Increased susceptibility to tuberculosis following HIV-1 seroconversion contributes significantly to the tuberculosis epidemic in sub-saharan Africa. Lung specific mechanisms underlying the interaction between HIV-1 and Mycobacterium (M.) tuberculosis infection are incompletely understood. This study addressed the effect of HIV-1 and latent M. tuberculosis infection on viral-entry receptors and ligands in bronchoalveolar lavage (BAL). Median fluorescence intensity (MFI) of entry receptor expression was measured by multiparameter flow cytometry and chemokine expression by multiplex bead array.
Irrespective of HIV-1 status, BAL T-cells expressed higher MFI for the beta-chemokine receptor (CCR)5 than peripheral blood T-cells (p<0.001), in particular the CD8+ T-cells of HIV-1 infected persons showed elevated CCR5 expression (p=0.026). The concentration of BAL CCR5 ligands, regulated upon activation normal T-cell expressed and secreted (RANTES; p<0.001) and macrophage inflammatory protein (MIP)-1β (p=0.004) were elevated in the BAL of HIV-1 infected persons compared to controls. CCR5 expression and RANTES concentration correlated strongly with HIV-1 viral load in BAL. By contrast these alterations were not associated with M. tuberculosis sensitization in vivo nor did M. tuberculosis infection of BAL cells ex vivo change RANTES expression.
These data suggest ongoing HIV-1 replication predominantly drives local pulmonary CCR5+ T-cell activation in HIV/latent M. tuberculosis co-infection.
BAL; CCR5; RANTES; TB; viral load
We investigated the relationship between varicella zoster virus (VZV) specific memory CD4+ T cells and CD4+Foxp3+ regulatory T cells (Tregs) that accumulate after intradermal challenge with a VZV skin test antigen. VZV-specific CD4+ T cells were identified with a MHC class II tetramer or by intracellular staining for either IFN-γ or IL-2 after antigen re-challenge in vitro. VZV-specific T cells, mainly of a central memory (CD45RA−CD27+) phenotype, accumulate at the site of skin challenge compared to the blood of the same individuals. This resulted in part from local proliferation since >50% of tetramer defined antigen-specific CD4+ T cells in the skin expressed the cell cycle marker Ki67. CD4+Foxp3+ T cells had the characteristic phenotype of Tregs, namely CD25hiCD127loCD39hi in both unchallenged and VZV challenged skin and did not secrete IFN-γ or IL-2 after antigenic re-stimulation. The CD4+Foxp3+ T cells from unchallenged skin had suppressive activity, since their removal led to an increase in cytokine secretion after activation. After VZV antigen injection, Foxp3+CD25hiCD127loCD39hi T cells were also found within the VZV tetramer population. Their suppressive activity could not be directly assessed by CD25 depletion since activated T cells in the skin were also CD25+. Nevertheless there was an inverse correlation between decreased VZV skin responses and proportion of CD4+Foxp3+ T cells present indicating indirectly, their inhibitory activity in vivo. These results suggest a linkage between the expansion of antigen-specific CD4+ T cells and CD4+ Tregs that may provide controlled responsiveness during antigen-specific stimulation in tissues.
Background. Improved vaccination strategies against tuberculosis are needed, such as approaches to boost immunity induced by the current vaccine, BCG. Design of these strategies has been hampered by a lack of knowledge of the kinetics of the human host response induced by neonatal BCG vaccination. Furthermore, the functional and phenotypic attributes of BCG-induced long-lived memory T-cell responses remain unclear.
Methods. We assessed the longitudinal CD4+ T-cell response following BCG vaccination of human newborns. The kinetics, function, and phenotype of these cells were measured using flow cytometric whole-blood assays.
Results. We showed that the BCG-specific CD4+ T-cell response peaked 6–10 weeks after vaccination and gradually waned over the first year of life. Highly activated T-helper 1 cells, predominantly expressing interferon γ, tumor necrosis factor α, and/or interleukin 2, were present at the peak response. Following contraction, BCG-specific CD4+ T cells expressed high levels of Bcl-2 and displayed a predominant CD45RA–CCR7+ central memory phenotype. However, cytokine and cytotoxic marker expression by these cells was more characteristic of effector memory cells.
Conclusions. Our findings suggest that boosting of BCG-primed CD4+ T cells with heterologous tuberculosis vaccines may be best after 14 weeks of age, once an established memory response has developed.
Bacille Calmette-Guérin; Vaccination; Newborns; Memory T cells; T cell kinetics
Innate cells are essential for host defense against invading pathogens, and the induction and direction of adaptive immune responses to infection. We developed and optimized a flow cytometric assay that allows measurement of intracellular cytokine expression by monocytes, dendritic cells (DC) and granulocytes, as well as cellular uptake of green-fluorescent protein (GFP)-expressing mycobacteria, in very small volumes of peripheral blood.
We show that innate cell stimulation resulted in increased granularity of monocytes and mDCs and decreased granulocyte granularity that precluded flow cytometric discernment of granulocytes from monocytes and myeloid DC by forward and side scatter gating. Anti-CD66a/c/e antibody staining allowed reliable identification and exclusion of granulocytes for subsequent delineation of monocytes and myeloid DC. Intracellular cytokine expression by granulocytes, monocytes and mDC was remarkably sensitive to the dose of mycobacterial inoculum. Moreover, activation of monocytes and mDCs with live BCG reduced expression levels of CD14 and CD11c, respectively, necessitating optimization of staining conditions to reliably measure these lineage markers. Finally, we characterized expression of IL-12/23p40, TNF-α, IL-6, and IL-10, by GFP+ and GFP− monocytes and mDC from 25 healthy adults.
This assay may be applied to the study of innate cell responses to any GFP-expressing pathogen, and can be performed on blood volumes as low as 200µL per condition, making the assay particularly suitable for pediatric studies.
Mycobacteria; flow cytometry; monocytes; dendritic cells; granulocytes; innate cytokines
Vaccination against tuberculosis (TB) should provide long-term protective immunity against Mycobacterium tuberculosis (M.tb). The current TB vaccine, Bacille Calmette-Guerin (BCG), protects against disseminated childhood TB, but protection against lung TB in adolescents and adults is variable and mostly poor. One potential reason for the limited durability of protection may be waning of immunity through gradual attrition of BCG-induced T cells. We determined if a MVA85A viral-vector boost could enhance the durability of mycobacteria-specific T cell responses above those induced by BCG alone.
We describe a long-term follow-up study of persons previously vaccinated with MVA85A. We performed a medical history and clinical examination, a tuberculin skin test and measured vaccine-specific T cell responses in persons previously enrolled as adults, adolescents, children or infants into three different Phase II trials, between 2005 and 2011.
Of 252 potential participants, 183 (72.6%) consented and completed the study visit. Vaccine-induced Ag85A-specific CD4+ T cell responses were remarkably persistent in healthy, HIV-uninfected adults, adolescents, children and infants, up to 6 years after MVA85A vaccination. Specific CD4+ T cells expressed surface markers consistent with either CD45RA−CCR7+ central memory or CD45RA−CCR7− effector memory T cells. Similarly durable Ag85A-specific CD4+ T cell responses were detected in HIV-infected persons who were on successful antiretroviral therapy when MVA85A was administered. By contrast, Ag85A-specific CD4+ T cell frequencies in untreated MVA85A-vaccinated HIV-infected persons were mostly undetectable 3–5 years after vaccination.
MVA85A induces remarkably durable T cell responses in immunocompetent persons. However, results from a recent phase IIb trial of MVA85A, conducted in infants from the same geographic area and study population, showed no vaccine efficacy, suggesting that these durable T cell responses do not enhance BCG-induced protection against TB in infants.
Rationale: Novel tuberculosis (TB) vaccines should be safe and effective in populations infected with Mycobacterium tuberculosis (M.tb) and/or HIV for effective TB control.
Objective: To determine the safety and immunogenicity of MVA85A, a novel TB vaccine, among M.tb- and/or HIV-infected persons in a setting where TB and HIV are endemic.
Methods: An open-label, phase IIa trial was conducted in 48 adults with M.tb and/or HIV infection. Safety and immunogenicity were analyzed up to 52 weeks after intradermal vaccination with 5 × 107 plaque-forming units of MVA85A. Specific T-cell responses were characterized by IFN-γ enzyme-linked immunospot and whole blood intracellular cytokine staining assays.
Measurements and Main Results: MVA85A was well tolerated and no vaccine-related serious adverse events were recorded. MVA85A induced robust and durable response of mostly polyfunctional CD4+ T cells, coexpressing IFN-γ, tumor necrosis factor-α, and IL-2. Magnitudes of pre- and postvaccination T-cell responses were lower in HIV-infected, compared with HIV-uninfected, vaccinees. No significant effect of antiretroviral therapy on immunogenicity of MVA85A was observed.
Conclusions: MVA85A was safe and immunogenic in persons with HIV and/or M.tb infection. These results support further evaluation of safety and efficacy of this vaccine for prevention of TB in these target populations.
tuberculosis; HIV-1; vaccine; MVA85A; clinical trial
One third of the world’s population is infected with Mycobacterium tuberculosis (M.tb). A vaccine that would prevent progression to TB disease will have a dramatic impact on the global TB burden. We propose that antigens of M.tb that are preferentially expressed during latent infection will be excellent candidates for post-exposure vaccination. We therefore assessed human T cell recognition of two such antigens, Rv2660 and Rv2659. Expression of these was shown to be associated with non-replicating persistence in vitro. After six days incubation of PBMC from persons with latent tuberculosis infection (LTBI) and tuberculosis (TB) disease, Rv2660 and Rv2659 induced IFN-γ production in a greater proportion of persons with LTBI, compared with TB diseased patients. Persons with LTBI also had increased numbers of viable T cells, and greater specific CD4+ T cell proliferation and cytokine expression capacity. Persons with LTBI preferentially recognize Rv2659 and Rv2660, compared with patients with TB disease. These results suggest promise of these antigens for incorporation into post-exposure TB vaccines.
Mycobacterium tuberculosis; LTBI; TB disease; latency antigens; post-infection vaccine
The lack of an effective TB vaccine hinders current efforts in combating the TB pandemic. One theory as to why BCG is less protective in tropical countries is that exposure to non-tuberculous mycobacteria (NTM) reduces BCG efficacy. There are currently several new TB vaccines in clinical trials, and NTM exposure may also be relevant in this context. NTM exposure cannot be accurately evaluated in the absence of specific antigens; those which are known to be present in NTM and absent from M. tuberculosis and BCG. We therefore used a bioinformatic pipeline to define proteins which are present in common NTM and absent from the M. tuberculosis complex, using protein BLAST, TBLASTN and a short sequence protein BLAST to ensure the specificity of this process. We then assessed immune responses to these proteins, in healthy South Africans and in patients from the United Kingdom and United States with documented exposure to NTM. Low level responses were detected to a cluster of proteins from the mammalian cell entry family, and to a cluster of hypothetical proteins, using ex vivo ELISpot and a 6 day proliferation assay. These early findings may provide a basis for characterising exposure to NTM at a population level, which has applications in the field of TB vaccine design as well as in the development of diagnostic tests.
Rationale: Immunogenicity of new tuberculosis (TB) vaccines is commonly assessed by measuring the frequency and cytokine expression profile of T cells.
Objectives: We tested whether this outcome correlates with protection against childhood TB disease after newborn vaccination with bacillus Calmette-Guérin (BCG).
Methods: Whole blood from 10-week-old infants, routinely vaccinated with BCG at birth, was incubated with BCG for 12 hours, followed by cryopreservation for intracellular cytokine analysis. Infants were followed for 2 years to identify those who developed culture-positive TB—these infants were regarded as not protected against TB. Infants who did not develop TB disease despite exposure to TB in the household, and another group of randomly selected infants who were never evaluated for TB, were also identified—these groups were regarded as protected against TB. Cells from these groups were thawed, and CD4, CD8, and γδ T cell–specific expression of IFN-γ, TNF-α, IL-2, and IL-17 measured by flow cytometry.
Measurements and Main Results: A total of 5,662 infants were enrolled; 29 unprotected and two groups of 55 protected infants were identified. There was no difference in frequencies of BCG-specific CD4, CD8, and γδ T cells between the three groups of infants. Although BCG induced complex patterns of intracellular cytokine expression, there were no differences between protected and unprotected infants.
Conclusions: The frequency and cytokine profile of mycobacteria-specific T cells did not correlate with protection against TB. Critical components of immunity against Mycobacterium tuberculosis, such as CD4 T cell IFN-γ production, may not necessarily translate into immune correlates of protection against TB disease.
mycobacteria immunity; pediatric settings
MVA85A is a new tuberculosis vaccine aimed at enhancing immunity induced by BCG. We investigated the safety and immunogenicity of MVA85A in healthy adolescents and children from a tuberculosis 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. Antigen-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 tuberculosis. This includes induction of novel Th1 cell populations which have not been previously described in humans.
MVA85A; tuberculosis; vaccine; polyfunctional; IL-17
Rationale: The current tuberculosis (TB) vaccine, bacille Calmette-Guérin (BCG), does not provide adequate protection against TB disease in children. Furthermore, more efficacious TB vaccines are needed for children with immunodeficiencies such as HIV infection, who are at highest risk of disease.
Objectives: To characterize mycobacteria-specific T cells in children who might benefit from vaccination against TB, focusing on responses to antigens contained in novel TB vaccines.
Methods: Whole blood was collected from three groups of BCG-vaccinated children: HIV-seronegative children receiving TB treatment (n = 30), HIV-infected children (n = 30), and HIV-unexposed healthy children (n = 30). Blood was stimulated with Ag85B and TB10.4, or purified protein derivative, and T-cell cytokine production by CD4 and CD8 was determined by flow cytometry. The memory phenotype of antigen-specific CD4 and CD8 T cells was also determined.
Measurements and Main Results: Mycobacteria-specific CD4 and CD8 T-cell responses were detectable in all three groups of children. Children receiving TB treatment had significantly higher frequencies of antigen-specific CD4 T cells compared with HIV-infected children (P = 0.0176). No significant differences in magnitude, function, or phenotype of specific T cells were observed in HIV-infected children compared with healthy control subjects. CD4 T cells expressing IFN-γ, IL-2, or both expressed a CD45RA−CCR7−CD27+/− effector memory phenotype. Mycobacteria-specific CD8 T cells expressed mostly IFN-γ in all groups of children; these cells expressed CD45RA−CCR7−CD27+/− or CD45RA+CCR7−CD27+/− effector memory phenotypes.
Conclusions: Mycobacteria-specific T-cell responses could be demonstrated in all groups of children, suggesting that the responses could be boosted by new TB vaccines currently in clinical trials.
pediatric; HIV-1; tuberculosis; vaccine; T cells
Antigen-specific proliferation is a critical function of memory T cells that is often utilised to measure vaccine immunogenicity and T cell function. We proposed that measurement of intracellular expression of the nuclear protein, Ki67, could reliably assess specific T cell proliferation in vitro.
Ki67 was expressed in CD4+ and CD8+ T cells that had undergone in vitro proliferation after 6-day culture of human whole blood or PBMC with antigens. T cells cultured with no antigen did not express Ki67. When compared to current flow cytometry based proliferation assays, Ki67 detected proliferating cells with greater sensitivity than BrdU incorporation, whereas its sensitivity was similar to dye dilution of Oregon Green (OG), a CFSE derivative. Overall, the magnitude and cytokine expression profile of proliferating T cells detected by Ki67 expression correlated strongly with T cells detected with BrdU or OG. The intra-assay variability of Ki67 proliferation was 2–3% for CD4+ T cells, and 10–16% for CD8+ T cells. Finally, we demonstrate that the Ki67 assay detects tetanus toxoid-specific CD4+ T cell proliferation after infant vaccination with tetanus toxoid (TT).
Overall our data suggest that intracellular Ki67 expression provides a specific, quantitative and reproducible measure of antigen-specific T cell proliferation in vitro.
PPD, purified protein derivative; TT, tetanus toxoid; OG, Oregon Green; Ki67; T cells; Cellular proliferation; Vaccine; Clinical immunology
In most tuberculosis (TB) endemic countries, bacillus Calmette Guérin (BCG) is usually given around birth to prevent severe TB in infants. The neonatal immune system is immature. Our hypothesis was that delaying BCG vaccination from birth to 10 weeks of age would enhance the vaccine-induced immune response.
In a randomized clinical trial, BCG was administered intradermally either at birth (n=25) or at 10 weeks of age (n=21). Ten weeks after vaccination, and at 1 year of age, vaccine-specific CD4 and CD8 T cell responses were measured with a whole blood intracellular cytokine assay.
Infants who received delayed BCG vaccination demonstrated higher frequencies of BCG-specific CD4 T cells, particularly polyfunctional T cells co-expressing IFN-γ, TNF-α and IL-2, and most strikingly at 1 year of age.
Delaying BCG vaccination from birth to 10 weeks of age enhances the quantitative and qualitative BCG-specific T cell response, when measured at one year of age.
BCG; vaccination; birth; delayed; polyfunctional CD4 T cells
Rationale: The risk of developing active tuberculosis in persons with latent Mycobacterium tuberculosis infection is substantially increased shortly after HIV-1 seroconversion. Immune responses in the lung are important to restrict the growth of M. tuberculosis to prevent the development of disease.
Objectives: To investigate innate and adaptive immune responses to M. tuberculosis in bronchoalveolar lavage from HIV-1–infected persons without active tuberculosis.
Methods: Peripheral blood was drawn and bronchoalveolar lavage (BAL) performed on healthy, HIV-1–uninfected (n = 21) and HIV-1–infected (n = 15) adults. Growth of M. tuberculosis was assessed in monocytes and alveolar macrophages. Cytokine expression by mycobacteria-specific CD4 and CD8 T cells was measured by intracellular cytokine staining or IFN-γ ELISpot.
Measurements and Main Results: Mycobacterial growth in monocytes or alveolar macrophages from HIV-1–infected and –uninfected persons did not differ. Total CD4 T-cell frequencies in BAL were lower in HIV-1–infected than in HIV-1–uninfected persons (P < 0.001). Mycobacteria (bacillus Calmette-Guérin)-specific CD4 T-cell responses in BAL were severely impaired: Frequencies of cells expressing IFN-γ or tumor necrosis factor (TNF)-α, as well as polyfunctional cells, expressing IFN-γ, TNF-α, and IL-2 together, were lower in HIV-1–infected persons than in uninfected controls (P < 0.01 for all).
Conclusions: In addition to a total CD4 T-cell deficit, the function of mycobacteria-specific CD4 T cells is significantly impaired in the lung of HIV-1–infected persons, which may account for the HIV-1–associated elevated risk for developing tuberculosis.
HIV-1; tuberculosis; immunity mucosal; T-cells; macrophages
The immune response to vaccination with Bacillus Calmette-Guerin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-week old infants, routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 hours induced expression of predominantly IFN-γ, IL-2 and TNF-α in CD4+ T cells, in 7 distinct cytokine combinations. IL-4 and IL-10 expression were detected in CD4+ T cells at low frequencies, and only in cells that did not co-express Type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells, and produced mainly IFN-γ and/or IL-2, and less TNF-α, IL-4 and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-γ. The predominant phenotype of BCG-specific Type 1 T cells was that of effector cells, i.e., CD45RA–CCR7–CD27+, which may reflect persistence of M. bovis BCG in infants until 10 weeks of age. Among 5 phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+, and effector cells more likely to be IFN-γ+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-γ production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination, and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.
Human; Th1/Th2 Cells; T cells; Cytokines; Memory; Vaccination
HIV-1 infection causes a severe T cell compromise; however, little is known about changes in naïve, memory, effector and senescent T cell subsets during the first year of life. T cell subsets were studied over the first year of life in blood from 3 infant cohorts: untreated HIV-infected, HIV-exposed but uninfected, and HIV-unexposed. In HIV-infected infants, the frequency of CCR7+CD45RA+ naïve CD8+ T cells was significantly decreased, whilst the frequency of CCR7−CD45RA− effector memory CD8+ T cells was increased, compared with the control cohorts. A larger population of CD8+ T cells in HIV-infected infants displayed a phenotype consistent with senescence. Differences in CD4+ T cell subset frequencies were less pronounced, and no significant differences were observed between exposed and unexposed HIV-uninfected infants. We concluded that the proportion of naïve, memory, effector and senescent CD8+ T cells during the first year of life is significantly altered by HIV-1 infection.
CD4; CD8; memory; HIV-1; infants
The efficacy of BCG may be enhanced by heterologous vaccination strategies that boost the BCG-primed immune response. One leading booster vaccine, MVA85A, has shown promising safety and immunogenicity in UK human trials. We investigated the safety and immunogenicity of MVA85A in mycobacteria-exposed, but Mycobacterium tuberculosis-uninfected, healthy adults from a TB-endemic region of South Africa.
Twenty-four adults were vaccinated with MVA85A. All subjects were followed up for one year for adverse events and for immunological assessment.
MVA85A vaccination was well tolerated and induced potent T cell responses, measured by IFN-γ ELISPOT assay, which exceeded pre-vaccination levels up to 364 days after vaccination. BCG-specific CD4+ T cells boosted by MVA85A comprised of multiple populations expressing combinations of IFN-γ, TNF-α, IL-2 and IL-17, as measured by polychromatic flow cytometry. IFN-γ expressing and polyfunctional IFN-γ+TNF-α+IL-2+ CD4+ T cells were boosted during the peak BCG-specific response 7 days post-vaccination.
The excellent safety profile and quantitative and qualitative immunogenicity data strongly support further trials to assess the efficacy of MVA85A as a boosting vaccine in TB endemic countries.
Vaccination; tuberculosis; T cells; MVA85A; South Africa
World-wide, most infants born to HIV-infected mothers receive BCG. Tuberculosis is a major cause of death of HIV-infected infants in sub-Saharan Africa, and should be prevented. However, BCG may itself cause disease (BCGosis) in these infants. Information regarding the immunogenicity of BCG is imperative for the risk/benefit assessment of BCG vaccination in HIV-infected infants; however, no such data exists.
We compared BCG-induced CD4 and CD8 T cell responses, assessed by flow cytometry, in HIV-infected (n = 20), HIV-exposed but uninfected (n = 25), and HIV-unexposed (n = 23) infants, over their first year of life.
BCG vaccination of the 2 HIV-uninfected groups induced a robust response, characterized by IFN-γ, TNF-α, and/or IL-2-expressing CD4 T cells. In contrast, HIV-infected infants had a markedly lower response, throughout the first year of life. These infants also had significantly reduced numbers of IFN-γ, TNF-α and IL-2 co-expressing polyfunctional CD4 T cells, thought to indicate T cell quality.
HIV-1 infection severely impairs the BCG-specific T cell response during the first year of life. BCG may therefore provide little, if any, vaccine-induced benefit in HIV-infected infants. Considering the significant risk of BCGosis, these data strongly support not giving BCG to HIV-infected infants.
Tuberculosis; BCG; HIV; infants; T cells; immune response; polyfuntional; Th1; Th17
The extracellular adhesion protein (Eap) secreted by the major human pathogen Staphylococcus aureus is known to have several effects on human immunity. We have recently added to knowledge of these roles by demonstrating that Eap enhances interactions between major histocompatibility complex molecules and human leukocytes. Several studies have indicated that Eap can induce cytokine production by human peripheral blood mononuclear cells (PBMCs). To date, there has been no rigorous attempt to identify the breadth of cytokines produced by Eap stimulation or to identify the cell subsets that respond. Here, we demonstrate that Eap induces the secretion of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) by CD14+ leukocytes (monocytes and macrophages) within direct ex vivo PBMC populations (note that granulocytes are also CD14+ but are largely depleted from PBMC preparations). Anti-intercellular adhesion molecule 1 (CD54) antibodies inhibited this induction and implicated a role for this known Eap binding protein in cellular activation. IL-6 and TNF-α secretion by murine cells exposed to Eap was also observed. The activation of CD14+ cells by Eap suggests that it could play a significant role in both septic shock and fever, two of the major pathological features of S. aureus infections.
We investigated whether the pro-inflammatory T cell cytokines interleukin-17 (IL-17) and IL-22 are induced by human mycobacterial infection. Remarkably, >20% of specific cytokine-producing CD4+ T cells in peripheral blood of healthy, mycobacteria-exposed adults expressed IL-17 or IL-22. Specific IL-17 and IL-22 producing CD4+ T cells were distinct from each other and from Th1 cytokine-producing cells. These cells had phenotypic characteristics of long-lived central memory cells. In patients with tuberculosis disease, peripheral blood frequencies of these cells were reduced, while bronchoalveolar lavage fluid (BALF) contained higher levels of IL-22 protein, compared with healthy controls. IL-17 was not detected in this fluid, which may be due to suppression by Th1 cytokines, as PBMC IL-17 production was inhibited by IFN-γ in vitro. However, Th1 cytokines had no effect on IL-22 production in vitro. Our results imply that the magnitude and complexity of the anti-mycobacterial immune response have historically been underestimated. IL-17 and IL-22-producing CD4+ T cells may play important roles in the human immune response to mycobacteria.
human; T cells; cytokines; bacterial infection
In this study, we report the use of peptide-major histocompatibility complex tetramer technology to study the interactions that occur between Staphylococcus aureus proteins and human leukocytes. We demonstrated that this technology can be used to study the activity of superantigens such as toxic shock syndrome toxin 1 and also found that despite similarities to known proteins (i.e., major histocompatibility complex [MHC] class II molecules and superantigens), the S. aureus Eap protein does not block MHC-T-cell receptor interactions and is not a superantigen. Instead, it has nonspecific cross-linking activity that is dependent upon having at least two of its six 110-amino-acid repeats.
Human immunodeficiency virus type 1 (HIV-1) evokes a strong immune response, but the virus persists. Polymorphisms within known antigenic sites result in loss of immune recognition and can be positively selected. Amino acid variation outside known HLA class I restricted epitopes can also enable immune escape by interfering with the processing of the optimal peptide antigen. However, the lack of precise rules dictating epitope generation and the enormous genetic diversity of HIV make prediction of processing mutants very difficult. Polymorphism E169D in HIV-1 reverse transcriptase (RT) is significantly associated with HLA-B*0702 in HIV-1-infected individuals. This polymorphism does not map within a known HLA-B*0702 epitope; instead, it is located five residues downstream of a HLA-B*0702-restricted epitope SPAIFQSSM (SM9). Here we investigate the association between E169D and HLA-B*0702 for immune escape via the SM9 epitope. We show that this single amino acid variation prevents the immune recognition of the flanked SM9 epitope by cytotoxic T cells through lack of generation of the epitope, which is a result of aberrant proteasomal cleavage. The E169D polymorphism also maps within and abrogates the recognition of an HLA-A*03-restricted RT epitope MR9. This study highlights the potential for using known statistical associations as indicators for viral escape but also the complexity involved in interpreting the immunological consequences of amino acid changes in HIV sequences.
HIV-specific CD4+ T helper lymphocytes are preferred targets for infection. Although complete interruption of combination antiretroviral therapy (ART) can form part of therapeutic manipulations, there is grave concern that the resumption of viral replication might destroy, perhaps irreversibly, these T helper populations. High viremia blocks the proliferation capacity of HIV-specific helper cells. However, cytokine production assays imply that some antigen-specific effector function is retained. Despite this careful work, it remains unclear whether the return of HIV-1 replication physically destroys HIV-1–specific T helper cells in the peripheral blood. Difficulties in producing stable peptide-MHC class II complexes and the very low frequencies of antigen-specific CD4+ T cells have delayed the application of this powerful technique. Here we employ HLA class II tetramers and validate a sensitive, quantitative cell-enrichment technique to detect HIV-1 T helper cells. We studied patients with early-stage HIV infection who were given a short, fixed course of ART as part of a clinical study. We did not find significant deletion of these cells from the peripheral circulation when ART was stopped and unfettered HIV replication returned. The turnover of these virus-specific cells increased and they adopted an effector phenotype when viremia returned.
Increased susceptibility to tuberculosis following HIV-1 seroconversion contributes significantly to the tuberculosis epidemic in sub-Saharan Africa. Lung-specific mechanisms underlying the interaction between HIV-1 and Mycobacterium tuberculosis infection are incompletely understood. Here we address these questions by examining the effect of HIV-1 and latent M. tuberculosis co-infection on the expression of viral-entry receptors and ligands in bronchoalveolar lavage (BAL) of HIV-1-infected and -uninfected patients with and without latent M. tuberculosis infection. Irrespective of HIV-1 status, T cells from BAL expressed higher levels of the beta-chemokine receptor (CCR)5 than peripheral blood T cells, in particular the CD8+ T cells of HIV-1-infected persons showed elevated CCR5 expression. The concentrations of the CCR5 ligands RANTES and MIP-1β were elevated in the BAL of HIV-1-infected persons compared with that in HIV-1-uninfected controls. CCR5 expression and RANTES concentration correlated strongly with HIV-1 viral load in the BAL. In contrast, these alterations were not associated with M. tuberculosis sensitisation in vivo, nor did M. tuberculosis infection of BAL cells ex vivo change RANTES expression. These data suggest ongoing HIV-1 replication predominantly drives local pulmonary CCR5+ T-cell activation in HIV/latent M. tuberculosis co-infection.
BAL; CCR5; RANTES; TB; Viral load
The inflammatory response to Mycobacterium tuberculosis (M.tb) at the site of disease is Th1 driven. Whether the Th17 cytokines, IL-17 and IL-22, contribute to this response in humans is unknown. We hypothesized that IL-17 and IL-22 contribute to the inflammatory response in pleural and pericardial disease sites of human tuberculosis (TB).
We studied pleural and pericardial effusions, established TB disease sites, from HIV-uninfected TB patients. Levels of soluble cytokines were measured by ELISA and MMP-9 by luminex. Bronchoalveolar lavage or pericardial mycobacteria-specific T cell cytokine expression was analyzed by intracellular cytokine staining.
IL-17 was not abundant in pleural or pericardial fluid. IL-17 expression by mycobacteria-specific disease site T cells was not detected in healthy, M.tb-infected persons, or patients with TB pericarditis. These data do not support a major role for IL-17 at established TB disease sites in humans.
IL-22 was readily detected in fluid from both disease sites. These IL-22 levels exceeded matching peripheral blood levels. Further, IL-22 levels in pericardial fluid correlated positively with MMP-9, an enzyme known to degrade the pulmonary extracellular matrix. We propose that our findings support a role for IL-22 in TB-induced pathology or the resulting repair process.
Pleural tuberculosis; Pericardial tuberculosis; IL-17; IL-22; Inflammation