Deborah K. Dunn-Walters

B-cell diversity decreases in old age and is correlated with poor health status.

Older people suffer from a decline in immune system, which affects their ability to respond to infections and to raise efficient responses to vaccines. Effective and specific antibodies in responses from older individuals are decreased in favour of non‐specific antibody production. We investigated the B‐cell repertoire in DNA samples from peripheral blood of individuals aged 86–94 years, and a control group aged 19–54 years, using spectratype analysis of the IGHV complementarity determining region (CDR)3. We found that a proportion of older individuals had a dramatic collapse in their B‐cell repertoire diversity. Sequencing of polymerase chain reaction products from a selection of samples indicated that this loss of diversity was characterized by clonal expansions of B cells in vivo. Statistical analysis of the spectratypes enabled objective comparisons and showed that loss of diversity correlated very strongly with the general health status of the individuals; a distorted spectratype can be used to predict frailty. Correlations with survival and vitamin B12 status were also seen. We conclude that B‐cell diversity can decrease dramatically with age and may have important implications for the immune health of older people. B‐cell immune frailty is also a marker of general frailty.

B cells and aging: molecules and mechanisms.

Recent advances allow aging-associated changes in B-cell function to be approached at a mechanistic level. Reduced expression of genes crucial to lineage commitment and differentiation yield diminished B-cell production. Moreover, intrinsic differences in the repertoire generated by B-cell precursors in aged individuals, coupled with falling B-cell generation rates and life-long homeostatic competition, result in narrowed clonotypic diversity. Similarly, reductions in gene products crucial for immunoglobulin class switch recombination and somatic hypermutation impact the efficacy of humoral immune responses. Together, these findings set the stage for integrated analyses of how age-related changes at the molecular, cellular and population levels interact to yield the overall aging phenotype.

Vaccination‐induced changes in human B‐cell repertoire and pneumococcal IgM and IgA antibody at different ages

It is well known that older people are more susceptible to morbidity and mortality from infectious diseases, particularly from pulmonary diseases such as pneumococcal pneumonia where vaccines do not provide efficient protection as in younger populations. We have previously shown that the B‐cell repertoire in the old is reduced and hypothesise that this may contribute to the impaired humoral responses of the elderly. Here, we investigated the repertoire and antibody responses to winter vaccination in two age groups, aged 18–49 and 65–89. We found that the serum IgM and IgA pneumococcal responses were significantly impaired in the older group, with no difference in IgG levels. IGHM spectratype analysis seems to be the most promising in terms of its predictive ability for vaccine responses. Spectratypes showed a clear change in the repertoire at day 7 after vaccination, with a return to the baseline levels at day 28. The changes at day 7 reflected expansion of IGH sequences that have smaller, more hydrophilic, CDR3 regions, and these changes were attenuated in the older group. The older group was more likely to have spectratypes indicative of a reduced diversity at day 0 and day 28. On average, the baseline repertoire in the older group was comprised of larger CDR3 regions than in the younger group. In conclusion, IgA and IgM responses are significantly impaired in the elderly pneumococcal response and are likely key mediators of protection. Hydrophilicity and/or small size of the IGH CDR3 appear to be important in these responses.

The ageing B cell population: Composition and function

Age related changes in the structure and function of the immune system, collectively termed immunosenescence, result in poor responses to infections, increased susceptibility to cancers and increased incidence of autoimmune diseases. The humoral immune response, maintained by the B cell compartment, has a key role in an effective immune system—not only in producing high affinity antibodies that are crucial for vaccination strategies, but in assisting other components of the immune system in their function. Hence an understanding of B cell immunosenescence in particular is vital in designing strategies to combat the effects of age on immune function. Numerous studies have been undertaken using small animal models in order to understand immunosenescence, and these have contributed greatly to our understanding of the events that underpin impaired immune responses. However, there are key differences between the human and the mouse and a clear understanding of these differences is required when extrapolating from one species to the other. In this article we present an overview of B cell development and summarise current data on age-related B cell changes, at both the population level and at the individual mechanistic level. Areas of similarity and difference between human and mouse models are highlighted.

Redemption of autoantibodies on anergic B cells by variable-region glycosylation and mutation away from self-reactivity.

Significance Antibodies are selected to bind microbial but not self-antigens, because binding to self would compete with binding microbes, shorten antibody half-life, and cause autoimmunity. Self-tolerance is actively acquired in part by discarding self-binding antibodies before the body is exposed to a microbe or vaccine. The experiments here provide evidence of an opposite mechanism, allowing antibodies that initially bind both foreign and self-antigens to acquire self/non-self discrimination during the course of an immune response through somatic hypermutation away from self-reactivity. In addition to selection for lower-affinity binding to self, antibody variants were selected with fewer binding sites available to bind self-antigen because most were occupied by N-linked carbohydrate, possibly explaining the frequent occurrence of N-linked glycosylation of antibody variable domains. The best-understood mechanisms for achieving antibody self/non-self discrimination discard self-reactive antibodies before they can be tested for binding microbial antigens, potentially creating holes in the repertoire. Here we provide evidence for a complementary mechanism: retaining autoantibodies in the repertoire displayed as low levels of IgM and high IgD on anergic B cells, masking a varying proportion of autoantibody-binding sites with carbohydrates, and removing their self-reactivity by somatic hypermutation and selection in germinal centers (GCs). Analysis of human antibody sequences by deep sequencing of isotype-switched memory B cells or in IgG antibodies elicited against allogeneic RhD+ erythrocytes, vaccinia virus, rotavirus, or tetanus toxoid provides evidence for reactivation of anergic IgMlow IgD+ IGHV4-34+ B cells and removal of cold agglutinin self-reactivity by hypermutation, often accompanied by mutations that inactivated an N-linked glycosylation sequon in complementarity-determining region 2 (CDR2). In a Hy10 antibody transgenic model where anergic B cells respond to a biophysically defined lysozyme epitope displayed on both foreign and self-antigens, cell transfers revealed that anergic IgMlow IgD+ B cells form twice as many GC progeny as naïve IgMhi IgD+ counterparts. Their GC progeny were rapidly selected for CDR2 mutations that blocked 72% of antigen-binding sites with N-linked glycan, decreased affinity 100-fold, and then cleared the binding sites of blocking glycan. These results provide evidence for a mechanism to acquire self/non-self discrimination by somatic mutation away from self-reactivity, and reveal how varying the efficiency of N-glycosylation provides a mechanism to modulate antibody avidity.

The Relationship between CD27 Negative and Positive B Cell Populations in Human Peripheral Blood

CD27 expression has been used to distinguish between memory and naive B cells in humans. However, low levels of mutated and isotype-switched CD27−IgD− cells are seen in healthy adults, and these are increased in some autoimmune diseases and in the elderly. Thus CD27 is not a universal marker of memory B cells in humans. Various hypotheses have been put forward as to the function of the CD27− memory population. Since we have previously found high-throughput IGHV repertoire analysis useful to distinguish “innate-like” memory B cells (CD27+IgD+), we have employed similar analyses to elucidate the relationship between CD27− and CD27+ memory B cells. IgM+IgD− memory cells in both the CD27+ and CD27− compartments share the unique characteristics of the “innate-like” IgM+IgD+CD27+ cells. The switched CD27+ and CD27− memory cells share a similar IGHV repertoire, having more in common with each other than with “innate-like” memory cells, although it is interesting that IgG2 and IgA2 subclasses of antibody in both switched memory populations have a more “innate-like” repertoire. Clonality analysis shows evidence of a close clonal relationship between the two populations in that both CD27− and CD27+ switched memory cells can be found in the same genealogical tree. The expression of CD27 does not appear to occur in a linear developmental fashion, since we see CD27− cells as precursors of CD27+ cells and vice versa. Despite the similarities, the CDR-H3 repertoire of the CD27− cells is significantly different from both the CD27+IgD+ and CD27+IgD− populations, indicating that perhaps the lack of CD27 might be related to binding properties of the Ig CDR-H3 region.

Age- and tissue-specific differences in human germinal center B cell selection revealed by analysis of IgVH gene hypermutation and lineage trees.

The elderly produce increased levels of antibodies to autologous antigens and are less able to make high‐affinity antibodies to foreign antigens. Ig gene hypermutation is integral to the affinity maturation process but previous studies of hypermutation with age have yielded conflicting results. The cells studied have represented post‐germinal center (GC) populations and, therefore, the results may be complicated by possible differences in activation history. We studied Ig genes from GC B cells to elucidate which factors in the affinity maturation process change with age. Age‐related changes in the pattern of hypermutation were seen, although the analysis of variable region heavy chain (VH) genes and their lineage trees shows that an alteration in the mechanism of somatic hypermutation is unlikely. The changes are due to founder cell effects and/or the process of selection. Striking tissue‐specific differences were seen. All measurements indicated that selectionof Ig genes may decrease in Peyers patch GC but increase in splenic GC with age. These tissue‐specific differences highlight the importance of considering the activation and effector sites when studying immune senescence.

B cell repertoire and ageing

A diverse B cell repertoire is essential for an effective immune response. Not only to provide a variety of antibodies to recognise the multiplicity of likely pathogen challenge, but also because B cells are important regulators of the immune response. In addition to their excellent capabilities as antigen presenting and activating cells, recent work shows that some subpopulations of B cells can have suppressive functions. The diversity of the B cell population as a whole decreases with age, and is associated with ill health. Whether decreased diversity is a feature of all B cells or a reflection of altered subpopulations is not clear, since different subsets of B cells have different functions and their repertoires are shaped by different selection pressures.

Age-related changes in human peripheral blood IGH repertoire following vaccination

Immune protection against pulmonary infections, such as seasonal flu and invasive pneumonia, is severely attenuated with age, and vaccination regimes for the elderly people often fail to elicit effective immune response. We have previously shown that influenza and pneumococcal vaccine responses in the older population are significantly impaired in terms of serum antibody production, and have shown repertoire differences by CDR-H3 spectratype analysis. Here we report a detailed analysis of the B cell repertoire in response to vaccine, including a breakdown of sequences by class and subclass. Clustering analysis of high-throughput sequencing data enables us to visualize the response in terms of expansions of clonotypes, changes in CDR-H3 characteristics, and somatic hypermutation as well as identifying the commonly used IGH genes. We have highlighted a number of significant age-related changes in the B cell repertoire. Interestingly, in light of the fact that IgG is the most prevalent serum antibody and the most widely used as a correlate of protection, the most striking age-related differences are in the IgA response, with defects also seen in the IgM repertoire. In addition there is a skewing toward IgG2 in the IgG sequences of the older samples at all time points. This analysis illustrates the importance of antibody classes other than IgG and has highlighted a number of areas for future consideration in vaccine studies of the elderly.

B cell immunosenescence: different features of naive and memory B cells in elderly

Elderly people show a reduced protection against new infections and a decreased response to vaccines as a consequence of impairment of both cellular and humoral immunity. In this paper we have studied memory/naïve B cells in the elderly, evaluating surface immunoglobulin expression, production of the pro- and anti-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-10, and presence of somatic hypermutation, focusing on the IgG+IgD−CD27− double negative (DN) B cells that are expanded in the elderly. Our results show that naïve B cells from young donors need a sufficiently strong stimulus to be activated “in vitro”, while naïve B cells from old subjects are able to produce IL-10 and TNF-α when stimulated “physiologically” (α-CD40/IL-4), suggesting that these cells might play a role in the control of the immuno-inflammatory environment in the elderly. In addition, in the elderly there is an accumulation of DN B cells with a reduced rate of somatic hypermutation. Thus, DN B lymphocytes may be exhausted cells that are expanded and accumulate as a by-product of persistent stimulation or impaired germinal center formation.