A Functional Role for Antibodies in Tuberculosis

Abstract

While a third of the world carries the burden of tuberculosis, disease control has been hindered by a lack of tools, including a rapid, point-of-care diagnostic and a protective vaccine. In many infectious diseases, antibodies (Abs) are powerful biomarkers and important immune mediators. However, in Mycobacterium tuberculosis (Mtb) infection, a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach, we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses, such that Ltb infection is associated with unique Ab Fc functional profiles, selective binding to FcγRIII, and distinct Ab glycosylation patterns. Moreover, compared to Abs from Atb, Abs from Ltb drove enhanced phagolysosomal maturation, inflammasome activation, and, most importantly, macrophage killing of intracellular Mtb. Combined, these data point to a potential role for Fc-mediated Ab effector functions, tuned via differential glycosylation, in Mtb control.

Keywords: Fc-receptors; antibodies; inflammasome; innate immunity; tuberculosis.

Figure 1. Individuals with Ltb and Atb exhibit divergent humoral profiles

A least absolute shrinkage and selection operator (LASSO) (Tibshirani, 1997) identified nine Ab features (^ in Table 2) from the original 69 that best discriminate Ltb from Atb humoral profiles. Principal component analysis (PCA) using these nine features alone demonstrates the (A) dot plot of nearly non-overlapping Ab profiles in Ltb (n=22, blue) compared to Atb (n=20, red) individuals. The nine Ab features are represented in the Loadings Plot (B), a mirror image of the PCA dot plot, where the location of the Ab features reflects the distribution of the individuals in the dot plot (A).

Figure 2. Abs from Ltb and Atb individuals exhibit distinct Fc effector functional profiles

IgG purified from the serum of individuals with Ltb (n=22) and Atb (n=20) was assayed for its ability to mediate Fc effector functions including ADCC (A), Ab-dependent NK cell activation (B), Ab-dependent neutrophil phagocytosis (ADNP), (C) Ab-dependent cellular phagocytosis by THP1 monocytes (D), phagocytosis of uncoated beads by THP1 monocytes (E), and phagocytosis of influenza HA-coated beads (F) by THP1 monocytes. Functional differences were not due to endotoxin levels (Supplemental Figure 2L). Binding of polyclonal IgG to (G) FcγRIIa, (H) FcγRIIb, and (I) FcγRIIIa was evaluated by surface plasmon resonance. Integrated ratios of binding are plotted in (J) FcγRIIIa:FcγRIIb and (K) FcγRIIa:FcγRIIb. The error bars represent mean ± SEM of the individuals. p values were calculated using Mann Whitney tests and adjusted for age and sex. All experiments were run in triplicate. * p≤0.05; ** p≤0.01.

Figure 3. Abs are differentially glycosylated in individuals with Ltb and Atb

The heatmap illustrates the unsupervised clustering using one minus Pearson correlation of the Ab glycan profiles of Ltb (n=20, blue) and Atb (n=16, red) individuals from South Africa (A). Glycan structures are represented on the y-axis as the percent of all glycoform variants (z-scored). Dot plots represent differences between Ltb and Atb with respect to the major glycan categories: bisecting N-acetylglycosamine (GlcNAC-B: all structures containing a bisecting GlcNAc) (B and G), fucose (F: all structures containing fucose) (C and H), agalactosylated structures (G0: all structures lacking galactose) (D and I), digalactosylated structures (G2: all structures containing two galactose structures) (E and J), and sialylated structures (S: all structures with at least one sialic acid) (F and K). Corresponding diagrammatic representation of the common N-linked glycans quantified are shown. Dot plots in (B-F) derive from individuals from South Africa. Dot plots in (G-K) derive from individuals with Ltb (n=10) and Atb (n=10) from Texas/Mexico. Bars show mean of individuals ± SEM. p values were calculated using Mann Whitney tests, adjusted for age and sex, with a post-hoc Holms correction (Holm, 1979). ^& denotes the glycan features that remained significant following Holms correction. * p≤0.05; ** p≤0.01; *** p≤0.001.

Figure 4. Ltb compared to Atb Abs enhance macrophage activation and intracellular Mtb restriction

The dot plot represents Mtb uptake following Ltb or Atb Ab treatment compared to the no Ab control, with lines linking results from the same macrophage donor (A). Violin plots show the distribution of percent co-localization of bacteria within lysosomes measured in 200–600 macrophages per well (B). Representative ASC staining demonstrating the formation of perinuclear structure ASC specks (green) and Mtb (red) in the presence of Ltb Abs (C). ASC-speck numbers were quantified in 200–600 macrophages per well as a ratio to control non-specific pooled seronegative immunoglobulin (D). Inflammasome activity was further examined by IL1β release by ELISA following Mtb-infected macrophage treatment with Ltb or Atb Abs (E). Macrophage numbers were quantified by DAPI enumeration (F). Bacterial burden was ascertained by colony forming units in sextuplicate (G). Representative confocal microscopy panels show, following anhydrotetracycline induction of GFP expression in transcriptionally active bacteria, the constitutive mCherry fluorophore marking total bacterial burden and a GFP+ bacterial subpopulation within macrophages (DAPI) (H). Bacterial survival is plotted in the dot plot (live_GFP/total_MCH) in Mtb-infected macrophages derived from eight different macrophage donors. Lines depict comparison between same donor macrophages (I). Differences were not due to endotoxin levels (Supplemental Figure 2L and 5D). Wilcoxon matched-pairs signed rank (A, F and I), student t (D, E and G) and Mann Whitney (B) tests were used to calculate p values. Bars represent the mean of technical replicates ± SEM. * p≤0.05; ** p≤0.01; *** p≤0.001; ns= not significant.