Human macrophage model for selective evaluation of CD8⁺ and γδ⁺ cytotoxic T cell function in tuberculosis

The human macrophage cell line U937 was investigated as an in vitro model for human macrophage function in mycobacterial infections. This involved evaluating the ability of differentiated U937 cells to phagocytose Mycobacterium tuberculosis, control intracellular mycobacterial growth, and present mycobacterial antigens to human HLA class I-matched cytotoxic T lymphocytes (CTLs). Differentiation of U937 cells usmg IFN-γ, 1,25-(OH)₂ vitamin D₃, or PMA significantly enhanced their ability to phagocytose M. tuberculosis but failed to induce a subsequent respiratory burst response. Following infection, U937 cells were found to be permissive to the intracellular growth of both the virulent H37Rv strain of M. tuberculosis and the attenuated vaccine strain of M. bovis BCG. U937 cells have been shown to constitutively express high levels of cell surface HLA class I while expressing undetectable levels of HLA class II both at the mRN A level and at the cell surface. HLA class II expression was neither up-regulated following infection with M. tuberculosis nor inducible using IFN-γ, 1,25-(OH)₂ vitamin D₃, PMA, GM-CSF or a combination of these agents. In contrast, chronic infection of U937 cells with virulent H37Rv M. tuberculosis (but not with BCG) resulted in the cell surface expression of HLA class I being significantly up-regulated. Taken together, these characteristics made U937 cells a very attractive model for further investigations into their ability to present mycobacterial antigens to human HLA class I-restricted CTLs. Differentiation of U937 cells was found to completely abrogate their sensitivity to non-antigen specific cytolysis mediated by NK or LAK cells. Following infection with M. tuberculosis, U937 target cells were lysed by M. tuberculosis-primed CTLs from HLA class I-matched donors in an antigen-specific manner and with a similar efficiency to autologous macrophage targets. This cytolytic activity was restricted to live organisms since only U937 cells infected with virulent H37Rv M. tuberculosis and BCG but not those pulsed with soluble PPD were lysed by the HLA class I-matched effector cells. On the other hand, M. tuberculosisstimulated but ALA-mismatched CTLs failed to lyse infected U937 cells in an antigen-specific manner. T cell subset fractionation of the HLA class I-matched M. tuberculosis-primed CTL population and limiting dilution cloning demonstrated that the cytolytic activity was mediated by CD8⁺ cytolytic T cells and confirmed that CD4⁺ T cells showed no significant ability to lyse infected U937 target cells. Furthermore, this study found that M. tuberculosis-infected U937 target cells were lysed by CD8⁺ CTLs more rapidly and strongly than similarly infected autologous macrophage targets demonstrating the sensitivity of this in vitro model as an indicator for CD8⁺ cytolytic function in mycobacterial infections. M. tuberculosis-infected U937 cells were found to be highly sensitive to mycobacterial antigenspecific cytolysis mediated by γδ⁺ CTL. Mycobacterial antigen-specific γδ⁺ CTLs consistently showed stronger cytolytic activity against infected U937 target cells than γδ⁺ CTL but were not restricted to classical HLA class I or class II molecules. A panel of cytolytic human M. tuberculosis-reactive γδ⁺ CTL clones was established to investigate more thoroughly the role of γδ⁺ CTL lytic activity in human mycobacterial infections. This study examined the mechanism of cellular cytotoxicity used by these mycobacterial-specific γδ⁺ CTL clones against infected U93 7 targets and further investigated the effect of γδ⁺ T cell-mediated cytolysis on intracellular mycobacterial survival. Cytolysis mediated by the γδ⁺ T cell clones was found to be dependent on cell-to-cell contact. Furthermore, the ability of the γδ⁺ CTL clones to lyse infected targets was found to be strongly Ca²⁺-dependent, sensitive to cyclosporine A (a specific inhibitor of granule exocytosis ), and completely abrogated following Sr²⁺-induced de-granulation of the γδ⁺ T cell effectors, indicating that cytoxicity was mediated predominantly by the granule exocytosis/ perforin pathway. Despite being strongly cytolytic against infected U937 cells, however, the γδ⁺ CTL clones did not have any impact on the survival of intracellular M. tuberculosis. The major conclusions of this study are that U937 cells not only provide a useful in vitro human macrophage model allowing for selective evaluation of HLA class I-restricted CD8⁺ CTL function in mycobacterial infections but also provided a highly sensitive indicator for γδ⁺ CTL cytolytic activity

Vaginal microbiomes associated with aerobic vaginitis and bacterial vaginosis

A healthy vaginal microbiota is considered to be significant for maintaining vaginal health and preventing infections. However, certain vaginal bacterial commensal species serve an important first line of defense of the body. Any disruption of this microbial barrier might result in a number of urogenital conditions including aerobic vaginitis (AV) and bacterial vaginosis (BV). The health of the vagina is closely associated with inhabitant microbiota. Furthermore, these microbes maintain a low vaginal pH, prevent the acquisition of pathogens, stimulate or moderate the local innate immune system, and further protect against complications during pregnancies. Therefore, this review will focus on vaginal microbial “health” in the lower reproductive tract of women and on the physiological characteristics that determine the well-being of reproductive health. In addition, we explore the distinct versus shared characteristics of BV and AV, which are commonly associated with increased risk for preterm delivery

Genital inflammation, immune activation and risk of sexual HIV acquisition.

CAPRISA, 2016.Abstract available in pdf

Modulation of female genital tract-derived dendritic cell migration and activation in response to inflammatory cytokines and toll-like receptor agonists.

CAPRISA, 2016.Abstract available in pdf

Interleukin-10 Promoter Polymorphisms Influence HIV-1 Susceptibility and Primary HIV-1 Pathogenesis.

Interleukin (IL)–10 directly inhibits human immunodeficiency virus type 1 (HIV-1) replication, but it may also promote viral persistence by inactivation of effector immune mechanisms. Here, we show in an African cohort that individuals with genotypes associated with high IL-10 production at 2 promoter single-nucleotide polymorphisms ( 1082 and 592) were less likely to become HIV-1 infected but had significantly higher median plasma viral loads during the acute phase ( 3 months after infection). However, as the infection progressed, the association between genotype and median viral load was reversed. Thus, IL-10 may influence HIV-1 susceptibility and pathogenesis, but effects on the latter may differ according to the infection phase

Cervicovaginal inflammation facilitates acquisition of less infectious HIV variants.

CAPRISA, 2017.Abstract available in pdf

Genital—systemic chemokine gradients and the risk of HIV acquisition in women.

CAPRISA, 2017.Abstract available in pdf

Randomized cross-sectional study to compare HIV-1 specific antibody and cytokine concentrations in female genital secretions obtained by menstrual cup and cervicovaginal lavage.

CAPRISA, 2015.Abstract available in pdf

Broadly neutralizing antibody specificities detected in the genital tract of HIV-1 infected women.

CAPRISA, 2016.Abstract available in PDF file.

Inflammatory cytokine biomarkers to identify women with asymptomatic sexually transmitted infections and bacterial vaginosis who are at high risk of HIV infection.

CAPRISA, 2016.Abstract available in pdf