Abstract
Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody.
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Acknowledgements
We thank R. Karaffa and S. Durham for technical assistance. Supported by the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health (HHSN266200700006C, 1P01AI097092 and U19AI117891 to R.A.; T32AI074492 to A.E.; and U19AI09525801, UM1AI100663 and U01AI104342 to S.D.B.), Advanced Immunization Technologies (280873), the European Union (R.A.), the National Center for Advancing Translational Sciences (UL1TR000454 to A.K.M.) and The National Council for Scientific and Technological Development of Brazil (H.I.N.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health or that of the Centers for Disease Control and Prevention.
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A.H.E., K.J.L.J., S.D.B. and R.A. designed the study, interpreted data and wrote the paper; A.H.E. performed most of the experiments; K.J.L.J. performed next-generation sequencing of the IGH repertoire and devised, undertook and interpreted repertoire-data analysis; H.T.K. and H.I.N. analyzed the microarray data; C.W.D. and A.K.M. helped in data analysis and interpretation; K.M.R. processed repertoire-sequence data; and C.M.O., R.E., S.T., G.M.L., C.F.S., A.K.M. and P.G.T. helped in collecting and processing the clinical samples.
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Supplementary Figure 1 ABCs do not spontaneously secrete antibodies.
(a) Ex-vivo ELISPOT assay of day 7 ASCs plated on ELISPOT plates coated with either the 2013/14 TIV or anti-human IgG antibodies. The numbers on the bottom-left side of some of the wells represent the number of spots counted in that well. (b) ELISPOT assay where day 7 ABCs were cultured for five days (see Methods) and plated on ELISPOT plates coated as in (a). Shown are the spots when only feeder (CD19–) cells or feeder cells and ABCs were plated.
Supplementary Figure 2 Sorting B cell subsets 7 d after vaccination against influenza virus.
A FACS plot showing the gating strategy used to sort naïve B cells (grey), MBCs (green), ABCs (blue) and ASCs (red).
Supplementary Figure 3 HA-positive ABCs do not spontaneously secrete antibodies.
(a) Ex vivo ELISPOT assay of day 7 sorted HA-positive ASCs and HA-positive ABCs plated on ELISPOT plates coated with either the 2009 pandemic H1 HA or anti-human IgG antibodies. The numbers on the down-left side of some of the wells represent the number of spots counted in that well. (b) ELISPOT assay where day 7 HA-positive ABCs were cultured for five days (see Methods) and then washed and plated on ELISPOT plates coated as in (a).
Supplementary Figure 4 Sorting strategy for the BCR-repertoire analysis of ABC and ASC clonal lineages.
FACS plots (gated on isotype-switched B cells) showing the different B cell populations sorted at (a) day 7 and (b) day 14 after TIV vaccination (n = 3).
Supplementary Figure 5 Outlines of sorted cell populations for long-term analysis of the BCR repertoire.
(a) Outline of the 2013/14 season study where RNA was purified from total PBMCs at days 0, 28 and 90 after vaccination, and from total ABCs and ASCs at day 7 and from HA-positive ABCs at day 14 after vaccination (n = 3). (a) Outline of the 2014/15 season study where RNA was purified from memory B cells and total PBMCs at days 0, 28 and 90 after vaccination, and from HA-positive and total ABCs and total ASCs at day 7 and from HA-positive ABCs, total ABCs and memory B cells at day 14 after vaccination (n = 3).
Supplementary Figure 6 Clonal tracking of day-14 H1-positive ABC lineages in the 2013–2014 season study.
Percentage of day 14 HA-positive ABC clonal lineages identified among day 7 ABCs (green) or MBC (orange) populations at days 0, 28 and 90 after vaccination for donors 157, 162 and 163.
Supplementary Figure 7 Tracking of IGH SHM of the ABC clonal lineages by B cell repertoire sequencing.
(a) Longitudinal tracking of median of mean IGHV SHM frequencies of B cell clonal lineages that included HA-specific ABC members at day 14 post-vaccination with TIV during the 2013-14 season. Bars show the 95% confidence interval. (b) Each panel represents the mean IGHV SHM frequencies for individual clonal lineages from donors 157, 162, 163 and 8. The color of each point indicates the B cell subset, the point size corresponds to the number of reads and linear regression lines are shown in blue.
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Supplementary Figures 1–7 and Supplementary Table 2 (PDF 1580 kb)
Supplementary Table 1
192 genes that were differentially upregulated in the ABC subset (XLSX 46 kb)
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Ellebedy, A., Jackson, K., Kissick, H. et al. Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination. Nat Immunol 17, 1226–1234 (2016). https://doi.org/10.1038/ni.3533
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DOI: https://doi.org/10.1038/ni.3533
