Antibody responses to viral infections are sustained for decades by long-lived plasma cells (LLPCs). However, LLPCs have yet to be characterized in humans. Here we used CD19, CD38, and CD138 to identify four PC subsets in human bone marrow (BM). We found that the CD19−CD38hiCD138+ subset was morphologically distinct, differentially expressed PC-associated genes and exclusively contained PCs specific for viral antigens to which the subjects had not been exposed for over 40 years. Protein sequences of measles- and mumps-specific circulating antibodies were encoded for by CD19−CD38hiCD138+ PCs in the BM. Finally, we found that CD19−CD38hiCD138+ PCs had a distinct RNA transcriptome signature and human immunoglobulin heavy chain (VH) repertoire that was relatively uncoupled from other BM PC subsets and likely represents the B cell response’s “historical record” of antigenic exposure. Thus, our studies define human LLPCs and provide a mechanism for the life-long maintenance of anti-viral antibodies in the serum.

Background Advances in multi-parameter flow cytometry (FCM) now allow for the independent detection of larger numbers of fluorochromes on individual cells, generating data with increasingly higher dimensionality. The increased complexity of these data has made it difficult to identify cell populations from high-dimensional FCM data using traditional manual gating strategies based on single-color or two-color displays. Methods To address this challenge, we developed a novel program, FLOCK (FLOw Clustering without K), that uses a density-based clustering approach to algorithmically identify biologically relevant cell populations from multiple samples in an unbiased fashion, thereby eliminating operator-dependent variability. Results FLOCK was used to objectively identify seventeen distinct B cell subsets in a human peripheral blood sample and to identify and quantify novel plasmablast subsets responding transiently to tetanus and other vaccinations in peripheral blood. FLOCK has been implemented in the publically available Immunology Database and Analysis Portal – ImmPort (http://www.immport.org) for open use by the immunology research community. Conclusions FLOCK is able to identify cell subsets in experiments that use multi-parameter flow cytometry through an objective, automated computational approach. The use of algorithms like FLOCK for FCM data analysis obviates the need for subjective and labor intensive manual gating to identify and quantify cell subsets. Novel populations identified by these computational approaches can serve as hypotheses for further experimental study.

Upon vaccination, B cells differentiate into antibody secreting cells (ASCs) that migrate via the circulation to tissues. The kinetics of this response and the relationship of circulating ASCs to protective antibody titers have not been completely explored.Methods-Influenza-specific and total-IgG ASCs were enumerated by Elispot and flow cytometry daily in the blood in 6 healthy adults after trivalent influenza vaccination (TIV).Results-Peak H1-specific IgG ASC frequencies occurred variably from day 5 to 8 and correlated with the fold-rise rise in hemagglutination inhibition (HAI titers); r=0.91, p=0.006. H3-specific IgG ASC frequencies correlated less well, perhaps due to a mismatch of the H3 protein in the vaccine and that used in the Elispot assay. Peak frequencies of vaccine-specific and total-IgG ASCs were 0.3% and 0.8%, respectively, of peripheral blood mononuclear cells (PBMC). Peak TIV-, H1-, H3-, and total-IgG ASC frequencies were 1736 ± 1133, 626 ± 520, 592 ± 463, and 4091 ± 2019 spots/10 6 PBMC, respectively. Peak TIV-, H1-, and H3-specific IgG ASC of totalIgG ASC frequencies constituted 63% ± 21, 26% ± 10, 22% ± 17, respectively. Conclusion-After immunization with inactivated influenza vaccine the peak in influenzaspecific ASC frequencies is variable but correlates well with the magnitude of protective HAI responses.

Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutininspecific antibody production and changes in the size and character of hemagglutininreactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection "imprints" for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus. IMPORTANCE Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs).Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalkreactive, as well as to head-reactive, MBCs. Our findings provide experimental