Effect of Antiretroviral Therapy on the Memory and Activation Profiles of B Cells in HIV-Infected African Women.

Human immunodeficiency virus infection induces a wide range of effects in B cells, including skewed memory cell differentiation, compromised B cell function, and hypergammaglobulinemia. However, data on the extent to which these B cell abnormalities can be reversed by antiretroviral therapy (ART) are limited. To investigate the effect of ART on B cells, the activation (CD86) and differentiation (IgD, CD27, and CD38) profiles of B cells were measured longitudinally in 19 HIV-infected individuals before (median, 2 mo) and after ART initiation (median, 12 mo) and compared with 19 age-matched HIV-uninfected individuals using flow cytometry. Twelve months of ART restored the typical distribution of B cell subsets, increasing the proportion of naive B cells (CD27-IgD+CD38-) and concomitantly decreasing the immature transitional (CD27-IgD+CD38+), unswitched memory (CD27+IgD+CD38-), switched memory (CD27+IgD-CD38- or CD27-IgD-CD38-), and plasmablast (CD27+IgD-CD38high) subsets. However, B cell activation was only partially normalized post-ART, with the frequency of activated B cells (CD86+CD40+) reduced compared with pre-ART levels (p = 0.0001), but remaining significantly higher compared with HIV-uninfected individuals (p = 0.0001). Interestingly, unlike for T cell activation profiles, the extent of B cell activation prior to ART did not correlate with HIV plasma viral load, but positively associated with plasma sCD14 levels (p = 0.01, r = 0.58). Overall, ART partially normalizes the skewed B cell profiles induced by HIV, with some activation persisting. Understanding the effects of HIV on B cell dysfunction and restoration following ART may provide important insights into the mechanisms of HIV pathogenesis.

Residual T cell activation and skewed CD8+ T cell memory differentiation despite antiretroviral therapy-induced HIV suppression.

HIV infection results in excessive T cell activation and dysfunction which may persist even during effective antiretroviral therapy (ART). The dynamics of immune 'deactivation' and extent to which T cell memory subsets normalize after ART are unclear. We longitudinally assessed the influence of 1 year of ART on the phenotype of T cells in HIV-infected African women, relative to matched HIV-uninfected women, using activation (CD38, HLA-DR) and differentiation markers (CD27, CD45RO). ART induced a substantial reduction in T cell activation, but remained higher than HIV-uninfected controls. ART largely normalized the distribution of CD4+ T cell memory subsets, while the distribution of CD8+ T cell memory subsets remained significantly skewed compared to HIV-uninfected individuals. Thus, there was a considerable but only partial reversal of T cell defects upon ART. Understanding T cell impairment may provide important insights into mechanisms of HIV pathogenesis in the era of ART.

HIV Skews the Lineage-Defining Transcriptional Profile of Mycobacterium tuberculosis-Specific CD4+ T Cells.

HIV-infected persons are at greater risk of developing tuberculosis (TB) even before profound CD4 loss occurs, suggesting that HIV alters CD4(+) T cell functions capable of containing bacterial replication. An effective immune response to Mycobacterium tuberculosis most likely relies on the development of a balanced CD4 response, in which distinct CD4(+) Th subsets act in synergy to control the infection. To define the diversity of M. tuberculosis-specific CD4(+) Th subsets and determine whether HIV infection impacts such responses, the expression of lineage-defining transcription factors T-bet, Gata3, RORγt, and Foxp3 was measured in M. tuberculosis-specific CD4(+) T cells in HIV-uninfected (n = 20) and HIV-infected individuals (n = 20) with latent TB infection. Our results show that, upon 5-d restimulation in vitro, M. tuberculosis-specific CD4(+) T cells from healthy individuals have the ability to exhibit a broad spectrum of Th subsets, defined by specific patterns of transcription factor coexpression. These transcription factor profiles were skewed in HIV-infected individuals where the proportion of T-bet(high)Foxp3(+) M. tuberculosis-specific CD4(+) T cells was significantly decreased (p = 0.002) compared with HIV-uninfected individuals, a change that correlated inversely with HIV viral load (p = 0.0007) and plasma TNF-α (p = 0.027). Our data demonstrate an important balance in Th subset diversity defined by lineage-defining transcription factor coexpression profiles that is disrupted by HIV infection and suggest a role for HIV in impairing TB immunity by altering the equilibrium of M. tuberculosis-specific CD4(+) Th subsets.

Effect of HIV on the Frequency and Number of Mycobacterium tuberculosis-Specific CD4+ T Cells in Blood and Airways During Latent M. tuberculosis Infection.

Human immunodeficiency virus type 1 (HIV) infection substantially increases the risk of developing tuberculosis. There is extensive depletion of Mycobacterium tuberculosis-specific CD4+ T cells in blood during early HIV infection, but little is known about responses in the lungs at this stage. Given that mucosal organs are a principal target for HIV-mediated CD4+ T-cell destruction, we investigated M. tuberculosis-specific responses in bronchoalveolar lavage (BAL) from persons with latent M. tuberculosis infection and untreated HIV coinfection with preserved CD4+ T-cell counts. M. tuberculosis-specific CD4+ T-cell cytokine (interferon γ, tumor necrosis factor α, and interleukin 2) responses were discordant in frequency and function between BAL and blood. Responses in BAL were 15-fold lower in HIV-infected persons as compared to uninfected persons (P = .048), whereas blood responses were 2-fold lower (P = .006). However, an increase in T cells in the airways in HIV-infected persons resulted in the overall number of M. tuberculosis-specific CD4+ T cells in BAL being similar. Our study highlights the important insights gained from studying M. tuberculosis immunity at the site of disease during HIV infection.

Restoration of CD4+ Responses to Copathogens in HIV-Infected Individuals on Antiretroviral Therapy Is Dependent on T Cell Memory Phenotype.

Antiretroviral therapy (ART) induces rapid suppression of viral replication and a progressive replenishment of CD4(+) T cells in HIV-infected individuals. However, the effect of ART on restoring pre-existing memory CD4(+) T cells specific for common copathogens is still unclear. To better understand the dynamics of Ag-specific CD4(+) T cells during ART, we assessed the frequency, functional capacity, and memory profile of CD4(+) T cells specific for Mycobacterium tuberculosis and CMV in 15 HIV-infected individuals before and 1 y after ART initiation. After ART initiation, the frequency of M. tuberculosis-specific CD4(+) T cells showed little change, whereas CMV-specific CD4(+) T cells were significantly lower (p = 0.003). There was no difference in the polyfunctional or memory profile of Ag-specific CD4(+) T cells before and after ART. The replenishment of Ag-specific CD4(+) T cells correlated with the memory differentiation profile of these cells prior to ART. Pathogen-specific CD4(+) T cells exhibiting a late differentiated profile (CD45RO(+)CD27(-)) had a lower capacity to replenish (p = 0.019; r = -0.5) compared with cells with an early differentiated profile (CD45RO(+)CD27(+); p = 0.04; r = 0.45). In conclusion, restoration of copathogen-specific memory CD4(+) T cells during treated HIV infection is related to their memory phenotype, in which early differentiated cells (such as most M. tuberculosis-specific cells) have a higher replenishment capacity compared with late differentiated cells (such as most CMV-specific cells). These data identify an important, hitherto unrecognized, factor that may limit restoration of copathogen immunity in HIV-infected individuals on ART.

The novel capripoxvirus vector lumpy skin disease virus efficiently boosts modified vaccinia Ankara human immunodeficiency virus responses in rhesus macaques.

Poxvirus vectors represent promising human immunodeficiency virus (HIV) vaccine candidates and were a component of the only successful HIV vaccine efficacy trial to date. We tested the immunogenicity of a novel recombinant capripoxvirus vector, lumpy skin disease virus (LSDV), in combination with modified vaccinia Ankara (MVA), both expressing genes from HIV-1. Here, we demonstrated that the combination regimen was immunogenic in rhesus macaques, inducing high-magnitude, broad and balanced CD4(+) and CD8(+) T-cell responses, and transient activation of the immune response. These studies support further development of LSDV as a vaccine vector.

Longitudinal changes in CD4(+) T-cell memory responses induced by BCG vaccination of newborns.

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 CD45RACCR7 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.

Dysregulation of the Immune Environment in the Airways During HIV Infection.

HIV-1 increases susceptibility to pulmonary infection and disease, suggesting pathogenesis in the lung. However, the lung immune environment during HIV infection remains poorly characterized. This study examined T cell activation and the cytokine milieu in paired bronchoalveolar lavage (BAL) and blood from 36 HIV-uninfected and 32 HIV-infected participants. Concentrations of 27 cytokines were measured by Luminex, and T cells were phenotyped by flow cytometry. Blood and BAL had distinct cytokine profiles (p=0.001). In plasma, concentrations of inflammatory cytokines like IFN-γ (p=0.004) and TNF-α (p=0.004) were elevated during HIV infection, as expected. Conversely, BAL cytokine concentrations were similar in HIV-infected and uninfected individuals, despite high BAL viral loads (VL; median 48,000 copies/ml epithelial lining fluid). HIV-infected individuals had greater numbers of T cells in BAL compared to uninfected individuals (p=0.007); and BAL VL positively associated with CD4+ and CD8+ T cell numbers (p=0.006 and p=0.0002, respectively) and CXCL10 concentrations (p=0.02). BAL T cells were highly activated in HIV-infected individuals, with nearly 2-3 fold greater frequencies of CD4+CD38+ (1.8-fold; p=0.007), CD4+CD38+HLA-DR+ (1.9-fold; p=0.0006), CD8+CD38+ (2.8-fold; p=0.0006), CD8+HLA-DR+ (2-fold; p=0.022) and CD8+CD38+HLA-DR+ (3.6-fold; p<0.0001) cells compared to HIV-uninfected individuals. Overall, this study demonstrates a clear disruption of the pulmonary immune environment during HIV infection, with readily detectable virus and activated T lymphocytes, which may be driven to accumulate by local chemokines.

Qualification of a whole blood intracellular cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity by flow cytometry.

BACKGROUND: Qualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity. METHODS: Whole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period. RESULTS: The limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-γ- and IL-2-expressing T cells and less than 0.01% for TNF-α- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4 °C or 20-23 °C) and time (45, 60 or 90 min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells. CONCLUSIONS: The whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.

Bacillus Calmette-Guérin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles.

The immune response to vaccination with bacillus Calmette-Guérin (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-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-gamma, IL-2, and TNF-alpha in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-gamma and/or IL-2 and less TNF-alpha, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-gamma. 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 Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-gamma+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-gamma 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.