We observed that even in the absence of challenge, there are differences in the B-cell spectratypes between individuals (). These can represent changes in B-cell repertoire because of prior antigen exposure or changes in the naïve repertoire. With regard to the latter, the IGHM
spectratypes are particularly interesting, because the assumption is that they largely represent naïve B cells. This would imply that there is an element of interindividual variation in factors affecting repertoire generation in central B-cell development. These could be factors related to gene rearrangement, such as preferential use of different IGH
genes or terminal deoxynucleotidyl activity. Alternatively, they could be factors associated with tolerance selection of the repertoire such as occurs in the bone marrow and in the transitional B-cell stages (Wardemann et al., 2003
). These factors may also be responsible for the age-related differences in IGHM
CDR3 size that we see before vaccine challenge.
Vaccine challenge results in cellular changes in the peripheral blood at day 7 (Wrammert et al., 2008
; Baxendale et al., 2010
). These can be detected both by spectratype metrics showing a decreased diversity (lower CGD value of spectratype) and by sequence analysis indicating an increase in clonality. Although the sequence analysis only compared six old and six young, and there was interindividual variability (), we could still show a decrease in the extent of clonal expansion at day 7 in the old compared with the young. The older group also has less diverse repertoires at days 0 and 28, and they do not return to day 0 CGD values at day 28 as the younger group do. This may be related to the slower time to reach the peak IgA antibody response in the serum. Without the benefit of further time points, it is difficult to judge the kinetics of the response. It is entirely possible from these data that the IgA response in the old follows the same pattern as the young but that it is delayed as a whole so that it peaks later and resolves even later. Alternatively, there may be different factors affected in the activation and resolution phases of the response to cause these observations. Inadequate resolution of immune responses may be just as important as a failure to respond in the first place, and although literature is sparse on this topic, there are indications that regulatory functions of cells may be impaired with age (Hwang et al., 2009
). A delayed resolution could be a significant contributory factor to the observations that older people have less diverse repertoires, because if the effects of a single challenge can still be seen a month later, then the multiple challenges we encounter as part of everyday life could present a significant compromise to the baseline diversity.
While we have shown that we can use spectratyping of the B-cell repertoire in an isotype-specific manner, it is clear that the high levels of interindividual variability in prior antigen exposure are such that it would not be useful as a predictor in a study of IgG responses. The subjects in the study had both influenza and pneumococcal vaccinations, as part of their normal healthcare, thus were exposed to multiple antigens simultaneously. In spite of the complicated challenge, there are indications that some measures of the spectratypes can be correlated with the PPS-specific responses. The serum IgM and IgA antibody levels at day 28 were associated with more leptokurtotic IGHM and IGHA spectratypes at day 7. This peakedness seems to be a result of the expansion of clones containing smaller CDR3 values. In looking for a prevaccine predictor, we could see promising associations between the spectratype shape (CGD, ) and the IgM response. There was a significant difference in CGD values between the young and the old groups, and the individuals with the highest levels of IgM have CGD values near to 1. If an individual is in the over 65 age bracket and has a CGD value of lower than 0.9, then they will not produce a high level of PPS-specific IgM antibody.
It is clear, from data here and elsewhere, that the IgM response is severely diminished in the older population. Whether this is because of a failure of the TD or the TI pathway of B-cell activation cannot be determined from these data, but the nature of the PPV23 vaccine would lead one to expect the latter. In the light of these data, and the recent report that removal of IgM antibody from serum diminishes opsonophagocytic activity against two common PPS serotypes (Park & Nahm, 2011
), it would seem appropriate that future studies aimed at improving PPS responses in the elderly should include consideration of the IgM response. In addition, we found that although the IgA serum response was not significantly different in the two age groups at day 28, there was a significant difference at day 7 (). This implies that there might be an age-related temporal difference in either production or trafficking of IgA. Temporal differences such as this in the face of bacterial infection could have severe consequences for an individual, and these data have highlighted the need for analysis at different time points after vaccination. Although we saw a significant increase in IgG ANA autoantibodies with age, we did not see any evidence that vaccination caused an increase as has previously been reported (Huang et al., 1992
). This may be due to differences in adjuvant use in vaccines, but it is comforting to know that the recent commonly used vaccines have no deleterious effects in this respect.
Perhaps the most striking observation from these spectratype data is that the mean size of the CDR3 region is significantly decreased after challenge in both young and old age groups for all isotypes. The extent of the change in the CDR3 size appears to be the same for both age groups, but the old group starts at a higher size in the first instance, so that there are significant age-related differences in CDR3 size both before and after vaccination (). The precise effect of these differences is not known, because we could not find any clear correlations between CDR3 size and serum antibody. The lack of correlations with antibody might reflect the noise in the experiment caused by having multiple vaccine challenges at the same time, or it may be due to some other factor such as differential locations for responding cells vs. responding antibody. The sequencing data confirm the spectratype data, showing an overall decrease in mean CDR3 size, and also show that when the effect of clonal expansion is removed the challenge-induced CDR3 size shift is similar between the two groups. Selection for smaller CDR3 size has previously been reported in the germinal centre response to hapten NP in mice (Lacroix-Desmazes et al., 1998
), and a study of 256 peripheral blood IGHV6-1
IgM genes in humans has noted that antigen-experienced genes have shorter CDR3 than those that are unmutated (Rosner et al., 2001
). More recently, we have shown for the whole repertoire of IGHV
genes in three young individuals that memory B cells have shorter CDR3 regions than naïve or transitional B cells (Wu et al., 2010
). This is also apparent in these data, where class-switched (and therefore antigen-experienced) genes have shorter CDR3 regions than IgM genes even at day 0. A small CDR3 size appears to be important in relation to the hydrophobic nature of the antigen-binding site of the antibody; lower sizes of CDR3 regions tend to have a lower hydrophobicity and within this, even when sequences of the same size are compared, those that belong to expanded clones have a further decrease in hydrophobicity compared with the unique sequences. So it looks very much as if sequences with a smaller, more hydrophilic, antigen-binding site are being selected and expanded in the younger population more so than in the old.
In summary, even though the level of serum IgA and IgM antibodies is much lower than that of IgG antibodies, it is in the two former isotypes that the significant age-related defects in the response to pneumococcal vaccine are found. B-cell spectratype analysis of IGHM sequences seems to be the most promising avenue for predicting responses to pneumococcal vaccination, although it appears that there are still factors other than B-cell repertoire that are involved in the senescence of the humoral immune response. We have also shown clear evidence that expansion of IGH sequences that have smaller, more hydrophilic, CDR3 regions is a feature of these common vaccinations and that the clonal expansion is reduced in the older group. Given the apparent importance of the CDR3 region in this respect, it is of note that the baseline repertoire of older people comprises larger CDR3 regions, on average, than the younger group and therefore may be at a disadvantage in the initiation of appropriate immune responses.