Age related differences in dynamics of specific memory B cell populations after clinical pertussis infection

Abstract

For a better understanding of the maintenance of immune mechanisms to Bordetella pertussis (Bp) in relation to age, we investigated the dynamic range of specific B cell responses in various age-groups at different time points after a laboratory confirmed pertussis infection. Blood samples were obtained in a Dutch cross sectional observational study from symptomatic pertussis cases. Lymphocyte subpopulations were phenotyped by flowcytometry before and after culture. Memory B (Bmem) cells were differentiated into IgG antibody secreting cells (ASC) by polyclonal stimulation and detected by an ELISPOT assay specific for pertussis antigens pertussis toxin (Ptx), filamentous haemagglutinin (FHA) and pertactin (Prn). Bp antigen specific IgG concentrations in plasma were determined using multiplex technology. The majority of subjects having experienced a clinical pertussis episode demonstrated high levels of both Bp specific IgG and Bmem cell levels within the first 6 weeks after diagnosis. Significantly lower levels were observed thereafter. Waning of cellular and humoral immunity to maintenance levels occurred within 9 months after antigen encounter. Age was found to determine the maximum but not base-line frequencies of Bmem cell populations; higher levels of Bmem cells specific for Ptx and FHA were reached in adults and (pre-) elderly compared to under-fours and schoolchildren in the first 6 weeks after Bp exposure, whereas not in later phases. This age effect was less obvious for specific IgG levels. Nonetheless, subjects' levels of specific Bmem cells and specific IgG were weakly correlated. This is the first study to show that both age and closeness to last Bp encounter impacts the size of Bp specific Bmem cell and plasma IgG levels.

Figure 1. Flow cytometry analysis of B cell populations before and after culture.

Shown are bar charts of B cell frequencies of our five age groups with geometric means and 95% CI. Graphs show relative frequencies of total B cells (A) as well as memory B cells (C) and plasma cells (D) as percentage of all lymphocytes and memory B cells as percentage of all CD19⁺ B cells (B) per age group. The factor of enrichment of memory B cells due to in vitro stimulation is likewise shown per age-group (E) and exemplified by one donor in two flow cytometry dot plots (F). Age groups are designated as follows; u4s (under-fours), sch (schoolchildren), ado (adolescents), adu (adults) and eld ((pre-) elderly).

Figure 2. Pertussis specific IgG levels decrease with time.

Box plots of specific IgG concentrations of Ptx (left), FHA (middle) and Prn (right) in symptomatic cases in acute (≤1.5 months), intermediate (1.6–8.9 months) and maintenance phase (≥9 months) after the last known immunizing event. Box plots show first and third quartiles and medians and whiskers represent 5–95% CI. Below the x-axis geometric mean values are given for each group. ∧ =  p<0.1; * = p<0.05; ** =  p< 0.01; *** =  p<0.001, **** =  p<0.0001

Figure 3. Pertussis specific levels of B_mem decrease with time.

Box plots of specific B_mem frequencies of Ptx (left), FHA (middle) and Prn (right) in symptomatic cases in acute (≤1.5 months), intermediate (1.6–8.9 months) and maintenance phase (≥9 months) after the last known immunizing event. Box plots show first and third quartiles and medians and whiskers represent 5–95% CI. Below the x-axis geometric mean values are given for each group. ∧ =  p<0.1; * = p<0.05; ** =  p< 0.01; *** =  p<0.001, **** =  p<0.0001

Figure 4. Comparing pertussis specific IgG levels between age-groups and per time after exposure.

Box plots representing age stratified levels of Ptx-, (A) FHA- (B) and Prn- (C) specific IgG concentrations in acute (≤1.5 months), intermediate (1.6–8.9 months) and maintenance phase (≥9 months) after last known immunizing event. Age groups are designated as follows; u4s (under-fours), sch (schoolchildren), ado (adolescents), adu (adults) and eld ((pre-) elderly). Below the x-axis geometric mean values are given for each group. ∧ =  p<0.1; * = p<0.05; ** =  p< 0.01; *** =  p<0.001, #  = significantly lower than same age group in acute phase, ⊥  = significantly lower than same age group in intermediate phase

Figure 5. Age-related differences in pertussis specific B_mem cell levels in acute phase.

Box plots representing age stratified levels of Ptx-, (A) FHA- (B) and Prn- (C) specific B_mem frequencies in acute (≤1.5 months), intermediate (1.6-8.9 months) and maintenance phase (≥9 months) after last known immunizing event. Age groups are designated as follows; u4s (under-fours), sch (schoolchildren), ado (adolescents), adu (adults) and eld ((pre-) elderly). Below the x-axis geometric mean values are given for each group. ∧ =  p<0.1; * = p<0.05; ** =  p< 0.01; *** =  p<0.001, #  = significantly lower than same age group in acute phase

Figure 6. Spearman correlations between pertussis specific B_mem and corresponding IgG levels.

Dots represent paired antigen specific B_mem cell frequencies and plasma IgG levels for Ptx (A), FHA (B) and Prn (C) for each participant.

Figure 7. Longitudinal analysis of levels of specific IgG and B_mem cells in nine pertussis patients.

Antigen specific IgG levels at two time points per patient for Ptx (A), FHA (B) and Prn (C) and corresponding B_mem frequencies for Ptx (D), FHA (E) and Prn (F) are depicted.