Antibody-secreting plasma cells persist for decades in human intestine

Abstract

Plasma cells (PCs) produce antibodies that mediate immunity after infection or vaccination. In contrast to PCs in the bone marrow, PCs in the gut have been considered short lived. In this study, we studied PC dynamics in the human small intestine by cell-turnover analysis in organ transplants and by retrospective cell birth dating measuring carbon-14 in genomic DNA. We identified three distinct PC subsets: a CD19⁺ PC subset was dynamically exchanged, whereas of two CD19^- PC subsets, CD45⁺ PCs exhibited little and CD45^- PCs no replacement and had a median age of 11 and 22 yr, respectively. Accumulation of CD45^- PCs during ageing and the presence of rotavirus-specific clones entirely within the CD19^- PC subsets support selection and maintenance of protective PCs for life in human intestine.

Figure 1.

PCs survive for 1 yr and comprise three distinct subsets in human SI. (A) Immunofluorescence confocal micrograph of endoscopic biopsy from (female) donor duodenum 1 yr after Ptx into male recipient. Tissue sections were probed with X/Y chromosome fluorescent in situ hybridization probes (Y, green; X, red) and stained with anti-CD38 (red) and anti-CD45 (blue). Hoechst (gray) stains individual nuclei. The micrograph is representative of five gender-mismatched transplants. (B) Representative flow cytometric analysis of PCs (top) and B cells (bottom) from resected SI obtained during Whipple procedure or donor/recipient SI during Ptx. Dot plots and histograms are representative of all (CD27, CD38, CD19, and CD45), 4 (CD138), 19 (HLA-DR), and 5 (CD20) subjects. (C) The lengthwise representation of the PC subsets was determined by flow cytometric analysis of biopsies taken at intervals along resected duodenum-proximal jejunum from individual subjects after Whipple procedure. n = 5; paired Student’s t test 0–20 cm (n = 3). (D) Blimp-1 expression among PC subsets was analyzed by intracellular staining and flow cytometry. The histogram is representative for 12 subjects (Whipple procedure). (E) Representative micrographs of memory B cells and PC subsets sorted by flow cytometry and stained with Hemacolor reagent. n = 2; Whipple procedure. (F) The percentage of memory B cells and PC subsets expressing cell surface IgA (black dots; n = 14) or IgM (red dots; n = 15) was determined by flow-cytometric analysis. Bars indicate median values. Dots represent individual subjects, SI resections from Whipple procedure, and Ptx. (G) IgA secretion from flow-sorted IgA⁺ (black dots) or IgM⁻ (red dots) memory B and PC subsets was determined by ELISA after 40-h culture in vitro and was normalized to the total for each subject. n = 5; Whipple procedure and Ptx. (F and G) One-way repeated measures ANOVA (RM-ANOVA), IgA⁺ versus IgM⁺ memory B cells in F; paired Student’s t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Bc, B cell; mB, memory B cell; Rel., relative.

Figure 2.

SI-PC subsets have different replacement kinetics and in vivo stability. (A) Representative flow cytometric analysis of memory B cell (mB) and PC subsets in endoscopic biopsies from donor duodenum 52 wk after Ptx (donor HLA-A2⁻/A3⁺; recipient HLA-A2⁺/A3⁻). (B) The percentage of recipient (Rec.) cells in each subset 3, 6, and 52 wk after Ptx was determined by HLA class I expression as in A. Each dot represents an individual donor. n = 10. One-way RM-ANOVA was used. (C) Chimerism was determined in endoscopic biopsies from donor duodenum (closed circles) and adjacent recipient duodenum (open circles) 52 wk after Ptx. n = 5. (D and E) The relative representation irrespective of origin of CD19⁺ PCs among total PCs (D) and CD45⁻ among CD19⁻ PCs (E) was determined before and 52 wk after Ptx in donor (closed circles; n = 4) and adjacent recipient duodenum (open circles; n = 3). (F) Donor–recipients pairs with different CD19⁻ PC subset representation before Ptx were examined 52 wk after transplantation (n = 3). Data points represent individual subjects; lines connect data from the same patients at different time points (D and E) or donor and recipient tissue at the same time point (F). Paired Student’s t test was used. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.

Figure 3.

CD19⁻ PCs exhibit features of advanced maturity and are enriched for rotavirus-specific clones. (A) The percentage of CD95⁺ cells was determined relative to isotype control by flow cytometric analysis. n = 5; Whipple procedure. (B) The mean fluorescence intensity (MFI) of Bcl2 relative to isotype control staining (ΔMFI) on PC subsets was determined by flow cytometric analysis and presented as the fraction of total value for each subject. n = 12; Whipple procedure. (C–F) The percentage of Ki67⁺ (n = 9), BCMA⁺ (n = 10), CD56⁺ (n = 14), and CD28⁺ (n = 7) was determined relative to isotype control staining in SI resections (Whipple procedure and Ptx). (G) Rotavirus-reactive PCs were detected using eGFP-labeled VLPs relative to eGFP by flow cytometric analysis (SI resections from Whipple procedure [n = 3] or Ptx [n = 4]; median age 49 yr; range 7–74 yr; 2 female). Data points represent individual subjects. Red bars indicate mean values. FSC, forward side scatter. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; one-way RM-ANOVA.

Figure 4.

Clonal relationships indicate selection and sequential development of SI PCs. (A–D) High throughput sequencing was performed on FACS IgA⁺ PC subsets from SI resections (n = 4; 2 female; mean age 70 yr, range 61–77 yr; Whipple procedure) and analyzed as described in the High throughput sequencing of IGHV section of Materials and methods. (A) The frequency of IGHV gene segments used by the PC subsets is shown for two representative subjects (S1 and S3). (B) The level of somatic hypermutation among PC subsets for sequenced subjects (S1–S4) is presented in violin plots. The median number of mutations is indicated on plots in red bars and numbers. (C) The data table gives Morisita-Horn indices for clonal similarity between PC subsets in sequenced subjects (S1–S4). (D) Clonally related sequences assigned between PC subsets illustrated in a Circos plot. Lines represent clones shared between CD19⁺ and CD45⁺ (red), CD45⁺ and CD45⁻ (black), CD19⁺, and CD45⁻ (blue) or all PC subsets (gray) above a threshold of ≥10 unique sequences for one representative subject (S3). The width of the lines is proportional to the number of unique sequences within single clones. The asterisk marks the zoomed in region (left) describing figure elements in the Circos plot.

Figure 5.

CD45⁻ PCs accumulate with age and persist for decades in human SI. (A) The representation of each SI-PC subset in SI resections (n = 72) obtained during Whipple procedure or donors and recipients during Ptx was determined by flow cytometric analysis and plotted against subject age. Lines represent nonlinear fitting (one-phase exponential decay). R² and two-tailed p-values of age and subset representation are shown on the graphs (Pearson correlation). (B) Dot plots show representative flow cytometric analysis of subjects with increasing age from A. (C) Carbon-14 concentrations in genomic DNA isolated from SI-PC subsets bead sorted from SI resections (Whipple procedure) were measured by accelerator mass spectrometry (n = 5; 1 female). The average age of cells in each PC subset was inferred from plotting the concentration of carbon-14 from each sample on the atmospheric carbon-14 curve, determining the corresponding year, and subtracting the year samples were acquired. Dots represent carbon-14 values and year of birth for individual donors. Vertical dashed red lines indicate data from subjects born before, yellow lines during the increase, and blue lines after the peak in atmospheric carbon-14. (D) Illustration of the strategy to infer age of cells based on amount of carbon-14 in genomic DNA relative to carbon-14 level in the atmosphere and subject age (hypothetical subject A [blue]; subscript gives age). Note that in subjects born prior to the bomb peak (red; B₀), cells generated during the bomb spike (i.e., B₇) will have higher carbon-14 concentration relative to their renewal rate, and cells generated post-bomb (i.e., B₂₀ and B₆₀) will have lower carbon-14 concentration relative to their renewal rate.