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Immunity. Author manuscript; available in PMC 2016 Jul 21.
Published in final edited form as:
Immunity. 2015 Jul 21; 43(1): 132–145.
Published online 2015 Jul 14. doi: 10.1016/j.immuni.2015.06.016
PMCID: PMC4680845
NIHMSID: NIHMS706000
PMID: 26187412

Long-lived Plasma Cells Are Contained Within the CD19−CD38hiCD138+ Subset in Human Bone Marrow

Jessica L. Halliley,1,15 Christopher Tipton,2,3 Jane Liesveld,4 Alexander F. Rosenberg,5 Jaime Darce,6 Ivan V. Gregoretti,6 Lana Popova,6 Denise Kaminiski,5 Christopher F. Fucile,5 Igor Albizua-Santin,1 Shuya Kyu,1 Kuang-Yueh Chiang,7 Kyle T. Bradley,8 Richard Burack,9 Mark Slifka,10 Erika Hammarlund,10 Hao Wu,11 Liping Zhao,11 Edward E. Walsh,12 Ann R. Falsey,12 Troy D. Randall,13 Wan Cheung Cheung,6,14 Iñaki Sanz,2,3,16 and F. Eun-Hyung Lee1,3,16,*
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Associated Data

Supplementary Materials

Abstract

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.

Keywords: human, long-lived plasma cells, antibody secreting cells, plasmablasts, plasma cells, measles, mumps infection, vaccine, heterogeneity, bone marrow, next generation sequencing, proteomics

INTRODUCTION

Circulating high-affinity antibodies provide protection against previously encountered pathogens. This serological memory is maintained for decades by long-lived plasma cells (LLPCs) without re-exposure to antigen (Amanna et al., 2007; Fairfax et al., 2008; Oracki et al., 2010). In mice, virus-specific LLPCs survive in the bone marrow (BM) and secrete antibody for essentially the animal’s lifespan (Manz et al., 1997; Slifka and Ahmed, 1998). These LLPCs are maintained in the absence of memory B cells (Ahuja et al., 2008; Slifka and Ahmed, 1998), suggesting that they are truly long-lived, rather than being replenished from memory precursors.

BM-resident LLPCs are also thought to be the main source of circulating IgG antibody in humans (McMillan et al., 1972). The striking longevity of at least some human PC is best illustrated by the life-long persistence of serum antibodies generated in response to natural infection by measles and mumps viruses, whose half-life has been estimated at 3,014 and 542 years respectively (Amanna et al., 2007). In contrast, serum antibody responses against tetanus vaccination have a half-life of 11 years and responses to influenza viruses are variable depending on whether exposure was by immunization or infection (Amanna et al., 2007). Such a wide range is consistent with differential participation of bona fide LLPC in different immune responses, a proposition also supported by the differential stability of diverse autoantibody responses and their variable susceptibility to B cell depletion therapy (Cambridge et al., 2006).

The expression of markers commonly used to define LLPC is heterogeneous and not limited to BM PC. Although CD138 is expressed by BM PC (Medina et al., 2002), its expression can also be induced on PCs in short-term cultures (Huggins et al., 2007; Jourdan et al., 2011) and approximately half of the circulating PCs generated during an acute immune response also express CD138 (Gonzalez-Garcia et al., 2006; Medina et al., 2002; Qian et al., 2010), even though most of these cells are short-lived. In turn, CD138-expressing PCs in the BM are heterogeneous in their expression of various markers, including CD19 and HLA-DR (Liu et al., 2012; Medina et al., 2002). Moreover, receptors important for PC homing and survival, such as CXCR4 and BCMA, also fail to distinguish between long-lived and short-lived PCs (Benson et al., 2008; Medina et al., 2002). Thus, the phenotype of human LLPCs remains undefined, and as a result, these cells have not been rigorously characterized.

Here we tested whether LLPCs could be definitively distinguished from other PCs in human BM. We found that PCs specific for viruses encountered more than 40 years prior to the study were exclusively contained within the CD19−CD38hiCD138+ subset. These cells have a distinct PC morphology, are predominantly non-cycling and express many of the gene products implicated in LLPC function, homing and survival. We also show that the CD19−CD138+CD38+ cells in the BM are the only ones to encode circulating serum antibodies specific for measles and mumps and that monoclonal antibodies reconstructed from these sequences exclusively bind to the appropriate antigens. Finally, we show that the CD19−CD138+CD38+ subset of BM PCs has a distinct RNA transcriptome signature and a broadly diverse VH repertoire that is not dominated by large, clonally related populations and contains sequences that are mostly unrelated to those in other PC populations in the BM, suggesting that these cells likely to represent the historical record of antigenic exposure.

RESULTS

Characterization of distinct PC subsets in human BM by frequency, Ig secretion, morphology, and isotypes

To understand PC heterogeneity in the human BM, iliac crest aspirates were obtained from 14 healthy adults without a history of infection or vaccination within the previous month and cell suspensions were analyzed by flow cytometry. We initially gated on live lymphocytes lacking CD3 or CD14 expression (non-T cells, non-monocytes) or IgD negative cells (to eliminate late transitional and naive B cells) (Figure 1a). The remaining cells were divided into CD19+ and CD19− populations and subsequently characterized by the expression of CD138 and CD38 (Figure 1a) as previously described (Liu et al., 2012; Medina et al., 2002; Mei et al., 2015; Rawstron et al., 2002). Since all PCs in human BM express high amounts of CD38 (Rawstron et al., 2002), we confined our subsequent analysis to 4 populations; CD19+CD38hiCD138− cells (subset A), CD19+CD38hiCD138+ cells (subset B), CD19−CD38hiCD138− cells (subset C) and CD19−CD38hiCD138+ cells (subset D) (Figure 1a). The frequencies of all the PC subsets were validated with the use of two separate anti-CD19 monoclonal antibodies (SJ25C1 and HIB19 clones) and alternative gating strategies (supplemental figure 1). The frequencies of each of the 4 populations varied between individuals (Figure 1b), but subset B was generally the largest and subset C was generally the smallest. The mean yields for subsets A, B, C, and D were 3666 ± 4589, 4785 ± 7307, 1910 ± 2767, and 2848 ± 4050 cell/mL respectively. The average percentages of BM mononuclear cells for subsets A, B, C, and D were 0.05 ± 0.02, 0.07 ± 0.06, 0.02 ± 0.02, and 0.04 ± 0.02 respectively.

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PC subsets in human BM

(a) PC subsets in the BM were sorted by excluding cells that express CD3, CD14, and IgD and separating cells into CD19+ and CD19− fractions (top panels). Lower panels represent subsets of CD19−IgD− (left) and CD19+IgD− (right) fractions with PC subsets A, B, C, and D identified by CD38 and CD138. (One representative example of 31 samples analyzed). Alternative gating strategies quantified similar frequencies of cells in each subset (supplemental figure 1). (b) Number of cells in each PC subset per mL of BM (top) and % of mononuclear cells from each BM aspirates (bottom) N= 14, bar represents the mean. (c) Total IgG ELISPOTs (top) and IgA ELISPOTs from subsets A–D and naïve B cells (N) from one healthy donor. Input cell numbers are indicated in right corner. (d) Frequency of IgG and IgA-secreting cells from subsets A–D and naïve B cells by unstimulated sorted cells. Respective number of subjects listed above each subset (p-value for pop D 0.03, other pop A, B, C, and N were not significant (NS)). (e) Morphology of sorted BM PC subsets (100× magnification) by cytospins and Wright-Geimsa stain. Representative images of 100 cells of each subset A–D and naïve B cells are shown. Representative of 3 separate BM samples. Additional images from one direct bone marrow aspirate (supplemental figure 2). (f) Quantitative RNA expression of 5,000 sorted cells from subsets (A–D) and naïve (N) and memory (M) B cells. Relative mRNA expression is shown in arbitrary units normalized to GAPDH. One representative example of 2 experiments.

To enumerate functional PCs within each subset, we performed IgG and IgA ELISPOTs. In contrast to naïve B cells (CD19+IgD+CD27−), all PC subsets contained IgG− and IgA-secreting cells (Figure 1c). The frequency of IgG-secreting cells was highest in subset D and lowest in subset C (Figure 1d), whereas the frequency of IgA-secreting cells was higher in subsets A and B than in subsets C and D (Figure 1d). Only in subset D was the ratio of IgG: IgA significantly different (p= 0.03, N=6). The combined frequencies of IgG− and IgA-secreting cells made up about 20–35% of the input population in the ELISPOT assays (Figure 1d). The low frequency of ELISPOTs is most likely due to reduced viability or functionality of sorted PCs, since control studies showed that only 24% of IgG-secreting cells in total PBMCs were recovered after the sorting process (data not shown). In addition, contamination with non-PC was less likely since post-sort purity was 92–95%. Moreover, similar frequencies of ELISPOTs (19–45% of input cells), were obtained from sorted multiple myeloma cell lines. Thus, the relatively low frequencies of ELISPOTs in our sorted subsets are likely explained by reduced viability or function and not by contamination with other cell types.

Given that PCs display characteristic morphological features, such as an eccentric oval nucleus and a prominent Golgi apparatus (Miller, 1931), we performed cytospins of cells from each subset, stained them with Wright-Giemsa, and examined 100 cells in each fraction by microscopy. Cells in each subset had distinctive features of PCs (Figure 1e). However, cells from subset D displayed unique features including a more condensed nucleus and a higher cytoplasm/nucleus ratio (Figure 1e). In addition, cells in subset D had the most homogeneous features, with consistently well-rounded nuclei and distinct Golgi. These distinct features were observed in cells from 3 separate patients (ages 40, 55, and 60 y.o.). Lastly, a large majority (74%) of cells in subset D prominently displayed large vacuoles, a feature rarely observed (<1% of cells) in other subsets, including the other CD138+ cells or pop B (Figure 1e). We also readily identified PCs with and without vacuoles (subsets D and B) in Wright-Giemsa stained BM aspirates from healthy patients (supplemental figure 2), suggesting that vacuoles are not artifacts of FACS sorting. Finally, none of our donors demonstrated any signs of malignancy for up to 2 years post-analysis. Thus, we conclude that cytoplasmic vacuoles are a physiological feature of healthy PC and are restricted to cells within subset D.

We next determined the relative expression of transcription factors involved in either maintaining B cell identity (PAX5) or licensing PC differentiation (BLIMP-1, XBP1). We found that PAX5 was poorly expressed in every PC subset compared to naïve and memory B cells (Figure 1f), whereas BLIMP-1 and XBP1 were both highly expressed by subsets A, B and D, but nearly undetectable in naïve and memory B cells. These data support the contention that subsets A, B and D are primarily composed of terminally differentiated PCs.

As B cells differentiate into PCs, they lose features of B cells (such as CD20 and surface Ig), exit cell cycle, gain expression of the transcription factor BLIMP-1, and upregulate receptors for homing and survival in the BM (Hauser et al., 2002; Tourigny et al., 2002; Turner et al., 1994). Accordingly, we used multiparameter flow cytometry to evaluate these characteristics (Figure 2; results summarized in Supplemental Table 1). As shown in figure 2a, CD20 was downregulated on nearly every cell in all four BM subsets, consistent with the loss of CD20 that begins as early as 7 days after vaccination in newly formed plasmablasts (PBs) (Qian et al., 2010). In contrast, surface Ig which is gradually downregulated during PC maturation was expressed on more than a third of the cells in subset A, to a lesser extent in subsets B and C, and on only 8% of the cells in subset D. This observation is in contrast to the high frequencies of circulating PBs that express surface Ig.

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Characterization of BM PC subsets and blood plasmablasts

Representative histograms of PC subsets A–D and naïve B cells (N) in BM and peripheral blood (CD138− and CD138+) plasmablasts (PBs) obtained 7 days after vaccination showing surface expression of (a) CD20, surface Ig, CD27, and HLA-DR (b) intracellular BLIMP-1 and IRF4, (c) intracellular Ki-67, and (d) surface expression of CXCR4, CD28, IL-6R, and FcγRIIb. Expression on control naïve B cells shown in gray. (Representative number of samples for each marker shown in supplemental table 1).

Circulating PBs as well as PCs in subsets B and D uniformly expressed high amounts of CD27, a member of the TNFR family that is upregulated during B cell activation and is linked to PC differentiation (Avery et al., 2005), whereas cells in subsets A and C had a bi-modal distribution of CD27 (Figure 2a). HLA-DR, a marker of cell activation previously shown to decrease during PC maturation (Medina et al., 2002), was expressed by the vast majority of blood PBs, but exhibited a range of expression in subset A and was nearly absent from cells in subsets B–D (Figure 2a).

We also observed nearly universal expression of BLIMP-1 in subsets A, B and D, which validates the qPCR results and further suggests that these subsets represent PC lineages (Figure 2b). In contrast, BLIMP-1 was only expressed by about half the cells in subset C (Figure 2b). Additionally, we found a consistently high amount of IRF4 expression in cells from subsets A, B, and D, but observed heterogenous staining in subset C (Figure 2b), again suggesting that subset C consists of a mixture of PCs and non-PCs.

We also found that Ki-67, a nuclear protein associated with recent cell division, was expressed by a large fraction of cells in subset A, a lower frequency of cells in subset B and a minority of subset D (Figure 2c). In direct contrast, nearly all post-vaccination PBs from peripheral blood expressed Ki67, consistent with the idea that these cells were recently generated.

The relative expression of molecules involved in PC homing and survival was also highly informative (Figure 2d). For example, CXCR4, a chemokine receptor implicated in PC homing and retention in the BM (Hauser et al., 2002; Nie et al., 2004), was expressed on about 70% of cells in subsets A, B and D, but only on about 42% of cells in subset C (Figure 2d). CD28, a co-stimulatory molecule associated with LLPC survival (Rozanski et al., 2011), was expressed at the highest frequency (20%) on BM cells in subset D and was virtually absent in blood PB. Despite the role on IL-6 in PC survival (Hilbert et al., 1995), the IL-6R was mostly expressed by blood PBs and at a much lower frequency by any BM subset (Figure 2d). Finally, the expression of the inhibitory FcγRIIb, which promotes PC apoptosis (Xiang et al., 2007), was widely variable from subject to subject but was typically lower on cells in subsets C and D (Figure 2d).

Together, these results demonstrate that, in contrast to other BM PC subsets, cells in subset D have a uniform gene expression pattern that is consistent with the expected features of LLPCs.

Identification of human LLPCs in the CD19−CD38hiCD138+ subset

Serum antibody responses against vaccination or infection can last for decades (Amanna et al., 2007), suggesting similar longevity for LLPCs. To test whether LLPCs were confined to a discrete population in the BM, we sorted cells from each PC subset from 7 healthy adults (mean age 45 years, range 33–55 years) and performed ELISPOTs using plates coated with tetanus or with anti-IgG to enumerate all IgG-secreting cells (Lee et al., 2011). We found that tetanus-specific responses were only present in subset D in 4 subjects (Figure 3a–b), with a mean frequency of 0.51% of total IgG ASC (range 0 – 1.8%). Variability of cell numbers of the PC subsets A, B, C, and D for each individual was common and thus, to account for different cell numbers in each well, a Fisher’s exact test was applied to evaluate the null hypothesis that the frequencies are equal in the two subsets. Comparisons between pop A–D and pop B–D are shown to be statistically different (figure 3b) but there was no statistical difference between pop C–D due to low cell numbers. Cell recovery from pop C was often lowest of all BM subsets due to low frequencies (figure 1b) and heterogeneity of this population (not all cell were PC, figure 2b). Thus, we concluded that long-lived responses reside predominantly in pop D.

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Identification of LLPCs by ELiSPOTs

(a) ELISPOTs of total IgG-secreting cells (top row), tetanus-specific IgG secreting cells at steady state (2nd row), tetanus-specific IgG secreting cells 21 d after tetanus vaccination (3rd row), measles-specific IgG secreting cells (4th row), mumps-specific IgG secreting cells (5th row), and influenza (flu)-specific IgG secreting cells (6th row). Input cell numbers at right corner. (b) Frequency of tetanus-specific IgG secreting cells in subsets A–D and naive B cells (N) from 7 different adults (mean age 45, range 33–55 years old) (filled symbols). Open symbols frequency of tetanus-specific IgG ASC in pop A–D and naïve B cells 21 days after tetanus vaccination. (c) Frequency of measles-specific (closed triangles) or mumps-specific (open triangles) IgG-secreting cells subsets A–D and naïve (N) B cells from 11 subsets PC frequencies in each BM PC subsets (pop A, B, C, D), and naïve (N) B cells from 11 adults (mean age 49 years; range 43–70 years). Gray triangle represents measles IgG PC frequency one year afterwards. (d) Frequency of influenza-specific IgG secreting cells in subsets A–D and naïve (N) B cells from 9 subjects (mean ages 45 years, range 30–56 years). A Fisher’s exact test was then applied * p value <1 × 10−2, ** p value <1 × 10−3, *** p value <1 × 10−4.

To understand the BM PC response after a recent tetanus vaccination, we enumerated tetanus-specific IgG ELISPOTs after vaccination on days 0, 7 and 21 in the blood and on day 21 in the BM. We found no tetanus-specific ASC in the blood prior to vaccination, but observed 76 ASC per 106 PBMC on day 7, which declined to 0 on day 21. In contrast, we observed tetanus-specific IgG ELISPOTs on day 21 in the BM in both subsets B (0.36% of total IgG) and pop D (0.20% of total IgG) (Figure 3a–b), suggesting that subsets B and D can be rapidly populated with ASCs following vaccination, but that PCs are maintained for longer periods in subset D.

To conclusively establish the longevity of PCs in subset D, we obtained BM aspirates from 11 older healthy adults (mean age 49 years; range 43–70 years) with a high serum antibody titer to either measles or mumps and no history of MMR vaccination to ensure exposure of these viruses by natural childhood infection. We sorted cells from each of the PC subsets in the BM and performed measles-specific and mumps-specific ELISPOTs. Similar to what we observed with tetanus responses, measles-specific and mumps-specific ELISPOTs were found prominently in subset D, with a mean frequency of 0.6% (range 0–1.8%) of total IgG ASC (Figure 3a and c). Comparisons between subsets A–D (p-value of 8.094e-5) and A–B (p-value of 3.06e-12) were found to be highly significant. Again, due to low cell numbers, comparison between pop C–D was not significant. Strikingly, measles-specific ELISPOT analysis repeated a year later in one subject confirmed the restriction of the response to subset D, with nearly identical frequencies (0.6% and 0.5% of total IgG ASC; figure 3c). Although we could not discriminate difference between pop D and pop C due to overall low cell numbers in pop C, we conclude that the LLPC reside in pop D since nearly all PC cells in the BM reside in pop A, B, and D.

In contrast to measles and mumps, healthy adults typically encounter influenza virus antigens annually either through vaccination or natural infection. Thus, we postulated that influenza-specific ASC, including long-lived PC elicited by natural infection and recently-generated, short-lived PC will likely be distributed between multiple subsets (Skowronski et al., 2008; Yu et al., 2008). To test this possibility, we sorted PC subsets from BM aspirates from 9 subjects (mean ages 45 years, range 30–56 years) and performed influenza-specific ELISPOTs using the 2010 seasonal vaccine (Halliley et al., 2010; Kyu et al., 2009). We observed influenza-specific PCs in subsets A, B and D (Figure 3a and d). Statistical comparisons showed differences between pop A–D and pop C–D but no differences were noted between pop B and D. Together, these results indicate that short-lived PCs can reside in subset B and possibly in subset A, whereas LLPCs are restricted to subset D.

Long-lived serum antibody responses are maintained by CD19−CD38hiCD138+ cells

Since the half-life of human serum IgG is 15–30 days (Morell et al., 1970), any measles-specific or mumps-specific circulating IgG present in adults who have not encountered these antigens since childhood must be recently produced by LLPCs. To formally demonstrate the cellular origin of long-lived anti-viral antibodies, we performed a coordinated analysis of serum and bone marrow of one 64-year-old adult who had been infected with measles and mumps as a child. Virus-specific IgG was affinity purified using measles or mumps antigens and interrogated by serum Ig proteomic methods described previously (Cheung et al., 2012; Sato et al., 2012). Virus-specific IgG antibodies were eluted, verified for measles or mumps activity by antigen-specific ELISA (Figure 4a), then digested and analyzed on the Liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) (Figure 4b). Mass spectra identified from each digested eluate were compared using SEQUEST to HCDR3 sequence databases (VH1-VH6), generated from cells in subsets A, B, D and naïve B cells. Top candidate HCDR3 sequences (VKGGNLLGIRPFDS for measles and HCDR3 sequence AKMIGSSAWYPFDY for mumps - Figure 4b), were identified based on the mapping of high confidence peptide spectra. A total of 55 (measles) and 67 (mumps) peptides were mapped covering 100% of the variable region of the gamma chains. Importantly, the unique measles and mumps HCDR3 peptide sequences were restricted to RNA sequences only present in subset D (117 VH sequences containing measles-specific HCDR3 and 59 VH sequences containing mumps-specific HCDR3 - Figure 4c). These virus-specific HCDR3 sequences were not found in subsets A or B or in naïve B cells (Figure 4c).

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Serum antibodies to measles and mumps restricted to BM subset D

(a) ELISA activity of affinity purified measles-specific and mumps-specific serum antibodies from a 64 year old adult. Original sera (blue), measles eluted fraction (red), and mumps eluted fraction (black) are shown. (b) LC-MS/MS spectra produced and matched by SEQUEST to the full V-region tryptic peptide GGNLLGIRPFDSWGQGTLVTVSSASTK (for measles) and MIGSSAWYPFDYWGQGTLVAVSSASTK (for mumps) contain heavy chain CDR3 (underlined) and their junction with the framework-4. (c) Number of HCDR3 sequences identified from a total of 24,003 gamma chain sequences from subset A, 90,061 from B, 158,114 from D, and 28,351 from naïve B cells that match the peptide sequences obtained in panel b. (d) Specificity of recombinant monoclonal antibody reconstituted from the of VH and VL sequences from the BM subsets D NGS data that aligned with VH and VL tryptic peptides HCDR3: AKMIGSSAWYPFDY and LCDR3: GTWDSSLGIVL from the mump-eluted fractions. Total anti-measles and mumps activities from the original serum are shown as controls.

We also mapped the VL peptides digested from the mumps-specific Ab and reconstructed the most confident full-length VL-region sequences (Cheung et al., 2012; Sato et al., 2012). Using full-length heavy and light chain sequences with HCDR3: AKMIGSSAWYPFDY and LCDR3: GTWDSSLGIVL, we constructed and expressed a recombinant monoclonal antibody (Cheung et al., 2012; Sato et al., 2012) and tested its specificity by ELISA. We found that the reconstructed monoclonal antibody was specific for mumps, but did not recognize measles antigens (Figure 4d). Together, these data demonstrate that LLPCs currently producing serum antibodies specific for antigens encountered decades previously originate only from subset D of the human BM.

LLPCs have a distinct VH repertoire that is uncoupled from other PC subsets

The antibody repertoires expressed by different PC subsets were analyzed by NGS of sorted BM populations (subset C was not included due to low cell numbers). Consistent with our ELISPOT data (Figure 1c), subset D was differentiated by a predominant expression of IgG sequences (76%). In contrast, IgG was lower than IgA in subset A (35% and 65%) and more evenly split in subset B (56% and 44%). Despite wide variability between subjects, the average number of somatic mutations was similar in each of the BM PC subsets (21.6 ± 9.9 in A, 21.8 ± 10.0 in B and 17.8 ± 9.3 in D).

Repertoire diversity within the different subsets was ascertained using several metrics of clonality. Unique clonotypes were comprised of all the sequences sharing the same V-D-J rearrangement and had a CDR3-H of identical length, with at least 70% sequence similarity within that region and conservation of the VH-D and D-JH junctional sequences. Of the 5719 sequences obtained from PCs in subset A, we identified 1037 clonotypes, of the 9411 sequences from subset B, we identified 3319 clonotypes, and of the 13811 sequences from subset D, we identified 2578 clonotypes (Figure 5a). We also found that PCs in all 3 subsets expressed a very diverse repertoire that was primarily composed of singletons and clonotypes of relatively small size. Specifically, the most abundant clonotype in each subset made up only 1.5% of total sequences in subset A, 2.2% in B, and 1.6% in D. A high degree of diversity within all the BM PC subsets was also indicated by very high D50 and D80 scores representing the number of clonotypes accounting for the top 50% or 80%, respectively, of sequences within a given population. Thus, the D50 scores for subsets A, B, and D were 156, 488, and 299 respectively and the D80 scores were 424, 1545 and 978 respectively (Figure 5a).

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Clonal diversity of BM PC subsets and blood PBs

The VH repertoire for VH families 1–6 was determined by next generation sequencing of cells in (a) BM subsets A, B and D and (b) CD138+ and CD138− PBs in peripheral blood after tetanus vaccination. The cumulative percentage of sequences (Y-axis) versus lineage (clonal) size (x-axis) ranked by abundance. D50 designates the number of clonotypes in the top 50% of the sequences and D80 represents the number of clonotypes in the top 80% of the sequences. Numbers above each plot indicate frequency of unique IgG clonotypes per total number of IgG sequences.

As a comparative counterpoint of oligoclonal PB populations (Qian et al., 2010; Wrammert et al., 2008), we analyzed CD138+ and CD138− PB sorted from healthy subjects 7 days after tetanus vaccination and sequenced the productively rearranged VH genes from cells that had switched to either IgG or IgA. Sequences from CD138− PBs were comprised of 57% IgG and 43% IgA, whereas sequences from CD138+ PBs were comprised of 63% IgG and 37% IgA. Of the 10,735 sequences from CD138− PBs, we identified 459 clonotypes, and of the 11,892 sequences from CD138+ PBs, we identified 486 clonotypes (Figure 5b). The most abundant single clonotype in CD138− PBs made up nearly 30% of the total sequences (Figure 5b), whereas the most frequent clonotype in the CD138+ PBs made up nearly 14% of the total sequences. Moreover, the D50 scores of CD138− and CD138+ PBs were 2 and 4 respectively, whereas the D80 scores of CD138− and CD138+ PBs were 10 and 25 respectively (Figure 5b). Thus, unlike the PCs in the BM, the acutely circulating PB subsets are dominated by a very small number of substantially expanded clones.

A separate LLPC compartment that contains unique antibody responses would be expected to express a repertoire that is distinct from other PC populations. Thus, we determined the degree of inter-relatedness of the PCs in subsets A, B and D. As illustrated by Circos plots (Figure 6a–b) for IgG and IgA sequences, a large fraction of subset D was indeed uncoupled from other BM populations. However, subset D shared 6% of IgG and 11% of IgA clonotypes with subset A and shared 15% of IgG and 16% of IgA with subset B (Figure 6ab). Thus, a relatively small segment of subset D sequences was shared with subsets A and B. In contrast, the repertoires of post-vaccination blood CD138+ and CD138− PBs were highly connected, as they shared 58% and 41% of IgG and IgA sequences, respectively (Figure 6c–d).

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Connectivity of VH repertoires of PC subsets in the BM

Connectivity is shown with circos plots for (a) IgG and (b) IgA sequences for PC subsets A, B, and D in the BM of an adult age 40 years old at steady state. Connectivity is indicated for (c) IgG and (d) IgA sequences from CD138+ and CD138− PB subsets in peripheral blood 7 days after tetanus vaccination (age 56 years old). The red line in the outer ring denotes the average number of mutations for each population and the gray trace represents the number of mutations found in each clone. The second track indicates the numbers of individual sequences. The divisions in the third track identify separate clonal populations. Clonal relationships are indicated by gray or colored lines and the thickness of the lines represents the number of clones related between populations. Colored lines in the middle represent relationships between clonotypes that consist of more than 50 sequences, whereas the gray lines denote relationships of clonotypes of less than 50 sequences. Boxes below show % of sequences or clonotypes that are connected between the indicated PC/PB subsets in the BM or blood.

Given the polyclonality of subset D and in order to correct for sampling efficiency, we performed a control experiment by splitting 20,000 cells sorted from subset D into two fractions of 10,000 cells and sequencing them in separate reactions. We found that only 44% of all sequences and 40% of the lineages were concordant between the two theoretically identical populations, a result consistent with a very polyclonal compartment and small average clone sizes. After applying a correction factor generated from these data, we conclude that the corrected level of repertoire connectivity between subset D and subset A is 13% for IgG and 25% for IgA, and the corrected level of repertoire connectivity between subset D and subset B is 34% for IgG and 36% for IgA. This result is in sharp contrast with the degree of connectivity between subsets A and B (43% of IgG and 30% of IgA prior to correction). Therefore, the application of the same correction factor would indicate almost complete identity between subsets A and B. Correction factors are at best estimates to distinguish polylclonal vs oligoclonal repertoires. In all, PC subsets in the BM are highly polyclonal with subset D including a largely independent segment, whereas recently expanded peripheral PBs after acute antigen exposure are highly inter-related and are primarily comprised of a limited number of expanded clones.

Pop D has a distinct RNA transcriptome with genes involved in autophagy

To further understand the properties and mechanisms of survival of pop D as the LLPC compartment, we performed RNA sequencing in BM pop A, B, and D at steady state in 5 separate healthy adults (mean ages 44 ± 12 years, 25 to 54 years). (Pop C was not collected due to variable BLIMP-1 staining or low numbers). Gene expression was log-transformed and selected based on pairwise comparisons from 20,692 available genes for false discovery rate (FDR) <0.00001 and absolute value of fold change > 1.5. In the BM, 74, 768, and 154 genes were differentially expressed between pop A&B, A&D, and B&D respectively (figure 7a, b). Initial IPA canonical pathway analysis identified several pathways important for BM survival including CD28 and mTOR signaling.

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Transcriptome analysis of PC subsets and Upregulation of Autophagy pathways

(a) Numbers of selected genes are shown from 5 BM aspirates sorted for pop A, B, and D. Genes were selected based on pairwise comparisons from a total of 20692 available genes. False discovery rates (FDR) were computed from p-values as described by Storey (2002). Heat map shows per-gene z-score of log10-transformed, replicate-averaged data (zeros set to non-zero minimum of profiles).). The selection criteria for a pairwise comparison were as follows: FDR < 0.00001, absolute value of fold change > 1.5, and the maximum group mean > −0.5 (roughly equivalent to non-log expression of 0.3). Directionality corresponds to the first group relative to the second. For example, “Up” in A vs B means A > B. (b) numbers of genes differentially expressed between pop A & B, pop A & D, and pop B & D. (c) Principal component analysis for pops A, B and D based on 788 selected genes. (d) LC3BII staining of pop B and D by confocal microscopy. (e) Electron microscopy of pops A, B and D. Red arrow shows lipid droplets, green arrows show autophagosomes (AP) (f) Percentage of AP in BM PC subsets by EM (number of cells with AP /total cells) pop A: 11/22, pop B: 34/62, and pop D: 47/55.

PCA analysis showed that pop D clearly segregated from pop A and B following a pattern of sequential differentiation progressing from pop A to B to D along PC1 (figure 7c). Of significant interest, genes involved in activation of autophagy, a process shown to be critical for the survival and function of mouse PC (Pengo et al., 2013), were a major contributor to PCA separation of pop D relative to the other BM PC populations (differentially expressed genes involved in autophagy are listed in supplemental figure 3).

To further explore autophagy activation, we performed LC3BII staining by confocal microscopy and enumerated autophagosomes by electron microscopy (EM). Punctate LC3BII is a well-established marker of activated autophagy resulting from conversion of the autophagy factor LC3BI (ATG8) to its phosphatidylethanolamine-conjugated form LC3BII, leading to its association with the autophagosome membrane. Punctuate LC3BII expression is unique to population D (figure 7d). In addition, EM also showed a higher frequency of autophagosomes in subset D (85% of all pop D PC) compared to 50% in subset A or 55% in subset B PC (figure 7e, f). Combined, transcriptome analysis and microscopy results indicate that activated autophagy is a distinctive feature of pop D and identify this pathway as an important mechanism of survival of human LLPC.

DISCUSSION

Acute recall immune responses generate short-lived proliferative plasmablasts that produce a transient burst of antigen-specific antibodies. Thereafter, pathogen-specific antibodies are sustained by resting, terminally differentiated PC that in mouse models may survive for the life of the animal. Similarly, LLPC have been proposed in humans to explain the persistence, in the absence of antigen-specific stimulation, of antimicrobial antibodies with very long half-lives (10 to >3,000 years). However, direct proof for this mechanism and the actual identity of human LLPC have remained elusive (Gonzalez-Garcia et al., 2006; Gonzalez-Garcia et al., 2008; Hargreaves et al., 2001; Liu et al., 2012; Mei et al., 2015; Odendahl et al., 2005; Qian et al., 2010; Radbruch et al., 2006), and alternative models, most notably the polyclonal by-stander stimulation of memory cells, have been proposed to explain serological memory (Bernasconi et al., 2002). These critical gaps stem from the inability to determine the longevity of multiple PC subsets previously reported in the human BM and to ascribe the generation of long-lived antibodies to any singular population. Instead, location in the bone marrow, a resting state and the expression of markers associated with ASC maturation (such as expression of CD138, and downregulation of HLA-DR or CD19), are frequently used as surrogate markers for LLPC.

Here we identified in the human BM a population of CD19−CD38hiCD138+ cells (subset D), that unambiguously contains LLPCs. These cells have a distinctive morphology, express BLIMP-1, IRF4 and XBP-1 and are non-cycling. Critically, as determined by Elispot, subset D are the only cells to secrete antibodies specific for measles and mumps viruses more than 40 years after infection. Such a definitive link was demonstrated by exclusive matching of the serum anti-viral antibody proteome and the BCR repertoire expressed by subset D but not by any other BM PC compartment. These findings are validated by the demonstration that recombinant antibodies reconstructed from subset D sequences recapitulate the anti-viral specificity of the serum antibody fraction specific for the corresponding virus.

In contrast to subset D, other BM PC subsets produce antibodies against more recent antigens, such as influenza and tetanus, but not measles or mumps, thereby suggesting that they are relatively short-lived. Although these subsets share PC features, such as low expression of PAX5 and CD20 and high expression of BLIMP-1, XBP-1 and IRF4, other characteristics were indicative of a more short-lived phenotype. Thus, consistent with recent differentiation from B cells, significant fractions of subsets A–C retained sIg and MHC class II and expressed lower levels of CD27. Similarly, substantial Ki-67 expression in A–C is in keeping with recent derivation from proliferating precursors.

The longevity of measles and mumps-specific antibody responses is attributed to the intrinsic lifespans of LLPCs generated following viral infection. However, despite being generated by vaccination, the half-life of the tetanus-specific antibody response is remarkably long at 11 years (Amanna et al., 2007), compared to the much shorter half-life of antibody responses to polysaccharides (Kelly et al., 2006). Thus, it is perhaps not surprising that, under steady-state conditions, we find tetanus-specific PCs in the LLPC compartment. Nevertheless, the faster decay of anti-tetanus responses relative to anti-measles and mumps responses demonstrates significant variability in the lifespan of LLPC generated under different conditions.

Antibody responses to influenza vaccination also decline relatively quickly, although detectable serum titers may persist for 90 years (Yu et al., 2008), suggesting that both short-lived and long-lived PCs are generated. Consistent with this idea, we find influenza-specific PCs in subsets A–B as well as in subset D. Given that influenza virus is constantly changing by yearly antigenic drift and the occasional shift in subtype (Palese, 2007), it could be predicted that influenza-specific PCs in subset D will produce antibodies against temporally distant serotypes, whereas cells in subsets A, B, and even D will contain cells reactive with more contemporary serotypes. In addition, the presence of anti-influenza antibodies in LLPC could represent responses to the conserved nucleoprotein antigens shared between influenza strains encountered decades apart (Kaminski and Lee, 2011). Differentiating between these possibilities will require comprehensive studies of the fine specificity of anti-influenza PCs.

LLPC could derive from B cell precursors shared by other BM PC subsets through a progressive differentiation process in which phenotypic changes in the different PC subsets would reflect the age of the cells in each population, with the oldest cells progressively losing CD19 and surface Ig. This sequential differentiation model is supported by the progressive transcriptional separation of the different PC populations demonstrated by PCA which would be consistent with sequential maturation of subset D from common precursors differentiating through stages A and B.

Alternatively, LLPC could originate from separate precursors which would activate specific differentiation, homing and/or survival programs that ultimately determine their longevity upon taking residence in the bone marrow microenvironment. Our current data cannot definitely distinguish between these models. Extended longitudinal studies after primary immunization with neoantigens will be required to clarify the ontogeny of human LLPC.

Nevertheless, human LLPC are distinguished from other BM PC, including other CD138+ cells, by several unique features that provide important mechanistic insights regarding their differentiation and survival programs. Thus, subset D cells include prominent cytoplasmic vacuoles representative of ongoing lysosomal autophagy, a process recently recognized as critical for PC homeostasis by regulating Ig secretion and optimizing energy metabolism and survival (Pengo et al., 2013). In keeping with this concept, the combination of transcriptional programs and microscopy analysis strongly point to active autophagy as a major mechanism of LLPC survival.

This study contributes the first high-throughput sequencing analysis of human BM PC. NGS is a powerful means of testing the expected properties of a separate LLPC. A central prediction for a distinct LLPC compartment is that, over a lifetime, it would progressively accumulate cells that have responded to a myriad of antigens. A corollary of this prediction is that in adults, a well-established LLPC would display a highly diversified repertoire, a property that is consistent with our results. Interestingly, repertoire analysis established the presence of two distinct components in subset D. We postulate that the larger component, which is uncoupled from other BM PC populations, represents steady-state LLPC containing the historical record of B cell responses, a conclusion supported by the exclusive presence of mumps and measles-specific PCs in subset D decades after antigen exposure. In contrast, a much smaller fraction of subset D is clonally connected with subsets A and B, a feature we interpret as indicative of new arrivals derived from more recent B cell responses. Newcomers into the D subset might eventually become bona fide LLPC and contribute to the steady-state fraction depending on their intrinsic longevity (Amanna and Slifka, 2010), and/or their ability to compete for survival niches. This model would also account for the presence of some Ki-67+ cells in subset D, as these cells may be derived from recently dividing precursors (Cassese et al., 2003; Radbruch et al., 2006). Alternatively, the presence of a low frequency of Ki-67+ cells in subset D could indicate that limited homeostatic proliferation may be important to maintain the LLPC pool, as shown for human memory cells (Macallan et al., 2005).

In summary, the identification of CD19−CD38hiCD138+ cells in human BM as a bona fide LLPC compartment will enable investigators to understand the cellular source of different types of protective and pathogenic antibodies. It will also pave the way for a precise understanding of the molecular roadmaps underlying the differentiation and survival of this critical compartment. In turn, this knowledge will be central to our ability to maximize the generation of long-lived protective responses in microbial vaccination and prevent the accumulation of pathogenic PC in autoimmune diseases and transplantation.

METHODS

Subjects

Bone marrow aspirates were obtained from 35 healthy adults (ages 22 – 70 years, mean 44 ± 13). Eleven of 35 adult subjects were older (age > 40 years range 43 to 70, mean 52 ± 8 years) were recruited due to high serum titers of measles or mumps from history of natural infection with measles and mumps viruses during childhood. All adult subjects were vaccinated to influenza vaccination within 1–11 months prior to BM aspirates. Blood and bone marrow aspirate was obtained from each patient and mononuclear cells were isolated by density gradient centrifugation. Blood for serum and BM were also obtained from one 64-year old man for proteomics studies. Vaccinated and healthy asymptomatic adults: Two healthy adult subjects (ages 27 & 56 years) were enrolled. Subjects received the tetanus toxoid vaccinations Td or combination Tdap as a part of routine medical care. PBMC were isolated pre-vaccine, and on days 6–7 for all vaccination subjects. All subjects in this study were recruited at the University of Rochester Medical Center or Emory University, and all studies were approved by the Institutional Review Boards at the University of Rochester Medical Center and Emory University.

VH next generational sequencing

Total cellular RNA was isolated from: blood CD19+CD138+and CD19+Cd138− and pop A, B, D from one blood after tetanus vaccination and 3 BM using the RNeasy Mini Kit (Qiagen, Inc. Valencia, CA) by following the manufacturer's protocol. Approximately 400 pg of RNA was subjected to reverse transcription using the iScript RT kit (BioRad, Inc., Hercules, CA). Resulting cDNA products were included with 50nM VH1-VH6 specific primers and 250nM Ca, Cm, and Cg specific primers in a 20 μl PCR reaction using High Fidelity Platinum PCR Supermix (Life Technologies, Carlsbad, CA) and amplified by 40 cycles. Nextera indices were added and products were sequenced on an Illumina MiSeq with a depth of approximately 300,000 sequences per sample. One BM sample was used as a control and 20,000 pop D cells were collected and RNA was isolated and NGS was performed as described above. For all sequences were aligned with IMGT.org/HighVquest (Alamyar et al., 2012). Sequences were then analyzed for V region mutations and clonality. All clonal assignments were based on matching V and J regions, matching CDR3 length, and 70% CDR3 homology. All sequences are plotted using Matlab or Circos visualization tools (Krzywinski et al., 2009).

Serum Proteomics

Measles- or mumps-specific polyclonal antibodies from one adult (age 64 years old) were purified by affinity chromatography using a custom column consisting of measles or mumps antigens and fractions were eluted, verified for measles or mumps activity, then digested with chymotrypsin, pepsin, elastase, and trypsin, and analyzed by the LC-MS/MS. MS/MS spectra were searched using SEQUEST against the V-region full peptides generated from the sequences provided by the NGS results of pop A, B, D, and naïve B cells. Top candidate V-region sequences including CDR3 peptides from measles and mumps antibodies were identified as previously described (Cheung et al., 2012; Sato et al., 2012).

Monoclonal mumps-specific antibody reconstruction

The top candidate gamma and kappa chains, containing HCDR3 AKMIGSSAWYPFDY and LCDR3: GTWDSSLGIVL, identified in pop D from the affinity purified mumps-specific antibodies through the use of proteomics were synthesized, cloned and expressed in 293T cells. Specificity of the monoclonal antibody was tested by ELISA against measles and mumps antigens.

​

HIGHLIGHTS

  • -
    The CD19−CD38hiCD138+ BM PC subset represent human LLPCs.
  • -
    CD19−CD38hiCD138+ BM LLPCs are responsible for long-lived viral antibodies in serum.
  • -
    BM LLPCs have a VH repertoire that is uncoupled from other BM PC subsets.
  • -
    BM LLPCs have a unique RNA transcriptome compared to other BM PC subsets.

Supplementary Material

2

Acknowledgements

The authors would like to thank Jennifer Scantlin, Claudine Nkurunziza, Deanna Maffett, and Julie Kozarsky for human subject recruitment. We also appreciate Edmund Waller, Jeremy Bechelli, Karen Rosell and the Atlanta Clinical & Translational Science Institute (ACTSI) for their assistance with the bone marrow aspirates, Chelsea Cook, Flow Cytometry Core at URMC, and Wayne Harris at Emory University, and Jennifer Hom, Linda Kippner, and Melissa Kemp for assistance in preparation of the manuscript.

Supported by:

NIH: K23 AI67501, R21AI109601, R21AI094218, P01 A1078907, R37AI049660, U01AI045969, Autoimmunity Center of Excellence- ARRA:AI056390-06S2, HHSN266200500030C (N01-AI50029), AI078907, AI049600, NIH/NCATS UL1 TR000454, UO1 AI082196, Oregon National Primate Research Center grant, 8P51 OD011092-53, R01 AI097357, and U19 AI109962.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflicts of Interests: F.E.L. has research grants with Genentech and is the founder of MicroBplex. I.S. consults for Pfizer,Genentech, and has research grants with Biogen and Takeda Pharmaceuticals. A.R.F. consults for AstraZeneca, Medimmune, Sanofi Pasteur, and Wyeth and has research grants from GSK and Sanofi Pasteur. E.E.W. has research grants from GSK and Sanofi Pasteur and consults for Astra Zeneca. J.L.H., C.T., J.L, A.F.R., J.D., I.V.G, L.P., D.K., C.F.F., I.A.S., S.K., K-Y.C., K.T.B., R.B., M.S., E.H., L.Z., T.D.R., and W.C.C. have no conflicts of interest.

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