Effects of aging, cytomegalovirus infection, and EBV infection on human B cell repertoires

Abstract

Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by CMV is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or EBV infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of IGH gene rearrangements to study the BCR repertoires over two successive years in 27 individuals ranging in age from 20 to 89 y. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG Ig genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, whereas CMV infection correlates with the proportion of highly mutated Ab genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.

Figures

Figure 1. V, D, J usage in young and older people is comparable

The panels show the frequency of major V genes (higher than 1%) (A), D genes (B), and J genes (C). The color-coded bars represent the sample average within different age groups, 20-31 years (gray, n=10), 61-69 years (green, n=7), 72-89 years (blue, n=10) from Y2. Error bar is standard error. Pairwise comparison was performed with two-sided student t-test.

Figure 2. Increased IGH CDR3 lengths in older individuals

Average CDR3 length, hydrophobicity, and net charge of unmutated sequences (middle panel) and mutated sequences (lower panel) of each sample are grouped by age. Sequences are pooled from two time points Y2 and Y3. Pairwise comparison was performed with two-sided student t-test. * P

Figure 3. V-mutation levels are increased in CMV positive individuals

A, Frequencies of gDNA sequences with different V-mutation levels are correlated strongly between samples from two consecutive years of the same participants. V-mutation frequencies are categorized to 3 levels: =10% (high) mutation. B, Frequencies of sequences with different mutation levels are grouped by age, gender, CMV or EBV status. Sequences are pooled from two years for each participant. P values are calculated by two-sided student t-test, including all data points for each category. * P

Figure 4. Frequencies of highly mutated IgM and IgG sequences are increased in some elderly individuals, and correlate with CMV infection regardless of age

A, Frequencies of IgM (upper panel) and IgG (lower panel) cDNA sequences with different levels of V-mutation grouped by age of participants. B, Frequencies of IgM (upper panel) and IgG (lower panel) cDNA sequences with different levels of V-mutation grouped by CMV status of participants. Sequences are pooled from two years. P values are calculated by two-sided student t-test, including all data points for each category. * P

Figure 5. Age and chronic EBV infections are correlated with persistent B cell clonal expansion

A, B, Clonality scores of the Ig repertoire of each sample are grouped by age for time point Y2 (A, n=27) and Y3 (B, n=25). The pairwise comparison between different age groups is performed with two-sided Wilcoxon tests. ** P Methods section.

Figure 6. Phylogenetic visualization of large clones

The large clones are visualized via immunitree. Each arrow points from an ancestral clone to a descendent clone. Sizes of the nodes correspond to the number of reads observed; the blue portion of the pie chart represents Y2, while the yellow portion represents Y3. The clones are labeled with the number of mutations from their immediate parent subclone. The root node's numerical label counts the number of mutations from the best matching germline reference V segment.