Long-Lived Plasma Cells Are Contained within the CD19(-)CD38(hi)CD138(+) Subset in Human Bone Marrow

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(-)CD38(hi)CD138(+) 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 more than 40 years. Protein sequences of measles- and mumps-specific circulating antibodies were encoded for by CD19(-)CD38(hi)CD138(+) PCs in the BM. Finally, we found that CD19(-)CD38(hi)CD138(+) PCs had a distinct RNA transcriptome signature and human immunoglobulin heavy chain (VH) repertoire that was relatively uncoupled from other BM PC subsets and probably 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: antibody secreting cells; bone marrow; heterogeneity; human; long-lived plasma cells; measles; mumps infection; next generation sequencing; plasma cells; plasmablasts; proteomics; vaccine.

Figure 1. 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.

Figure 2. 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).

Figure 3. Identification of LLPCs by ELiSPOTs

(a) ELISPOTs of total IgG-secreting cells (top row), tetanus-specific IgG secreting cells at steady state (2^nd row), tetanus-specific IgG secreting cells 21 d after tetanus vaccination (3^rd row), measles-specific IgG secreting cells (4^th row), mumps-specific IgG secreting cells (5^th row), and influenza (flu)-specific IgG secreting cells (6^th 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.

Figure 4. 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.

Figure 5. 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.

Figure 6. 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.

Figure 7. 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.