B-cells from TB patients as well as LTBI individuals have an impaired function.
Total B-cells were isolated from PBMCs using magnetic bead separation for CD19 and stimulated with the combination of anti-CD40 and anti IgG/ IgM activating antibodies to obtain maximal, polyclonal B-cell activation. Data on isolated B-cells were obtained from 17 controls, 14 LTBI individuals, 13 TB patients and 16 treated TB patients. Lines indicate the median values of all groups. Ethnicity of donors was indicated using the following symbols: ‘black circle’ = West Europe; ‘black diamond’ = Est Europe; ‘black square’ = Africa; ‘black triangle’ = Asia; ‘black star’ = Sud America. Mann-Whitney test was performed to compare infected groups to the uninfected controls and a p< 0.05 was considered significant. * marks differences that remained significant after multiple test correction using Kruskal-Wallis testing with Dunn’s post-test. A. Isolated total CD19+ B-cells were labelled with a violet cell proliferation dye, proliferation in unstimulated conditions was subtracted from the anti-CD40 + purified IgG/ IgM stimulated conditions to obtain the delta proliferation reflecting the stimulation induced proliferation. Proliferation was assessed after 6 days of stimulation. B. Cytokine production measured by intracellular cytokine staining by flow cytometry for IL-6 (B) and IL-10 (C). Boxes express the 25–75% of data, with a line at median and whiskers indicate 5–95% data points. Cytokine production was measured after 2 days of stimulation; cytokines in unstimulated conditions were subtracted from stimulated conditions. C. Immunoglobulin production was measured in supernatants of unstimulated, isolated B-cells on day 2 of culture using multiplex bead array and expressed as total pg/ml for all isotypes combined. Lines indicate median values for each group. D. Immunoglobulin production per isotype, expressed as mean production per group of individuals for IgA, IgM, IgE and IgG. E. HLA-DR expression on B-cells following stimulation with anti-CD40 + anti IgG/ IgM expressed within the total CD19+ B-cells. Lines indicate median values.
