Analysis of Normal Plasma Cell Distribution Across Distinct Age Cohorts Reveals Age-Dependent Changes

Abstract

Hematopoietic and stromal cells within the bone marrow (BM) provide membrane-bound and/or soluble factors that are vital for the survival of plasma cells (PCs). Recent reports in murine BM demonstrated the dynamic formation and dispersion of PC clusters. To date, PC clustering in normal human BM has yet to be thoroughly examined. The goal of this study was to determine whether PC clusters are present in human BM and whether clustering changes as a function of age. Quantification of PCs and clustering in BM sections across six different age groups revealed that fewer PCs and PC clusters were observed in the youngest and oldest age groups. PC clustering increased with age until the sixth decade and then began to decrease. A positive correlation between the number of PCs and PC clusters was observed across all age groups. PC clusters were typically heterogeneous for immunoglobulin heavy- and light-chain expression. Taken together, these data demonstrate that PC clusters are present in human BM and that PC clustering increases until middle adulthood and then begins to diminish. These results suggest the spatial distribution of BM PC-supportive stromal cells changes with age.

Keywords: aging; bone marrow; clustering; plasma cells.

Figure 1.

Detection of PCs in human bone marrow. (A) Kappa PCs (green), lambda (red) PCs, and DAPI (blue) were detected in BM from the following age groups: (I) 0–9 years of age; (II) 10–19 years of age; (III) 20–39 years of age; (IV) 40–59 years of age; (V) 60–79 years of age; (VI) 80–99 years of age. (B) Kappa (green) and lambda (red) PCs found in bone marrow of 60- to 79-year-old patients. Arrows point to representative PC clusters. Scale bar shown in white is 50 mm.

Figure 2.

Intra- and inter-patient heterogeneity among the number of PCs and PC clusters. (A, B) Examples of BM ROIs from the same patient in the 20–39 years age group. Kappa PCs (green), lambda PCs (red), and DAPI (blue). (C, D) Box and whisker plots illustrating the variability in the total number of PCs and PC clusters observed per ROI for each patient in the 20–39 years age group. Median is indicated by line, and mean is signified by + sign. Results from the 20–39 years age group are shown as representative data for the variability seen within each age group. Scale bar shown in white is 50 mm.

Figure 3.

Total PCs and total PC clusters in human BM vary with age. (A) The total number of PCs per ROI and (B) the total number of PC clusters per ROI, over the six age groups. (C) Correlation between the number of PCs and number of PC clusters observed in ROIs. Analysis included patients from all age groups. To account for BM cellularity, we calculated a cellularity multiplier and applied it to the total PC and total PC cluster counts from all patients in age groups of 10 years and older. The graphed results are shown in panels D–F. (D) The total number of PCs per ROI relative to BM cellularity. (E) The total number of PC clusters per ROI, over the six age groups, relative to BM cellularity. (F) Correlation between the number of PCs and number of PC clusters relative to BM cellularity observed in ROIs. Analysis included patients from all age groups. Significant differences (p<0.01) between age groups are indicated with an asterisk (*).

Figure 4.

The number and type of PC clusters in human BM varies with age. (A) The total number of PC doublets per ROI (includes kappa, lambda, and kappa/lambda doublets). (B) The total number of PC clusters comprised of 3–5 cells. (C) The total number of PC clusters (comprised of 6 or more cells) per ROI. Significant differences (p<0.01) between age groups are indicated with an asterisk (*). (D) Comparison of the percent of various PC clusters found in BM across the different age groups.

Figure 5.

Total number and percentage of single PCs. (A) The total number of single PCs and (B) the percentage of single PCs and PC clusters in relation to the total number of PCs. Significant differences (p<0.01) between age groups are indicated with an asterisk (*).

Figure 6.

Heavy- and light-chain analysis of PC clusters. (I) Seven PC clusters (labeled A–G) were initially analyzed for expression of Ig LCs, kappa (green)/lambda (red). (II) Subsequent antibody stripping and restaining of the same clusters for Ig HC expression, IgA (orange)/IgG (teal). (III) Resulting heavy- and light-chain Ig isotypes in PC clusters. Scale bar shown in white is 20 mm.

Figure 7.

Analysis of CD271, CXCL12, and TNFSF13 in human BM samples. (A) Staining of human BM from a patient in the 20–39 years age group for CD271 (red), PCs (green), and DAPI (blue). Scale bar shown in white is 20 mm. (B–E) Proximity of CD271+ cells and PCs from a patient in the 20–39 years age group. Scale bar shown in white is 10 mm. (F) Staining of human BM from a patient in the 20–39 years age group for CXCL12 (red), PCs (green), and DAPI (blue). (G) Staining of human BM from a patient in the 40–59 years age group for TNFSF13 (red), PCs (green), and DAPI (blue). Scale bar shown in white is 20 mm.

Appendix Figure 1.

Analysis of CD271 with age in human BM samples. Representative CD271 staining of human BM from patients of the following age groups: (A) 0–9 years, (B) 10–19 years, (C) 20–39 years, (D) 40–59 years, (E) 60–79 years, (F) 80–99 years. Scale bar shown is 50 mm.