Specific T cell frequency and cytokine expression profile do not correlate with protection against tuberculosis after bacillus Calmette-Guérin vaccination of newborns

Abstract

Rationale: Immunogenicity of new tuberculosis (TB) vaccines is commonly assessed by measuring the frequency and cytokine expression profile of T cells.

Objectives: We tested whether this outcome correlates with protection against childhood TB disease after newborn vaccination with bacillus Calmette-Guérin (BCG).

Methods: Whole blood from 10-week-old infants, routinely vaccinated with BCG at birth, was incubated with BCG for 12 hours, followed by cryopreservation for intracellular cytokine analysis. Infants were followed for 2 years to identify those who developed culture-positive TB-these infants were regarded as not protected against TB. Infants who did not develop TB disease despite exposure to TB in the household, and another group of randomly selected infants who were never evaluated for TB, were also identified-these groups were regarded as protected against TB. Cells from these groups were thawed, and CD4, CD8, and γδ T cell-specific expression of IFN-γ, TNF-α, IL-2, and IL-17 measured by flow cytometry.

Measurements and main results: A total of 5,662 infants were enrolled; 29 unprotected and two groups of 55 protected infants were identified. There was no difference in frequencies of BCG-specific CD4, CD8, and γδ T cells between the three groups of infants. Although BCG induced complex patterns of intracellular cytokine expression, there were no differences between protected and unprotected infants.

Conclusions: The frequency and cytokine profile of mycobacteria-specific T cells did not correlate with protection against TB. Critical components of immunity against Mycobacterium tuberculosis, such as CD4 T cell IFN-γ production, may not necessarily translate into immune correlates of protection against TB disease.

Figure 1.

Recruitment and enrollment of participants into the study.

Figure 2.

Gating strategy Flow cytometric analysis of bacillus Calmette-Guérin (BCG)–induced T cell cytokine production. Whole blood was incubated with BCG for 12 hours. Representative dot plots from a single participant are shown. (A) Gating strategy used to identify CD4, CD8, and γδ T cells. From left to right, leukocytes from whole blood were acquired and cell doublets excluded with forward scatter area versus forward scatter height parameters. T cells were identified by assessing CD3 expression against IFN-γ expression, which enables inclusion of any T cells that may have down-regulated CD3 expression upon activation. Subsequently, CD3⁺ T cells were differentiated into conventional T cells, which did not express γδ T cell receptor, and γδ T cells, by assessing γδ expression against CD8 expression. Finally, the conventional T cell population was divided into CD4⁺ and CD8⁺ T cells. (B) Representative dot plots of cytokine coexpression patterns in CD4 T cells from unstimulated, BCG, and staphylococcal enterotoxin B (SEB)–stimulated conditions. APC = allophycocyanin; Cy5.5PerCP-A = cyanine peridinin chlorophyll protein; FSC = forward scatter.

Figure 3.

Frequency and cytokine expression profile of bacillus Calmette-Guérin (BCG)–specific CD4 T cells. (A) Frequencies of total, IL-2, IFN-γ, tumor necrosis factor (TNF)–α, and IL-17 cytokine-expressing CD4 T cells, as detected by an intracellular cytokine assay after incubation of whole blood with BCG. Total cytokine frequencies incorporate all cytokine-positive CD4 T cells. One participant had a very high total and IFN-γ CD4 T cell response (4.010 and 3.797%, respectively), and these values are shown individually on the graph. (B) Frequencies of distinct subsets of specific CD4 T cells, based on combinations of cytokine expression. After selecting CD4 T cells, boolean gating was used to generate 15 distinct cytokine-expressing subsets. The Kruskal-Wallis test was used to assess differences between the groups of infants. None of the parameters assessed was different between groups (all: P > 0.05). TB = tuberculosis.

Figure 4.

Frequency and cytokine expression profile of bacillus Calmette-Guérin (BCG)–specific CD8 T cells. (A) Frequencies of total, IL-2, IFN-γ, tumor necrosis factor (TNF)–α, and IL-17 cytokine-expressing CD8 T cells, as detected by an intracellular cytokine assay after incubation of whole blood with BCG. Total cytokine frequencies incorporate all cytokine-positive CD8 T cells One participant had a very high total and IFN-γ CD4 T cell response (6.198 and 6.157%, respectively), and these values are shown individually on the graph. (B) Frequencies of distinct subsets of specific CD8 T cells, based on combinations of cytokine expression. After selecting CD8 T cells, boolean gating was used to generate 15 distinct cytokine-expressing subsets. One participant had a very high IL-2 CD4 T cell response (0.199%), and this value is shown individually on the graph. The Kruskal-Wallis test was used to assess differences between the groups of infants. None of the parameters assessed was different between groups (all: P > 0.05).

Figure 5.

Bacillus Calmette-Guérin (BCG)–induced γδ T cell responses. Frequencies of γδ T cells expressing cytokines after incubation of whole blood with BCG. The Kruskal-Wallis test was used to assess differences between the groups of infants. None of the parameters assessed was different between groups (all: P > 0.05). TNF = tumor necrosis factor.