The broad, long term objective of this project is to elucidate the mechanism or mechanisms underlying the relativedeficiencyofserologicalimmunityintheelderly,withparticularattentiontohumanB1-likecellsand naturalantibodiesderivedtherefrom.Thespecificfocusofthepresentapplicationistounderstandtheage- relatedfailureofimmunedefenseagainstinfectionwithS.pneumoniae,bothatrestandafterimmunization withpneumococcal vaccine.Akeymechanismfor disposing of infectious pneumococci lies in opsonization by serum antibody, and a principal target for opsonizing antibody is phosphorylcholine (PC). Thus, we will studyBcells,particularlyB1-likecells,thatbindPCandsecreteanti-PCantibodies. It is known that human serum contains antibodies against PC, that human B1-like cells recognize PC,andthatthetotalpopulationofhumanB1-likecellsdeclineswithage.Butitisnotknownwhathappens toPC-bindingB1-likecellsinolderindividualsascomparedtoyoungerindividuals.Inmice,whereadoptive transferexperimentsarepossible,serumIgMderivedfromB1cellsofoldermicefailstoalterthecourseof pneumococcal infection in mice lacking antibody, whereas an equal amount of serum IgM from younger miceiseffective.Thiscoincideswithanage-relatedchangeintheunderlyingnatureofmouseB1cellanti- PCantibodiesastranscribedimmunoglobulinbecomeslessgermlineinsequence.Thesereportsregarding age-related changes in total human B1-like cell number and in mouse B1 cell anti-PC antibody repertoire suggestmechanismsforthefailureofpneumococcaldefenseinolderpeople. WehypothesizethattherelativedeficiencyofserologicalimmunityagainstPC/pneumococciinolder 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- bindingB1-likecells/poorfunctionofB1-likecellanti-PCantibodies.Thesecharacteristicshaveeithernever been studied in human B1-like cells or never been examined in antigen-specific human B1-like cells with respecttoage.Wewilldeterminethevalidityofourhypothesesthroughthefollowingaimsinwhichwewill 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 thatwillsuggestnewwaystoimproveanti-pneumococcalantibodyimmunedefenseintheelderly.
Infectionwithpneumococcicausesdisproportionatelyhighmorbidityandmortalityintheelderlyduetopoor immune function and inadequate antibody defense. Human B1-like cells are a unique subpopulation of B cells that is responsible for producing antibodies that target pneumococcal bacteria, and we will study the number, nature and function of anti-pneumococcal B1-like cells from older donors and compare with anti- pneumococcal B1-like cells from younger donors. Our results will diagnose what aspect of anti- pneumococcal B1-like cells and/or their antibodies fails in older individuals, and this will guide novel therapeutic interventions to restore proper B1-like cell physiology and reverse the anti-pneumococcal immunedysfunctionofaging.
