Age-related waning of immune responses to BCG in healthy children supports the need for a booster dose of BCG in TB endemic countries

Abstract

In the absence of a more effective vaccine against TB and in the interest of developing one, it is essential to understand immune responses associated with BCG protection. We comprehensively characterized T cell populations in BCG-vaccinated children over time. Blood from 78 healthy, BCG-vaccinated children representing four age groups (<1 yr, ≥1 yr <2 yr, ≥2 yr <5 yr, ≥5 yr), was stimulated in vitro for 24 hours and 6 days with live BCG to induce effector and central memory responses. Antigen-specific CD4, CD8, γδ and regulatory T cell populations were phenotyped and intracellular and secreted cytokines measured by flow cytometry and multiplex ELISA respectively. Our results demonstrated that populations of naïve T cells predominated in infants, compared to older children. However, BCG-specific effector CD4 T cell responses were equivalent and antigen-specific CD4 T cell proliferative capacity was increased in infants compared to older children. Increases in innate immune responses including γδ T cell responses and secreted pro-inflammatory cytokines were noted with increasing age. In conclusion, we identified that the capacity to expand and differentiate effector T cells in response to BCG stimulation wanes with increasing age, which may indicate waning central memory immunity. Booster vaccination could be considered to maintain the antigen-specific central memory pool and possibly enhance the duration of protection.

Figure 1

Children of all ages have measurable BCG specific CD4+ IFNγ responses, but non-specific responses increase with increasing age. (A) Flow cytometric plots from a representative individual infant showing intracellular IFNγ, IL17 or IL22 cytokine secretion by BCG stimulated CD4+ T cells are shown. (B) Frequencies of IFNγ producing CD4+ T cells, (C) IL22 producing CD4+ T cells or (D) IFNγ producing γδ T cells, as detected by an intracellular cytokine assay following stimulation of whole blood with SEB or BCG for 20 hours, are shown. Horizontal bars represent median values. The dotted line represents a cut-off of 0.01% for a significant response. The Mann-Whitney test was used to calculate p values.

Figure 2

Secretion of IL1β, IL6 and TNFα was significantly lower in infants than older children. Cytokines were measured in supernatants from diluted whole blood cultures, stimulated with BCG for 6 days using a multiplex assay. Horizontal bars represent median values. Data shown on a log scale. A Mann Whitney test was used to calculate p values.

Figure 3

The frequencies of Ki67+ CD4 and IFNγ expressing Ki67 CD4+ T cells following stimulation with BCG (A) or SEB (B) for 6 days are shown. Ki67 proliferation and expression of IFNγ in response to BCG stimulation in vitro wanes with increasing age, however non-specific proliferation due to SEB increases with increasing age. Horizontal bars represent median values.

Figure 4

Regulatory T cells (A) Representative plots from a healthy control child are shown. From left to right, CD3 vs Time is shown to detect differences in flow. Cell doublets were excluded with forward scatter area versus forward scatter height parameters. Plotting FSC-A vs SSC-A allows identification of the lymphocyte population based on size and granularity. CD4+CD3+T cells were identified, followed by CD4+CD25+T cells. Using FMO gates, FOXP3 and CD39 gates were placed to identify the CD3+CD4+CD25+CD39+FOXP3+ population. Age-related frequency of regulatory T cells in (B) unstimulated whole blood and (C) whole blood incubated with BCG for 20 hours. Horizontal bars represent median values.

Figure 5

(A) The frequency of CD4+ T cell memory phenotypes as described by expression of CD27 and CD45RA in the four age groups is shown. (B) Memory phenotype of BCG specific IFNγ expressing CD4+ T cells in children of different age groups after 20 hours stimulation with BCG and (C) after 6 days stimulation with BCG.