The broad, long term objective of this project is to elucidate the mechanism or mechanisms underlying the relative deficiency of serological immunity in the elderly, with particular attention to human B1-like cells and natural antibodies derived therefrom. The specific focus of the present application is to understand the age- related failure of immune defense against infection with S. pneumoniae, both at rest and after immunization with pneumococcal vaccine. A key mechanism for disposing of infectious pneumococci lies in opsonization by serum antibody, and a principal target for opsonizing antibody is phosphorylcholine (PC). Thus, we will study B cells, particularly B1-like cells, that bind PC and secrete anti-PC antibodies. It is known that human serum contains antibodies against PC, that human B1-like cells recognize PC, and that the total population of human B1-like cells declines with age. But it is not known what happens to PC-binding B1-like cells in older individuals as compared to younger individuals. In mice, where adoptive transfer experiments are possible, serum IgM derived from B1 cells of older mice fails to alter the course of pneumococcal infection in mice lacking antibody, whereas an equal amount of serum IgM from younger mice is effective. This coincides with an age-related change in the underlying nature of mouse B1 cell anti- PC antibodies as transcribed immunoglobulin becomes less germline in sequence. These reports regarding age-related changes in total human B1-like cell number and in mouse B1 cell anti-PC antibody repertoire suggest mechanisms for the failure of pneumococcal defense in older people. We hypothesize that the relative deficiency of serological immunity against PC/pneumococci in older humans, before and after pneumococcal vaccination, is due to one or more of: age-related loss of PC- binding B1-like cells, erosion of B1-like cell anti-PC antibody sequence/repertoire, and dysfunction of PC- binding B1-like cells/poor function of B1-like cell anti-PC antibodies. These characteristics have either never been studied in human B1-like cells or never been examined in antigen-specific human B1-like cells with respect to age. We will determine the validity of our hypotheses through the following aims in which we will compare older vs younger healthy donors, and older donors before and after pneumococcal vaccination. SA1. We will evaluate the level of PC-binding B1-like cells, by immunofluorescent staining for B cell subpopulations and PC-binding, followed by flow cytometry/cell sorting. SA2. We will determine the sequence/repertoire of PC-binding B1-like cell antibodies by single cell PCR followed by sequencing, cloning, and ELISA for specificity. SA3. We will evaluate the...