SummaryThe immune system of aged individuals often produces antibodies that have lower affinity and are less protective than antibodies from young individuals. Recent studies in mice suggested that antibodies produced by old individuals may be encoded by distinct immunoglobulin (Ig) genes and that the somatic hypermutation process in these individuals is compromised. The present study employed Igh 6 scid mice reconstituted with normal lymphocytes from young (2-3-mo-old) and aged (20-25-mo-old) donors and immunized with a protein conjugate of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) to determine whether the molecular changes in antibody repertoire reflect senescence in the B cells or whether they are mediated by the aging helper T lymphocytes. The NP-reactive B cells from splenic germinal centers (GC) were recovered by microdissection of frozen tissue sections and their rearranged Ig heavy chain variable region (VH) genes of the V186.2/V3 families were sequenced. It was found that the V, gene repertoire of the GC B cells was strongly influenced by the source of the CD4 § T cells. When T cells were donated by young mice, the anti-NP response in GC was dominated by the canonical V186.2 gene, even if the responder B cells came from aged donors. However, when the mice were reconstituted with T cells from aged donors, the expression of the V186.2 gene by young B cells was diminished and the response was dominated by the C1H4 gene, another member of the V186.2/V3 family. In contrast, the somatic hypermutation process in the GC B cells followed a different pattern. The mutation frequencies in the animals that were reconstituted with both B and T cells from young donors (1/50 to 1/150 bp) were comparable to the frequencies previously reported for NP-immunized intact young/adult mice. However, when either lymphocyte subset was donated by the aged mice, the mutation frequencies dedined. Thus, mice reconstituted with T cells from the aged and B cells from the young had severely compromised mutational mechanism. Likewise, the recipients of aged B and young T cells had diminished mutations even though the repertoire of their anti-NP response was dominated by the canonical V186.2 gene. It appears that the change in germline-encoded repertoire and the decrease of somatic hypermutation represent distinct mechanisms of immunosenescence and that the aging of helper T cells plays a pivotal role in both of these processes.

SummaryThe development of memory B cells takes place in germinal centers (GC) of lymphoid follicles where antigen-driven lymphocytes undergo somatic hypermutation and affinity selection, presumably under the influence of helper T cells. However, the mechanisms that drive this complex response are not well understood. We explored the relationship between GC formation and the onset of hypermutation in response to the hapten phosphorylcholine (PC) coupled to antigenic proteins in mice bearing different frequencies of CD4 § T cells. PC-reactive GC were identified by staining frozen splenic sections with peanut agglutinin (PNA) and with monoclonal Abs against AB1-2, a dominant idiotope of T15 + anti-PC antibody. The nucleotide sequences of rearranged T15 V.1 genes were determined from polymerase chain reaction amplifications of genomic DNA from microdissected GC B cells. T15 + GC became fully developed by day 6-7 after primary immunization of euthymic mice with either PC-keyhole limpet hemocyanin (KLH) or PC-chicken gamma globulin (CGG). Yet the V.1 gene segments recovered from the primary GC as late as day 10-14 had low numbers of mutations, in contrast to responses to the haptens nitrophenyl or oxazolone that sustain high levels of hypermutation after GC formation. PCreactive B cells proliferate in histologically typical GC for considerable periods with no or little somatic hypermutation; the signals for GC formation are independent of those for the activation of hypermutation. We then examined GC 7 d after secondary immunization with PC-KLH in euthymic mice, in nu/nu mice reconstituted with limited numbers of normal CD4 § cells before priming (CD4 § and in nu/nu mice. All of these animals develop T15 + GC after antigen priming, however, the patterns of V gene mutations in the secondary GC reflected the levels of CD4 § cells present during the primary response. VDJ sequences from secondary GC of euthymic mice were heavily mutated, but most of these mutations were shared among all related (identical VDJjoints) sequences suggesting the proliferation of mutated, memory B cells, with little de novo somatic hypermutation. In contrast, the patterns ofV gene diversity in secondary GC from CD4 +-nu/nu mice suggested that there was ongoing mutation and clonal diversification during the first week after rechallenge. The secondary GC from T cell-deficient, nu/nu mice showed little evidence for mutational and/or recombinational diversity of T15 + B cells. We conclude that the participation of CD4 § helper cells is required for full activation of the mutator in GC and takes place in a dose-dependent fashion.

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