Bacillus Calmette-Guérin (BCG) Revaccination of Adults with Latent Mycobacterium tuberculosis Infection Induces Long-Lived BCG-Reactive NK Cell Responses

Abstract

One third of the global population is estimated to be latently infected with Mycobacterium tuberculosis We performed a phase I randomized controlled trial of isoniazid preventive therapy (IPT) before revaccination with bacillus Calmette-Guérin (BCG) in healthy, tuberculin skin test-positive (≥15-mm induration), HIV-negative South African adults. We hypothesized that preclearance of latent bacilli with IPT modulates BCG immunogenicity following revaccination. Frequencies and coexpression of IFN-γ, TNF-α, IL-2, IL-17, and/or IL-22 in CD4 T cells and IFN-γ-expressing CD8 T, γδ T, CD3(+)CD56(+) NKT-like, and NK cells in response to BCG were measured using whole blood intracellular cytokine staining and flow cytometry. We analyzed 72 participants who were revaccinated with BCG after IPT (n = 33) or without prior IPT (n = 39). IPT had little effect on frequencies or cytokine coexpression patterns of M. tuberculosis- or BCG-specific responses. Revaccination transiently boosted BCG-specific Th1 cytokine-expressing CD4, CD8, and γδ T cells. Despite high frequencies of IFN-γ-expressing BCG-reactive CD3(+)CD56(+) NKT-like cells and CD3(-)CD56(dim) and CD3(-)CD56(hi) NK cells at baseline, BCG revaccination boosted these responses, which remained elevated up to 1 y after revaccination. Such BCG-reactive memory NK cells were induced by BCG vaccination in infants, whereas in vitro IFN-γ expression by NK cells upon BCG stimulation was dependent on IL-12 and IL-18. Our data suggest that isoniazid preclearance of M. tuberculosis bacilli has little effect on the magnitude, persistence, or functional attributes of lymphocyte responses boosted by BCG revaccination. Our study highlights the surprising durability of BCG-boosted memory NKT-like and NK cells expressing antimycobacterial effector molecules, which may be novel targets for tuberculosis vaccines.

Copyright © 2016 by The American Association of Immunologists, Inc.

Figures

Fig. 1

(A) Consort diagram of participant recruitment and enrolment. (B and C) Study design: Schematic representation of treatments schedule of visits and blood draws in the two groups. (B) Group receiving no INH before BCG re-vaccination, observed for 6 months, then receiving 6 month dose of INH (in a maximum period of 7 months) for clinical equivalence (Observation-BCG-INH: OBI, n = 39). (C) Group receiving 6 months dose of INH, BCG re-vaccinated, observed for 12 months (INH-BCG-Observation: IBO, bottom, n = 33). Blood draws for whole blood ICS are indicated with inverted triangles for both groups. Blue and red shaded squares correspond to when the OBI and IBO groups received IPT, respectively. Stars denote time-points when additional experiments characterizing NK cells were performed.

Fig. 2. Effect of INH on ESAT-6/CFP-10-reactive cytokine-expressing lymphocyte subsets

(A) Frequencies of ESAT-6/CFP-10-reactive cytokine-expressing CD4+ (left) and CD8+ (right) T cells. All frequencies were background subtracted. Red and blue lines denote the group treated (IBO) or not treated (OBI) with INH for 6 months in the time window preceding BCG re-vaccination, respectively. No sample was collected at 22 weeks before BCG re-vaccination in the OBI group. Unadjusted p-values were calculated with the Mann-Whitney U-test, comparing frequencies of cytokine-positive cells between the two groups. To correct for multiple testing (Bonferroni method), P-values below 0.025 were considered statistically significant. (B) Median proportions of ESAT-6/CFP10 peptide pool-specific CD4 T cells co-expressing IFNγ, TNFα, IL-2, IL-17, and/or IL-22, either after isoniazid treatment (IBO) or observation (OBI). Arcs represent the cytokines expressed within each pie slice. (C) Same graphs as (A) for BCG-specific responses. (D) Proportions of BCG-specific CD4 T cells co-expressing IFNγ and/or IL-22 following isoniazid or observation. Unadjusted p-values were calculated with the Wilcoxon signed rank test before and after IPT or observation. To correct for multiple testing (Bonferroni method) P-values below 0.0125 (4 comparisons) were considered statistically significant.

Fig. 3. BCG-specific CD4 T cell responses after BCG re-vaccination

(A) Schematic diagram of blood draws to assess BCG-specific T cell responses after re-vaccination. (B) Representative flow cytometry plots of CD4 T cells in one OBI participant. First and second rows correspond to TNFα vs IFNγ profiles before BCG vaccination (pre-BCG vaccination, top) and 3 weeks post-vaccination (bottom) in the unstimulated (UNS, left) or BCG-stimulated (BCG, right) samples. (C) Total frequencies of BCG-specific cytokine-positive CD4 T cells in all participants from the OBI (top, blue) or IBO groups (bottom, red). P-values were calculated using Wilcoxon signed rank test, comparing the pre- and post-vaccination visits. Bonferroni-adjusted p-value threshold of 0.025 after correcting for the two performed comparisons was considered statistically significant. (D) Median (error bars represent IQR) frequencies of BCG-induced CD4 T cell responses. Blue and red lines correspond to group pre-treated (IBO) or not treated (OBI) with INH for 6 months. Unadjusted p-values were calculated using the Mann-Whitney U-test, comparing the two groups. (E) Median proportions of BCG-specific CD4 T cells co-expressing IFNγ, TNFα, IL-2, IL-17, and/or IL-22, either in groups observed only (OBI, top) or pre-treated with INH for 6 months (IBO, bottom). Numbers in the pie slices indicate the number of co-expressed cytokines. Arcs represent the cytokines expressed within each pie slice. P-values were calculated using a non-parametric paired permutation test comparing the proportions of subsets between two pies. (F) Median (error bars denote IQR) BCG-specific cytokine responses 1 year post-vaccination to pre-BCG baseline: total expression of at least one of 5 cytokines (IL-2, IL-17, IL-22, IFNγ and TNFα) in CD4 T cells. P-values were calculated using Wilcoxon signed rank test between baseline and 1-year post-vaccination responses within each group. Bonferroni adjusted p-value threshold of 0.025 was considered statistically significant.

Fig. 4. Cytokine-expression by BCG-reactive cell subsets before and after BCG re-vaccination

(A) Representative plots showing BCG-reactive IFNγ and TNFα expression by CD8 T cells, before BCG vaccination (pre-BCG vaccination, left) and 3-weeks post-vaccination (right) in unstimulated (UNS, top) or BCG-stimulated (BCG, bottom) samples. (B) BCG-specific IFNγ expression by CD8 T cells 3-5 weeks post vaccination in the OBI group. Each line represents an individual. P-values were calculated using Wilcoxon signed rank test between baseline and either 3 or 5 weeks post vaccination. Bonferroni adjusted p-value threshold of 0.025 was used to correct for the two comparisons. (C) Median (error bars denote IQR) responses in the IBO (red) or OBI (blue) groups 1 year after BCG re-vaccination. (D-F) BCG-reactive cytokine expression by γδ T cells, shown in identical graphs as those for CD8 T cells.

Fig. 5. Frequencies of cytokine-expressing BCG-reactive CD3+CD56+ NKT-like, CD3−CD56dim NK and CD3−CD56hi NK cells after BCG re-vaccination

(A) Representative flow cytometry plots depicting IFNγ-expressing cells in BCG-reactive CD3+CD56+ NKT-like cells. (B) Frequencies of BCG-reactive IFNγ-expressing cells within each cell subset in participants from the OBI group only. Unadjusted p-values, calculated using the Wilcoxon signed rank test, represent comparisons between timepoints (top right). (C) Comparison of median (error bars denote IQR) BCG-specific IFNγ responses 1 year post-vaccination to pre-BCG baseline. P-values were calculated using Wilcoxon signed rank test between baseline and 1-year post-vaccination responses. Bonferroni adjusted p-value threshold of 0.025 was used to correct for the two performed comparisons in B and C. Similar graphs are shown for CD3−CD56dim NK cells (D-F) and CD3−CD56hi NK cells (G-I).

Fig. 6. Functional and phenotypic characterization of BCG-reactive CD56dim and CD56hi NK cells

(A) Representative flow cytometry plots of CD16 and CD56 expression by CD3− lymphocytes to identify NK cell subsets (top left): CD56hiCD16lo and CD56dimCD16+ populations. Perforin expression by NK subsets and CD3−CD56−CD16− non-T or NK cells plotted against CD56 expression (bottom left). (B) Representative plots of total (left) CD56hiCD16− (blue plots, top) and CD56dimCD16+ (red plots, bottom), and BCG-reactive IFNγ-expressing cells (right panels), showing co-expression patterns of CD158b/CD57 (left panels) and CD8/CD161 (right panels). (C) Combinatorial expression of CD8, CD57, CD158b and CD161 as median proportions of CD56hiCD16lo cells (top) and CD56dimCD16+ (bottom), either in total NK cell populations before vaccination (left) or 3 weeks post BCG re-vaccination (middle), or only within BCG-reactive IFNγ-expressing NK cells 3 weeks post re-vaccination (right). Asterisks and arrow correspond to the predominant CD8−CD57−CD158b−CD161+ Boolean combination in both NK subsets. (D) Box and whisker plots of perforin median fluorescence intensities (MFI) in BCG-stimulated CD56hiCD16lo (blue) and CD56dimCD16+ (red) NK cells. Horizontal lines represent medians, the boxes the IQR and the whiskers represent the range. Unadjusted p-values were calculated with Wilcoxon signed rank test between the two subsets at vaccination baseline (pre-BCG), 3 weeks, and 1 year post re-vaccination. (E) Perforin expression, measured as MFI, in a paired analysis of unstimulated and BCG-stimulated samples in CD56hiCD16− (blue lines, top) and CD56dimCD16+ cells (red lines, bottom). Unadjusted p-values were calculated with Wilcoxon signed rank test. (F) Median proportions of the respective lymphocyte subsets within peripheral blood 3 weeks post-vaccination in the IBO group (left) or the median proportional contribution of these lymphocyte subsets to the total BCG-reactive IFNγ+ cells (right).

Fig. 7. IFNγ-expressing BCG-reactive CD3+CD56+ NKT-like, CD3−CD56dim NK and CD3−CD56hi NK cells are induced by BCG vaccination

(A) Study design of delayed BCG vaccination study in infants. Schematic representation of vaccination and blood draws for BCG-unvaccinated (delayed) and BCG-vaccinated infants. Blood was drawn at 5 weeks of age in both groups. (B) Representative flow cytometry plots depicting TNFα vs IFNγ expression in CD3+CD56+ NKT-like cells (green, left), CD3−CD56dimCD16+ NK (red, middle), and CD3−CD56hiCD16lo NK cells (blue, right) in unstimulated (top) or BCG-stimulated (bottom) samples from either a vaccinated (left panels) or unvaccinated infant (right panels). (C) Frequencies of BCG-reactive IFNγ+ CD3+CD56+ NKT-like cells (green, left), CD3−CD56dimCD16+ NK (red, middle), and CD3−CD56hiCD16lo NK (blue, right) cells. Horizontal lines represent medians, boxes represent the IQR, and whiskers represent the range. Unadjusted p-values were calculated with the Mann-Whitney U-test, comparing frequencies of cytokine-positive cells between the two groups. P-values below 0.05 were considered statistically significant.

Fig. 8. Bystander activation of BCG-reactive NK cell IFNγ expression

(A) Frequencies of BCG-specific IL2-expressing CD4+ T cells and IFNγ-expressing CD56dimCD16+ (red symbols, left) or CD56hiCD16− (blue symbols, middle) NK cells at 3 weeks post BCG re-vaccination in the BCG re-vaccination trial. Unadjusted p-values and correlation coefficients were calculated with Spearman’s non-parametric correlation test. The plot on the right depicts Spearman’s r coefficients before BCG-vaccination and at 3 weeks or 1 year following BCG re-vaccination. (B) Frequencies of IL2-expressing CD4+ T cells (green, left), IFNγ-expressing CD3−CD56dimCD16+ NK (red, middle), and CD3−CD56hiCD16lo NK (blue, right) cells in samples stimulated with BCG (top) or PHA (bottom). Horizontal lines represent medians, boxes the IQR, and whiskers represent the range. (C) Representative flow cytometry plots of IFNγ expression in CD3−CD56dimCD16+ NK (red) and CD3−CD56hiCD16lo NK (blue) in whole blood from healthy donors incubated with the shown cytokines, blocking antibodies or BCG. (D and E) Frequencies of IFNγ expressing CD3−CD56dimCD16+ NK (red, top) and CD3−CD56hiCD16lo NK (blue, bottom) in blood from 5 donors incubated with the shown cytokines, blocking antibodies and/or BCG. Unadjusted p-values were calculated with the Wilcoxon signed rank test.