of childbearing age. Results Infertile women have a significantly higher IC production of TNF‐α, IFN‐γ, IL‐2, and IL‐4 and higher ratios of TNF‐α/IL‐2, TNF‐α/IL‐4, and TNF‐α/IFN‐γ compared to the fertile women. Conclusion Cytokines produced by Th lymphocytes are important in orchestrating the immune response during conception, and Th‐cell dysregulation could be a reason for infertility.     

Modified vaccinia Ankara‐expressing Ag85A,a novel tuberculosis vaccine,is safe in adolescents and children,and induces polyfunctional CD4+ T cells

Modified vaccinia Ankara‐expressing Ag85A (MVA85A) is a new tuberculosis (TB) vaccine aimed at enhancing immunity induced by BCG. We investigated the safety and immunogenicity of MVA85A in healthy adolescents and children from a TB endemic region, who received BCG at birth. Twelve adolescents and 24 children were vaccinated and followed up for 12 or 6 months, respectively. Adverse events were documented and vaccine‐induced immune responses assessed by IFN‐γ ELISpot and intracellular cytokine staining. The vaccine was well tolerated and there were no vaccine‐related serious adverse events. MVA85A induced potent and durable T‐cell responses. Multiple CD4⁺ T‐cell subsets, based on expression of IFN‐γ, TNF‐α, IL‐2, IL‐17 and GM‐CSF, were induced. Polyfunctional CD4⁺ T cells co‐expressing IFN‐γ, TNF‐α and IL‐2 dominated the response in both age groups. A novel CD4⁺ cell subset co‐expressing these three Th1 cytokines and IL‐17 was induced in adolescents, while a novel CD4⁺ T‐cell subset co‐expressing Th1 cytokines and GM‐CSF was induced in children. Ag‐specific CD8⁺ T cells were not detected. We conclude that in adolescents and children MVA85A safely induces the type of immunity thought to be important in protection against TB. This includes induction of novel Th1‐cell populations that have not been previously described in humans.     

CD4+CD25highforkhead box protein 3+ regulatory T lymphocytes suppress interferon‐γ and CD107 expression in CD4+ and CD8+ T cells from tuberculous pleural effusions

Tuberculous pleural effusion is characterized by a T helper type 1 (Th1) profile, but an excessive Th1 response may also cause tissue damage that might be controlled by regulatory mechanisms. In the current study we investigated the role of regulatory T cells (T_reg) in the modulation of Th1 responses in patients with tuberculous (TB) pleurisy. Using flow cytometry we evaluated the proportion of T_reg (CD4⁺CD25^highforkhead box protein 3⁺), interferon (IFN)‐γ and interleukin (IL)‐10 expression and CD107 degranulation in peripheral blood (PB) and pleural fluid (PF) from patients with TB pleurisy. We demonstrated that the proportion of CD4⁺CD25⁺, CD4⁺CD25^highFoxP3⁺ and CD8⁺CD25⁺ cells were increased in PF compared to PB samples. Mycobacterium tuberculosis stimulation increased the proportion of CD4⁺CD25^low/negIL‐10⁺ in PB and CD4⁺CD25^low/negIFN‐γ⁺ in PF; meanwhile, CD25^high mainly expressed IL‐10 in both compartments. A high proportion of CD4⁺CD107⁺ and CD8⁺CD107⁺ cells was observed in PF. T_reg depletion enhanced the in‐vitro M. tuberculosis‐induced IFN‐γ and CD4⁺ and CD8⁺ degranulation responses and decreased CD4⁺IL‐10⁺ cells in PF. Our results demonstrated that in TB pleurisy T_reg cells effectively inhibit not only IFN‐γ expression but also the ability of CD4⁺ and CD8⁺ cells to degranulate in response to M. tuberculosis.     

Lack of Mycobacterium tuberculosis–specific interleukin‐17A–producing CD4+ T cells in active disease

Protective immunity to Mycobacterium tuberculosis (Mtb) is commonly ascribed to a Th1 profile; however, the involvement of Th17 cells remains to be clarified. Here, we characterized Mtb‐specific CD4⁺ T cells in blood and bronchoalveolar lavages (BALs) from untreated subjects with either active tuberculosis disease (TB) or latent Mtb infection (LTBI), considered as prototypic models of uncontrolled or controlled infection, respectively. The production of IL‐17A, IFN‐γ, TNF‐α, and IL‐2 by Mtb‐specific CD4⁺ T cells was assessed both directly ex vivo and following in vitro antigen‐specific T‐cell expansion. Unlike for extracellular bacteria, Mtb‐specific CD4⁺ T‐cell responses lacked immediate ex vivo IL‐17A effector function in both LTBI and TB individuals. Furthermore, Mtb‐specific Th17 cells were absent in BALs, while extracellular bacteria‐specific Th17 cells were identified in gut biopsies of healthy individuals. Interestingly, only Mtb‐specific CD4⁺ T cells from 50% of LTBI but not from TB subjects acquired the ability to produce IL‐17A following Mtb‐specific T‐cell expansion. Finally, IL‐17A acquisition by Mtb‐specific CD4⁺ T cells correlated with the coexpression of CXCR3 and CCR6, currently associated to Th1 or Th17 profiles, respectively. Our data demonstrate that Mtb‐specific Th17 cells are selectively undetectable in peripheral blood and BALs from TB patients.     

Functional deficits of pertussis-specific CD4(+) T cells in infants compared to adults following DTaP vaccination

Understanding the immune responses that explain why infants require multiple doses of pertussis vaccine to achieve protection against infection is a high priority. The objective of this study was to compare the function and phenotypes of antigen‐specific CD4⁺ T cells in adults (n = 12), compared to infants (n = 20), following vaccination with acellular pertussis (DTaP) vaccine. Peripheral blood mononuclear cells (PBMCs) were stimulated with pertussis toxoid (PT), pertactin (PRN) and filamentous haemagglutinin (FHA). Multi‐parameter flow cytometry was used to delineate CD4⁺ T cell populations and phenotypes producing interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐α and IL‐4. Based on surface CD69 expression, infants demonstrated activation of vaccine antigen‐specific CD4⁺ T cells similar to adults. However, among infants, Boolean combinations of gates suggested that type 1 (Th‐1) CD4⁺ T cell responses were confined largely to TNF‐α⁺IL‐2⁺IFN‐γ^‐ or TNF‐α⁺IL‐2^‐IFN‐γ^‐. A significantly lower percentage of polyfunctional T helper type 1 (Th1) responses (TNF‐α⁺IFN‐γ⁺IL‐2⁺) and type 2 (Th2) responses (IL‐4) were present in the infants compared to adults. Moreover, a significantly higher percentage of infants' functional CD4⁺ T cells were restricted to CD45RA^‐CCR7⁺CD27⁺ phenotype, consistent with early‐stage differentiated pertussis‐specific memory CD4⁺ T cells. We show for the first time that DTaP vaccination‐induced CD4⁺ T cells in infants are functionally and phenotypically dissimilar from those of adults.     

Multiple Antigen Peptide Containing B and T Cell Epitopes of F1 Antigen of Yersinia pestis Showed Enhanced Th1 Immune Response in Murine Model

Yersinia pestis is a facultative bacterium that can survive and proliferate inside host macrophages and cause bubonic, pneumonic and systemic infection. Apart from humoral response, cell‐mediated protection plays a major role in combating the disease. Fraction 1 capsular antigen (F1‐Ag) of Y. pestis has long been exploited as a vaccine candidate. In this study, F1‐multiple antigenic peptide (F1‐MAP or MAP)‐specific cell‐mediated and cytokine responses were studied in murine model. MAP consisting of three B and one T cell epitopes of F1‐antigen with one palmitoyl residue was synthesized using Fmoc chemistry. Mice were immunized with different formulations of MAP in poly DL‐lactide‐co‐glycolide (PLGA) microspheres. F1‐MAP with CpG oligodeoxynucleotide (CpG‐ODN) as an adjuvant showed enhanced in vitro T cell proliferation and Th1 (IL‐2, IFN‐γ and TNF‐α) and Th17 (IL‐17A) cytokine secretion. Similar formulation also showed significantly higher numbers of cytokine (IL‐2, IFN‐γ)‐secreting cells. Moreover, F1‐MAP with CpG formulation showed significantly high (P < 0.001) percentage of CD4⁺ IFN‐γ⁺ cells as compared to CD8⁺ IFN‐γ⁺ cells, and also more (CD4‐ IFN‐γ)⁺ cells secrete perforin and granzyme as compared to (CD8‐ IFN‐γ)⁺ showing Th1 response. Thus, the study highlights the importance of Th1 cytokine and existence of CD4⁺ and CD8⁺ immune response. This study proposes a new perspective for the development of vaccination strategies for Y. pestis that trigger T cell immune response.     

Lipoarabinomannan‐specific TNF‐α and IFN‐γ as markers of protective immunity against tuberculosis: a cohort study in an endemic setting

Lipoarabinomannan (LAM) is a virulent factor used for entry and survival of Mycobacterium tuberculosis (Mtb) in macrophages. Although the role of LAM for the diagnosis of tuberculosis (TB) has been extensively investigated, its cytokine response during natural Mtb infection in humans is largely unknown. In this study, LAM‐specific IFN‐γ, TNF‐α, and IL‐10 levels following whole blood assay were measured in untreated pulmonary TB patients, their contacts and community controls at baseline. In treated patients and contacts, cytokines were also measured at 6 and 12 months. At entry, 52.8% and 74.8% of controls and contacts were QFT‐GIT positive, respectively. At baseline, untreated TB patients and contacts had significantly lower IFN‐γ and TNF‐α response compared to community controls (p < 0.0001). Besides, untreated patients had significantly higher TNF‐α and IL‐10 response compared to their contacts (p < 0.0001). At 6 months, contacts and treated TB patients had significantly increased INF‐γ and TNF‐α response (p < 0.0001). In TB patients, IFN‐γ increased 10‐fold following chemotherapy suggesting its potential role for treatment monitoring. The data suggests that LAM might have an anti‐inflammatory effect during clinical TB and early Mtb infection. The data also suggests that LAM‐induced IFN‐γ and TNF‐α could be used as biomarkers of protective immunity.     

Effector T‐cell differentiation during viral and bacterial infections: Role of direct IL‐12 signals for cell fate decision of CD8+ T cells

To study the role of IL‐12 as a third signal for T‐cell activation and differentiation in vivo, direct IL‐12 signaling to CD8⁺ T cells was analyzed in bacterial and viral infections using the P14 T‐cell adoptive transfer model with CD8⁺ T cells that lack the IL‐12 receptor. Results indicate that CD8⁺ T cells deficient in IL‐12 signaling were impaired in clonal expansion after Listeria monocytogenes infection but not after infection with lymphocytic choriomeningitis virus, vaccinia virus or vesicular stomatitis virus. Although limited in clonal expansion after Listeria infection, CD8⁺ T cells deficient in IL‐12 signaling exhibited normal degranulation activity, cytolytic functions, and secretion of IFN‐γ and TNF‐α. However, CD8⁺ T cells lacking IL‐12 signaling failed to up‐regulate KLRG1 and to down‐regulate CD127 in the context of Listeria but not viral infections. Thus, direct IL‐12 signaling to CD8⁺ T cells determines the cell fate decision between short‐lived effector cells and memory precursor effector cells, which is dependent on pathogen‐induced local cytokine milieu.     

Dendritic cells from human mesenteric lymph nodes in inflammatory and non‐inflammatory bowel diseases: subsets and function of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDC) in mesenteric lymph nodes (MLN) may be important regulators of both inflammatory and non‐inflammatory mucosal immune responses but human studies are rare. Here we compare pDC from human MLN and peripheral blood (PB) by phenotype and function. MLN from patients with or without inflammatory bowel disease (IBD) undergoing colon surgery and PB from patients with IBD and from controls were used to isolate mononuclear cells. The pDC were analysed by flow cytometry for the expression of CD40, CD80, CD83, CD86, CCR6, CCR7, CX3CR1, CD103 and HLA‐DR. Purified pDC from MLN and PB were stimulated with staphylococcus enterotoxin B (SEB), CpG‐A, interleukin‐3 (IL‐3), SEB + IL‐3, CpG‐A + IL‐3 or left unstimulated, and cultured alone or with purified allogeneic CD4⁺ CD45RA⁺ HLA‐DR‐ T cells. Subsequently, concentrations of IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, IL‐17, interferon‐α (IFN‐α), IFN‐γ and tumour necrosis factor‐α (TNF‐α) in culture supernatants were determined by multiplex bead array. The PB pDC from IBD patients exhibited an activated and matured phenotype whereas MLN pDC and control PB pDC were less activated. CpG‐A and CpG‐A + IL‐3‐stimulated MLN pDC secreted less IL‐6 and TNF‐α compared with PB pDC from controls. Compared with co‐cultures of naive CD4 T cells with PB pDC, co‐cultures with MLN pDC contained more IL‐2, IL‐10 and IFN‐γ when stimulated with SEB and SEB + IL‐3, and less IFN‐α when stimulated with CpG‐A. MLN pDC differ phenotypically from PB pDC and their pattern of cytokine secretion and may contribute to specific outcomes of mucosal immune reactions.     

Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4+ T cells

Human type I interferons (IFNs) include IFN‐β and 12 subtypes of IFN‐α. During viral infection, infiltrating memory CD4 ⁺ T cells are exposed to IFNs, but their impact on memory T‐cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN‐α8 and ‐α10 most potently enhanced expression of IFN‐γ, IL‐2, and IL‐4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL‐2 and IL‐4 correlated with the time of preincubation with type I IFN, IFN‐γ production was enhanced best when IFN‐α was added immediately preceding or simultaneously with T‐cell stimulation. Comparison of T‐cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN‐α best enhanced cytokine expression when CD4 ⁺ T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T‐cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4 ⁺ T‐cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation.     

Selective effects of Lactobacillus casei Shirota on T cell activation, natural killer cell activity and cytokine production

Modulation of host immunity is an important potential mechanism by which probiotics confer health benefits. This study was designed to investigate the effects of a probiotic strain, Lactobacillus casei Shirota (LcS), on immune function using human peripheral blood mononuclear cells (PBMC) in vitro. In addition, the role of monocytes in LcS‐induced immunity was also explored. LcS promoted natural killer (NK) cell activity and preferentially induced expression of CD69 and CD25 on CD8 ⁺ and CD56 ⁺ subsets in the absence of any other stimulus. LcS also induced production of interleukin (IL)‐1β, IL‐6, tumour necrosis factor (TNF)‐α, IL‐12 and IL‐10 in the absence of lipopolysaccharide (LPS). In the presence of LPS, LcS enhanced IL‐1β production but inhibited LPS‐induced IL‐10 and IL‐6 production, and had no further effect on TNF‐α and IL‐12 production. Monocyte depletion reduced significantly the impact of LcS on lymphocyte activation, cytokine production and natural killer (NK) cell activity. In conclusion, LcS activated cytotoxic lymphocytes preferentially in both the innate and specific immune systems, which suggests that LcS could potentiate the destruction of infected cells in the body. LcS also induced both proinflammatory and anti‐inflammatory cytokine production in the absence of LPS, but in some cases inhibited LPS‐induced cytokine production. Monocytes play an important role in LcS‐induced immunological responses.     

Impaired activation of Stat1 and c‐Jun as a possible defect in macrophages of patients with active tuberculosis

Studies of patients with active tuberculosis (TB) and infected healthy individuals have shown that interferon (IFN)‐γ is present in sites of Mycobacterium tuberculosis infection in comparable levels. This suggests that there is a deficiency in the macrophage response to IFN‐γ in TB patients. We used recombinant human IFN‐γ to stimulate adherent monocyte‐derived macrophages from three groups of people: patients with active tuberculosis (TBP), their healthy household contacts (HHC) and healthy uninfected controls from the community (CC). We then evaluated the ability of the macrophages to inhibit the growth of M. tuberculosis H37Rv as well as their cytokine profile at early in infection (48 h). After IFN‐γ treatment, macrophages of healthy individuals (HHC and CC) controlled M. tuberculosis growth and produced mainly nitric oxide (NO) and interleukin (IL)‐12p70, whereas TBP macrophages did not kill M. tuberculosis. Additionally, TBP macrophages produced low levels of NO and IL‐12p70 and high levels of tumour necrosis factor (TNF)‐α and IL‐10. Transforming growth factor (TGF)‐β levels were similar among all three groups. M. tuberculosis infection had little effect on the cytokine response after IFN‐γ stimulus, but infection alone induced more IL‐10 and TGF‐β in TBP macrophages. There were no differences in Stat1 nuclear translocation and DNA binding between the groups. However, the phosphorylated Stat1 and c‐Jun (AP‐1) in nuclear protein extracts was diminished in TBP macrophages compared to macrophages of healthy individuals. These results indicate an impairment of Stat1‐dependent and Stat1‐independent IFN‐γ signalling in macrophages of people with active tuberculosis, suggesting a different molecular regulation that could impact macrophage functionality and disease outcome.     

Mucosal delivery of antigen‐coated nanoparticles to lungs confers protective immunity against tuberculosis infection in mice

Mucosal boosting of BCG‐immunised individuals with a subunit tuberculosis (TB) vaccine would be highly desirable, considering that the lungs are the principal port of entry for Mycobacterium tuberculosis (MTB) and the site of the primary infection and reactivation. However, the main roadblock for subunit TB vaccine development is the lack of suitable adjuvants that could induce robust local and systemic immune responses. Here, we describe a novel vaccine delivery system that was designed to mimic, in part, the MTB pathogen itself. The surface of yellow carnauba wax nanoparticles was coated with the highly immunogenic Ag85B Ag of MTB and they were directed to the alveolar epithelial surfaces by the incorporation of the heparin‐binding hemagglutinin adhesion (HBHA) protein. Our results showed that the i.n. immunisation of BCG‐primed BALB/c mice with nanoparticles adsorbed with Ag85B‐HBHA (Nano‐AH vaccine) induced robust humoral and cellular immune responses and IFN‐γ production, and multifunctional CD4⁺ T cells expressing IFN‐γ, IL‐2 and TNF‐α. Mice challenged with H37Rv MTB had a significantly reduced bacterial load in their lungs when compared with controls immunised with BCG alone. We therefore conclude that this immunisation approach is an effective means of boosting the BCG‐induced anti‐TB immunity.     

Activation of p38 mitogen-activated protein kinase is critical step for acquisition of effector function in cytokine-activated T cells, but acts as a negative regulator in T cells activated through the T-cell receptor

Peripheral blood CD4⁺ CD45RO⁺ T cells activated in vitro are able to induce expression of tumour necrosis factor‐α (TNF‐α) in monocytes via a contact‐dependent mechanism. Activation is achieved either with interleukin‐2 (IL‐2)/IL‐6/TNF‐α over an 8‐day period or cross‐linking CD3 using anti‐CD3 antibody for 48 hr. In this paper, we show that the p38 mitogen‐activated protein kinase (MAPK) signalling pathway played different roles in the generation of effector function in these two types of activated T cells. In anti‐CD3 activated T cells, p38 MAPK is a negative regulator for anti‐CD3 induced cell proliferation and has no significant effect on the acquisition of either the effector function (induction of monocyte‐derived TNF‐α) or production of T‐cell cytokines. In contrast, the p38 MAPK signalling pathway is required for the acquisition of cytokine‐induced effector function and promotes cell proliferation and cytokine production.