Prolonged exposure to <i>M. bovis</i> BCG affects NK cell cytoxicity.

<p>Human NK cells isolated from the blood of healthy donors were cultured in the presence or absence of interleukin-2 (IL-2) (100U/ml) and/or <i>M. bovis</i> BCG (MOI 1:4). A) Cell supernatants from a single donor were assayed for IFN-γ every 24h and for up to 96h post-culture. B) Recovered NK cells were assayed for cytotoxicity against the erythroleukemia line, K562, at an effector:target ratio of 1:1. C) and D) Bar graph showing significant reduction of NK cell cytotoxicity against K562 cells in the presence of IL-2 following 72h of exposure or not with mycobacteria from independent experiment and donor (Mean +/- SD of technical triplicates, unpaired t test, **p<0.01).</p

IL-2 induction of NKp44 expression on CD56^bright NK cells is inhibited by mycobacteria.

<p>A) FACS plot analysis of NKp44 expression from NK cells cultured in the presence or the absence of IL-2 (100U/ml) and or <i>M. bovis</i> BCG (MOI 1:5) for 5 days. B) Bar graphs showing B) significant increase of CD56 MFI of the NKp44⁺ NK cell population and C) significant decreased frequencies of IL-2 induced NKp44⁺ NK cell in the presence of mycobacteria from three independent experiments and donors (Paired t test, *p<0.05, **p<0.01).</p

IL-2 dependent induction of NK cell apoptosis by <i>M. bovis</i> BCG.

<p>A) Representative FACS dot-plot of purified NK cells co-cultured or not with <i>M. bovis</i> BCG (MOI 1:5) and IL-2 (100U/ml) for 4 days before BrdU incorporation, antibody detection and 7AAD staining (One of three technical replicates). B) Bar graph showing on the left, the frequency of replicating events (BrdU⁺/7AAD⁺) and, on the right, the frequency of apoptotic events (BrdU^-/7AAD^low) of NK cells from one donor exposed or not to mycobacteria and IL-2 (Mean +/- SD of technical triplicates, unpaired t test, *** p<0.001). C) Frequencies of early (PI⁺/Annex V^-) versus late apoptotic events (PI⁺/Annex V⁺) over the time of purified NK cells cultured or not with <i>M. bovis</i> BCG (MOI 1:1) and IL-2 (100U/ml). D) Bar graph summarizing frequencies of apoptotic events after 48h of culture with or without mycobacteria and IL-2 from independent experiments and donors (n=3, paired t test, *p<0.05).</p

<i>M. bovis</i> BCG inhibits IL-2 induced proliferation of CD56^bright NK cells.

<p>A) Purified NK cells from a healthy human donor were labelled with CFSE and cultured for 7 days in the presence of IL-2 (100U/ml) +/- <i>M. bovis</i> BCG at various MOI before flow cytometry analysis. Dose dependent inhibition of NK cell proliferation by mycobacteria was observed. B) Joined dot plot illustrating the reproducibility of NK cell proliferation inhibition by mycobacteria across different NK cell preparation across independent donors (n=4, paired t test, **p<0.01).</p

IFN-γ producing NK cells in response to mycobacteria express higher levels of CD56.

<p>Human NK cells were cultured in the presence of IL-2 (100U/ml) and/or <i>M. bovis</i> BCG (MOI 1:1) for a total of 24h including Brefeldin A treatment. A) FACS dot-plot showing the gating strategy to distinguish IFN-γ producing NK cells following BCG stimulation. B) CD56 overlay histogram comparing the expression of CD56 between IFN-γ producing and non-producing NK cells. C) Bar graph showing significant increase of CD56 mean fluorescence intensity (MFI) from IFN-γ producing NK cells following BCG stimulation across different donors (n=4, mean +/- SD, paired t test, **p<0.01).</p

CD27 and Mip-1β expression of CMV specific IFNγ producing CD4 T cell responses are influenced by HIV as well as tuberculosis co-infection in adults.

<p>Scatter-plots illustrating (A) the CD27 MFI fold-change to all CD4⁺ T cells and B) the proportion of cells producing Mip-1β of CMV specific IFNγ⁺-CD4⁺ T cell in patients under 10 or over 18 years old and according to HIV and TB infection status.</p

CD27 and Mip-1β expression of CMV specific IFNγ producing CD8 T cell responses are not significantly influenced by HIV as well as tuberculosis co-infection in adults.

<p>Scatter-plots illustrating (A) the CD27 MFI fold-change to all CD8⁺ T cells and B) the proportion of cells producing Mip-1β of CMV specific IFNγ⁺-CD8⁺ T cell in patients under 10 or over 18 years old and according to HIV and TB infection status.</p

Frequencies and absolute counts of IFNγ⁺-SEB–specific CD4⁺ or CD8⁺ T cell in children below 10 years of age and adults.

<p>Scatter-plots depicting A) the frequencies or B) absolute counts of SEB specific IFNγ⁺-CD4⁺ T cells s and C) frequencies of SEB specific IFNγ⁺-CD8⁺ T cells in the two-age groups and according to HIV status.</p

CD27 and Mip-1β expression of CMV specific IFNγ producing CD4 T cell responses are influenced by HIV as well as tuberculosis co-infection in adults.

<p>Scatter-plots illustrating (A) the CD27 MFI fold-change to all CD4⁺ T cells and B) the proportion of cells producing Mip-1β of CMV specific IFNγ⁺-CD4⁺ T cell in patients under 10 or over 18 years old and according to HIV and TB infection status.</p

Gating strategies and CMV responses evaluation.

<p>A) Morphological and CD marker expression gating strategy of CD4 and CD8 T cell. B) Representative CD4 and CD8 T cell IFNγ response from a significant responder. C) Gating strategy for the CD27 MFI Fold Change calculation of IFNγ producing cells compared to all CD4 or CD8 T cells. D) Gating strategy for the determination of IFNγ producing cells that were also positive for Mip1β production.</p