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 function of PC-binding B1-like cells and the
antibodies they produce, by determining B1-like cell secretion with ELISPOT assays and determining anti-
pneumococcal function with opsonophagocytic assays. This work will provide completely new information
that will suggest new ways to improve anti-pneumococcal antibody immune defense in the elderly.