Journal: Nature Medicine
Article Title: IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure
Figure Lengend Snippet: Qualitatively distinct PPD-specific antibody responses in ‘resisters’ compared with LTBI individuals. a , Graphs depict the AUCs calculated from the ratio of live to total intracellular bacterial burden in primary human monocyte-derived macrophages after treatment with purified IgG at 0.1 mg ml –1 , 0.01 mg ml –1 and 0.001 mg ml –1 (left) from ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI controls ( n = 39). Extension of analysis to additional donors was performed at a single concentration of purified IgG of 0.1 mg ml –1 due to sample availability (middle). Levels of secreted IL-1β from supernatants were measured by ELISA and are shown relative to no antibody treatment. Purified IgG from individuals in this study, with culture-confirmed pulmonary TB (ATB), is shown as a benchmark. Each line represents one healthy macrophage donor individual. For dot plots, lines are medians. The statistical significance was calculated using Wilcoxon’s matched-pairs signed rank, and two-tailed P values are shown. b , The calculated avidity against PPD from pooled plasma from ‘resisters’ ( n = 40), TST/IGRA-positive LTBI controls ( n = 39) and healthy, HIV-uninfected North Americans ( n = 10) are shown, with lines representing the fitted curves and dotted lines the 95% confidence intervals. Each plasma group was tested in triplicate, with associated calculated avidity represented in the dot plot. The statistical significance was calculated using the Student’s t -test, and a two-tailed P value is indicated. OD, optical density or absorbance. c – e , Plasma from ‘resisters’ ( n = 40) and LTBI controls ( n = 39) was assayed for the ability to mediate: PPD and ESAT6/CFP10-specific, antibody-dependent, monocyte-mediated cellular phagocytosis ( c ); PPD-specific, antibody-dependent neutrophil phagocytosis ( d ); and PPD-specific, NK cell activation by CD107a expression, macrophage inflammatory protein-1β and IFN-γ production ( e ). Data are representative of experiments performed in duplicate over three dilutions. Assays utilizing primary human neutrophils ( d ) and NK cells ( e ) were additionally performed utilizing three independent, healthy, HIV-negative donors. f , Affinity for FcγR2A(R), FcγR2A(H), FcγR3A(V) and FcγR3A(F) were determined using customized Luminex to PPD in ‘resisters’ ( n = 40) and LTBI individuals ( n = 39), using plasma diluted at 1:100. MFI is shown on the graph. The statistical significance was calculated using the Mann–Whitney U test, and P values are indicated. Dotted lines represent the median level detected in HIV-negative, healthy North American volunteers. g , Ratios of plasma levels of IgM, IgG and IgA1 reactive to PPD and LAM in ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI controls ( n = 39) are depicted with medians and interquartile ranges. The statistical significance was calculated using the Mann–Whitney U test, and two-tailed P values are indicated. h , Ratios of plasma levels of IgG1, IgG2, IgG3 and IgG4 reactive to PPD in ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI individuals ( n = 39) were measured by customized multiplex Luminex in serial dilutions. AUCs are depicted with medians and interquartile ranges. The statistical significance was calculated using the Mann–Whitney U test, and two-tailed P values are indicated. i , The relative distribution of glycoform substructures isolated from non-antigen-specific and PPD-specific IgG are depicted, with each column representing each individual. j , Principal component analysis demonstrates the overlapping profiles of ‘resisters’ ( n = 40) and TST/IGRA-positive LTBI individuals ( n = 39) in the dominant total glycans isolated from non-antigen-specific IgG compared with partially separating profiles from PPD-specific IgG. ADCP, antibody-dependent cellular phagocytosis; ADNP, antibody-dependent neutrophil phagocytosis; MIP, macrophage inflammatory protein.
Article Snippet: Briefly, plasma samples were diluted 1:50 in TSM buffer containing 0.05% Tween-20 and 1% BSA and incubated on the slides for 1 h. Slides were washed in TSM wash buffer (TSM-0.05%, Tween-20), and incubated simultaneously with secondary antibodies to IgG and IgM (Invitrogen, 5 μg ml–1 each) for 1 h to detect the presence of anti-glycan antibodies in the plasma.
Techniques: Derivative Assay, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Activation Assay, Expressing, Luminex, MANN-WHITNEY, Laser Capture Microdissection, Multiplex Assay, Isolation