High affinity antigen-specific antibodies form the basis of protective humoral immunity. Defective generation of such antibodies is a major cause of the increased susceptibility of the elderly to common infections. Antibody affinity maturation is the result of the selection of high affinity immunoglobulin (Ig)-expressing B-lymphocytes. Most Ig genes undergo antigen-driven somatic hypermutation (SHM) in specialized lymphoid germinal centers to generate higher affinity antibodies. Our preliminary data in mice with the model antigen nitrophenyl (NP) suggest that antibody affinity maturation continues in the late phase of the primary response (35-70 days post immunization) well after the involution of lymphoid germinal centers. Surprisingly, the highest affinity antibody producing cells are found predominantly in the bone marrow. Because the same bone marrow B-cells also exhibited higher rate of SHM in the canonical anti-NP Ig variable genes, we hypothesized that SHM may take place in situ in the bone marrow. SHM is absolutely dependent on the activation-induced cytidine deaminase (AICDA) which is thought to be expressed almost exclusively in germinal center B-cells. Our analysis indicate that bone marrow-derived, IgG-positive, mature B-cells also express substantial level of AICDA, supporting the possibility of in situ Ig hypermutation in bone marrow. Because reduced antibody affinity maturation in the elderly is due to low level AICDA expression and poor SHM we compared the level of AICDA expression in splenic and bone marrow B-cells from young (2 month old) and old (22 month old) mice. While AICDA expression was significantly lower in the old spleen, it was comparable between young and old in bone marrow IgG-positive B-cells. Based on these findings we propose that the bone marrow is an alternative site of antibody affinity maturation which may, potentially, be better preserved during aging than the other, peripheral lymphoid sites. In this exploratory application we will test this hypothesis by comparing the characteristics of affinity maturation in young and old bone marrow, by identifying the candidate B-cell population that migrates to the bone marrow and by performing reciprocal, adoptive transfers to establish the step(s) where translocation of the humoral response to the bone marrow may be impaired in aging. With these studies we hope to open a new area of research which we could pursue in subsequent larger applications to understand the cellular and molecular mechanisms of bone marrow-associated humoral response in both young and aged individuals and test novel vaccination strategies that would improve antibody responses in the elderly. ? ? ?
