Bacillus Calmette-Guérin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles

Abstract

The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-gamma, IL-2, and TNF-alpha in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-gamma and/or IL-2 and less TNF-alpha, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-gamma. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA-CCR7-CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-gamma+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-gamma production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.

Figure 1

Flow cytometric detection of CD4+ T cell cytokine expression in whole blood incubated with BCG for 12 hours, from a single 10-week old BCG-vaccinated infant. The cut-off gates for cytokine expression were determined using unstimulated T cells from whole blood incubated with co-stimulants only (A). Cytokine expression in CD4+ T cells from whole blood incubated with BCG (B), and with SEB (C) is shown. IL-2/TNF-α subset gating was based on patterns of cytokine expression in SEB-stimulated whole blood. Dotplots are gated on CD3+CD4+ T cells, and are representative of 29 infants.

Figure 2

Cytokine profiles of BCG-specific T cells in 10-week old infants, vaccinated at birth. (A) Frequency of CD4+ and of CD8+ T cells expressing individual Type 1 cytokines following incubation of blood with BCG for 12 hours, in 29 infants. Responses above 0.01% were considered positive. The horizontal line indicates the median and the whiskers the interquartile range. (B) Frequency of BCG-specific CD4+ T cells expressing different combinations of Type 1 cytokines. (C) Frequency of BCG-specific CD8+ T cells expressing different combinations of Type 1 cytokines. (D) Representative staining of intracellular Type 1 cytokines in BCG-specific CD4+ T cells and CD8+ T cells, from a single 10-week old infant. (E) Comparison of frequency of CD4+ and of CD8+ T cells expressing IFN-γ (dark bars) with frequency of T cells expressing IL-2 and/or TNF-α without IFN-γ (light bars), in 29 BCG-vaccinated infants.

Figure 3

Expression of IL-10 (A) and of IL-4 (C) in CD4+ T cells from whole blood from a single vaccinated infant, incubated with co-stimulants (UNS), BCG or SEB. Frequency of CD4+ T cells expressing IL-10 (B) and of IL-4 (D) following incubation of blood with co-stimulants only (UNS) (light bars) and BCG (dark bars) for 12 hours, in 29 infants.

Figure 4

Levels of IL-4 and IL-10, in plasma from whole blood incubated with BCG for 7 hours. The horizontal line represents the median. Background cytokine levels were subtracted.

Figure 5

Phenotype of BCG-specific CD4+ T cells in 10-week old infants, vaccinated at birth. Antigen-specific CD4+ T cells were identified by expression of intracellular IFN-γ (A), IL-2 (B), or both cytokines (C), and the expression of CD45RA, CCR7 and CD27 determined in each case. The plots illustrate the distribution of cytokine-expressing cells (in color; foreground) in relation to the entire CD4+ T cell population (grey; background, also shown in D).

Figure 6

Frequency of different subsets of BCG-specific CD4+ T cells, based on expression of CD45RA, CCR7 and CD27 among IFN-γ+ and IL-2+ (A), and IFN-γ+IL-2+ (B), expressing CD4+ T cells. (C) Frequency of these cells among cytokine-negative, i.e., mostly mycobacteria-non-specific, CD4+ T cells. The horizontal line represents the median and the whiskers the interquartile range. *, p<0.05; ns, p≥0.05.