Maturation of innate responses to mycobacteria over the first nine months of life.

Newborns and young infants are particularly susceptible to infections, including Mycobacterium tuberculosis. Further, immunogenicity of vaccines against tuberculosis and other infectious diseases appears suboptimal early in life compared with later in life. We hypothesized that developmental changes in innate immunity would underlie these observations. To determine the evolution of innate responses to mycobacteria early in life, whole blood or PBMC from newborns, as well as 10- and 36-wk-old infants, was incubated with viable Mycobacterium bovis bacillus Calmette-Guérin or TLR ligands. Innate cell expression of cytokines and maturation markers was assessed, as well as activation of the proinflammatory NF-κB- and MAPK-signaling pathways. Bacillus Calmette-Guérin-induced production of the proinflammatory cytokines TNF-α, IL-6, and IL-12p40 increased from the newborn period to 9 mo of age in monocytes but not in myeloid dendritic cells. No changes in production of anti-inflammatory IL-10 were observed. CD40 expression increased with age in both cell populations. Older infants displayed substantial activation of all three signal transduction molecules: degradation of NF-κB inhibitor IκBα and phosphorylation of MAPK Erk and p38 upon TLR1/2 triggering, compared with predominant activation of only one of any of these molecules in newborns. Maturation of innate proinflammatory responses during the first 9 mo of life may underlie more effective control of mycobacteria and other pathogens observed later in infancy and age-related differential induction of Th1 responses by vaccination.

Optimization of a whole blood intracellular cytokine assay for measuring innate cell responses to mycobacteria.

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 mDC 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 mDC 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 200 µL per condition, making the assay particularly suitable for pediatric studies.

Novel application of Ki67 to quantify antigen-specific in vitro lymphoproliferation.

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.