Research Project
9858268
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.����� �
