Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population

Abstract

The determinants of immunological protection against Mycobacterium tuberculosis (M.tb) infection in humans are not known. Mycobacterial growth inhibition assays have potential utility as in vitro surrogates of in vivo immunological control of M.tb. We evaluated a whole blood growth inhibition assay in a setting with high burden of TB and aimed to identify immune responses that correlate with control of mycobacterial growth. We hypothesized that individuals with underlying M.tb infection will exhibit greater M.tb growth inhibition than uninfected individuals and that children aged 4 to 12 years, an age during which TB incidence is curiously low, will also exhibit greater M.tb growth inhibition than adolescents or adults. Neither M.tb infection status, age of the study participants, nor M.tb strain was associated with differential control of mycobacterial growth. Abundance and function of innate or T cell responses were also not associated with mycobacterial growth. Our data suggest that this assay does not provide a useful measure of age-associated differential host control of M.tb infection in a high TB burden setting. We propose that universally high levels of mycobacterial sensitization (through environmental non-tuberculous mycobacteria and/or universal BCG vaccination) in persons from high TB burden settings may impart broad inhibition of mycobacterial growth, irrespective of M.tb infection status. This sensitization may mask the augmentative effects of mycobacterial sensitization on M.tb growth inhibition that is typical in low burden settings.

Fig 1. In vitro mycobacterial growth inhibition in M.tb-infected (QFT+) and uninfected (QFT-) individuals.

Inhibition of M.tb H37Rv growth by whole blood from QFT+ and QFT- adults (A), young adults (B) and children (C) assessed using a mycobacterial growth inhibition assay (MGIA). The growths of M. bovis BCG, M.tb H37Rv, M.tb isolate CDC1551 and M.tb isolate HN878 in the whole blood of adults (D) and young adults (E), respectively were compared. Spearman’s correlation analyses of the growth of different mycobacterial strains (F and G). The growth of BCG (H), HN878 and CDC1551 (I and J), was measured in the whole blood of adults and young adults, respectively. The red and blue circles represent QFT+ and QFT- individuals, respectively while the green and orange circles represent children and young adults respectively. The horizontal line represents the median, the boxes represent the interquartile range, and the whiskers represent the range. Differences in mycobacterial growth inhibition between both groups of individuals were evaluated with the Mann-Whitney test (shown P values).

Fig 2. In vitro mycobacterial growth inhibition in children and young adults.

Inhibition of M.tb H37Rv growth by whole blood from QFT- and QFT+ 8 and 18 year olds. Green and orange circles represent children and young adults, respectively. The horizontal line represents the median, the boxes represent the interquartile range, and the whiskers represent the range. Differences in mycobacterial growth inhibition between both groups of individuals were evaluated with the Mann-Whitney test (shown P values).

Fig 3. Detection of GFP-expressing BCG by innate cells and association between absolute numbers of innate cells and mycobacterial growth inhibition.

(A) Representative flow cytometry plot of IL-6, IL-12 and TNF-α cytokine expression by myeloid dentritic cells (mDCs), monocytes and neutrophils, measured in whole blood stimulated for 6 hours with BCG, BCG-GFP (shown) or LPS, relative to an unstimulated control sample. (B) Representative histograms indicating proportions of innate cells that phagocytosed BCG-GFP (green). (C) Absolute numbers of innate cell subsets per milliliter of unstimulated whole blood plotted against M.tb H37Rv growth. R and p values were calculated using Spearman’s correlation. (D) Absolute numbers of BCG-GFP-positive mDCs, monocytes or neutrophils per mL of whole blood in adult individuals, stratified by QFT status. The inclusion of TruCount beads during the cell staining steps of the innate whole blood assay allowed determination of the absolute number of each subset of cells per mL of whole blood. The red and blue circles represent QFT+ and QFT- adults, respectively. Horizontal lines represent medians and whiskers, the interquartile range. Differences in absolute counts of BCG-GFP-positive innate cells between the groups were evaluated with the Mann-Whitney test (shown P values). The pie charts show relative proportions of BCG-GFP-positive cells among each innate cell subset.

Fig 4. Mycobacteria-specific cytokine expression by innate cells in whole blood from adults and mycobacterial growth inhibition.

(A) Absolute numbers of total cytokine expressing innate cells per mL of whole blood plotted against M.tb H37Rv growth. R and p values were calculated using Spearman’s correlation analysis. (B) Numbers of BCG-specific mDCs, monocytes and neutrophils co-expressing IL-6, IL-12 and/or TNF-α in whole blood from QFT+ (red) and QFT- (blue) adults. Medians are represented by the horizontal line, interquartile ranges by the boxes, and ranges by the whiskers. The Mann-Whitney test was used to assess differences between QFT+ and QFT- adults and none were found to be different.

Fig 5. Representative flow cytometry plots of cytokine expression by CD4 T cells.

CD4 T cells expressing IFN-γ, IL-2, TNF-α and/or IL-17 upon stimulation with BCG or ESAT-6/CFP-10 peptide pools for 12 hours, compared to an unstimulated control sample. The plots represent cytokine-positive cells (red) overlayed onto the background of the entire CD4 T cell parent population (black). Similar assessment was also performed for CD8 and γδ T cells (data not shown).

Fig 6. Mycobacteria-specific T cells in whole blood from adults and mycobacterial growth inhibition.

(A) Absolute numbers of BCG-specific CD4, CD8 and γδ T cell subsets co-expressing IL-2, IFN-γ, TNF-α and/or IL-17, in whole bood from QFT+ (red) and QFT- (blue) adults. Medians are represented by the horizontal lines, interquartile ranges by the boxes, and ranges by the whiskers. The Mann-Whitney test was used to assess differences between QFT+ and QFT- adults and none were found to be different. (B) M.tb H37Rv growth plotted against frequencies of BCG-specific CD4, CD8 or γδ T cells expressing IFN-γ, or CD4 T cells expressing IL-17 in adults. R and p values were calculated using Spearman’s correlation analysis.