A unique population of IgG-expressing plasma cells lacking CD19 is enriched in human bone marrow by Henrik E. Mei, Ina Wirries, Daniela Frölich, Mikael Brisslert, Claudia Giesecke, Joachim R. Grün, Tobias Alexander, Stefanie Schmidt, Katarzyna Luda, Anja A. Kühl, Robby Engelmann, Michael Dürr, Tobias Scheel, Maria Bokarewa, Carsten Perka, Andreas Radbruch, and Thomas Dörner Blood Volume 125(11):1739-1748 March 12, 2015 ©2015 by American Society of Hematology

The human BM is the major site of PCs lacking expression of CD19. The human BM is the major site of PCs lacking expression of CD19. (A) CD19 expression by PB/PC (black histograms) was analyzed by flow cytometry in peripheral blood, tonsil, lymph node (LN), BM, and spleen samples (supplemental Figure 1). CD19 expression by total lymphocytes (dashed lines) and isotype control-stained PC (shaded) are shown for comparison in representative histograms. Horizontal bars indicate gates used to determine frequencies of PC lacking CD19. Frequencies of CD19− PC among total PC were determined (B, median values are indicated from 21 blood samples, 6 spleen samples, 1 LN/7 tonsil samples, and 34 femoral head BM samples), and numbers of CD19− and CD19+ PC per organ were extrapolated for 21 blood samples, 6 spleen samples, 1 LN/7 tonsil samples, and 25 femoral head BM based on20 (C, median values and interquartile range shown). Significance levels (***P < .001) obtained from Mann-Whitney U tests are shown for comparisons between BM data and other tissues. The grey data point in (B) indicates a parotid LN. (D) Percentages of PC lacking CD19 expression were analyzed in BM samples from iliac crest (36 samples), femoral head (34 samples), and sternum (9 samples). Kruskal-Wallis testing was used for statistical analysis. Consistent frequencies of CD19− PC were determined using various anti-CD19 clones (supplemental Figure 1F). Henrik E. Mei et al. Blood 2015;125:1739-1748 ©2015 by American Society of Hematology

CD19− BMPC express IgG with moderately mutated VH gene rearrangements and secrete antibodies commonly found in blood serum. CD19− BMPC express IgG with moderately mutated VH gene rearrangements and secrete antibodies commonly found in blood serum. (A) BMPC were analyzed for expression of CD19 and cyt IgG, IgM, and IgA. Summarized results from 11 donors are shown in (B) and were compared using the Mann-Whitney U test, ***P < .001. Median values are indicated. (C-D) Individual fluorescence-activated cell sorter (FACS)-sorted CD19+ and CD19− BMPC from 3 individuals were subjected to nested single-cell reverse transcriptase-polymerase chain reaction for amplification of VHDJHC gene rearrangements.18 A total of 82 sequences from CD19+ PC and 72 sequences from CD19− BMPC were analyzed: 33 CD19+IgG+, 44 CD19−IgG+, 46 CD19+IgA+, and 24 CD19−IgA+ sequences (and 7 IgM sequences, not separately shown). Sequences were analyzed for immunoglobulin isotype (supplemental Figure 2B) for VH and JH family use. Distributions were compared by χ2 test; respective P values are indicated (C), for absolute numbers and frequencies of somatic mutations per VH sequence, and for CDR3 length and mutations located within RGYW/WRCY hotspot motifs (D). Data from 65 tetanus toxoid (TT)-specific PB18 from blood 1 week after intramuscular TetDiph vaccination from 3 donors, respectively, are shown for comparison. After χ2 tests did not show significant differences in VHJHC segment use and CDR3 length distribution among different individuals’ samples, data were pooled and displayed together. Mann-Whitney U test was used to analyze data in (D) (*P < .05; **P < .01; ***P < .001). Median values are indicated. (E) Frequencies of antibody-secreting PC specific for TT were determined by Elispot among 4 pairs of FACS-sorted CD19+ and CD19− BMPC (donors were aged 37, 52, 62, and 72 years). Median values are indicated. Henrik E. Mei et al. Blood 2015;125:1739-1748 ©2015 by American Society of Hematology

The phenotype of CD19− BMPC indicates terminal PC differentiation and prosurvival capacity. The phenotype of CD19− BMPC indicates terminal PC differentiation and prosurvival capacity. (A) CD19+ and CD19− BMPC were isolated from 4 donors using a combined magnetic-activated cell-sorting/FACS protocol (supplemental Figure 4) and were subjected to global gene expression profiling. A heat map resulting from unsupervised clustering of data of 859 significantly differentially expressed probe sets reflecting 689 genes (supplemental Table IIIA) according to high-performance chip data analysis (HPCDA) after Bonferroni correction is shown. (B) CD19+ and CD19− BMPC (CD38highCD3−CD14−DAPI−) were analyzed by flow cytometry for the expression of CD28, CD56, HLA-DR, or CD95. Histograms representative of 8 (CD28), 14 (CD56), 18 (HLA-DR), or 19 (CD95) donors are shown. Numbers represent median values of the geometric mean fluorescence intensity values of all donors analyzed, frequencies represent median values of frequencies of PC expressing a marker gated as illustrated by the black bars in the histogram plots, where applicable (gray, CD19− BMPC; black CD19+ BMPC). Both median fluorescence intensity and frequency data were compared using the Wilcoxon test (**P < .01; ***P < .001). (C) Affymetrix signal intensity data for selected molecules involved in apoptosis regulation not contained in the list of differentially expressed genes as delivered by HPCDA, that is, BCL2 (B-cell lymphoma 2, probeset 203685_at), TNFRSF6 (CD95), TNFRSF10B (tumor necrosis factor-related apoptosis-inducing ligand receptor 2), BID (BH3 interacting-domain death agonist), and for caspases 8 and 3 (supplemental Table IIIB), were extracted from global gene expression analysis of CD19+ and CD19− BMPC. A distinct probeset for BCL2 (203685_at) was identified as differentially expressed by HPCDA. Student t test P values (BCL2, 0.05; TNFRSF6, 0.01; TNFRSF10B, 0.10; BID, 0.002; CASP8, 5.3 × 10−5; and CASP3, 1.1 × 10−5, were not considered significant according to the significance level of 0.05 corrected for multiple comparisons in the overall analysis (P = 2.29 × 10−8). Henrik E. Mei et al. Blood 2015;125:1739-1748 ©2015 by American Society of Hematology

CD19+ and CD19− BMPC subsets exhibit different ex vivo proliferation and in vivo persistence. CD19+ and CD19− BMPC subsets exhibit different ex vivo proliferation and in vivo persistence. (A) CD19+ and CD19− BMPC and peripheral blood PB/PC were analyzed for the expression of Ki-67 after cell fixation with formaldehyde and permeabilization with saponin. Frequencies of Ki-67+ cells are shown and were compared between blood PB/PC (6 samples, steady state) and BMPC subsets (Mann-Whitney U test; ***P < .001) and among pairs of BM CD19+ and CD19− PC (Wilcoxon test; *P < .05). Filled circles, Ki-67 staining (10 samples); open circles, control staining (available for 7 samples). Both BMPC subsets show frequencies of Ki-67+ cells significantly exceeding frequencies of isotype control stained cells (Mann-Whitney U test; both P < .01). Median values are indicated. (B) BM samples of 23 patients with rheumatoid arthritis treated with 2 × 1 g RTX (a chimeric anti-CD20 antibody) were analyzed for the presence of CD138+CD19+ and CD138+CD19− PC twice, once before, and once again 1 to 3 months after treatment. PC were detected by flow cytometry, and their numbers were extrapolated according to Trepel20 and were analyzed using the Wilcoxon test; *P < .05. Lines between data points indicate the trend shown by individual patients. Mean numbers of 3.10 × 108 (standard deviation [SD], 1.76 × 108) CD19+ and 1.36 × 108 (SD, 0.85 × 108) CD19− BMPC compare with 2.24 × 108 (SD, 1.69 × 108) CD19+ and 1.06 × 108 (SD, 0.93 × 108) CD19− BMPC after therapy, respectively. Henrik E. Mei et al. Blood 2015;125:1739-1748 ©2015 by American Society of Hematology

CD19+ but not CD19− PB and PC are detectable in peripheral blood after vaccination. CD19+ but not CD19− PB and PC are detectable in peripheral blood after vaccination. (A) TT-specific PB, IgG+HLA-DRhigh PB and IgG+HLA-DRlow PC were detected in peripheral blood by flow cytometry as shown (1 week after vaccination) and were analyzed for expression of CD19 in individuals before and at indicated time points after intramuscular vaccination against TetDiph. The analysis shown is representative of 9 individuals immunized with TetDiph vaccine. Numbers of TT-specific PB/mL blood are indicated to illustrate kinetics of the TT-specific PB response. (B) CD19 expression by PB/PC from 4 individuals 1 week after vaccination against 2009 H1N1 influenza with Pandemrix. (C) CD56+ PB and PC are absent from peripheral blood before and after vaccination. Representative analyses are shown for circulating PB/PC during steady state (#1 to #4), 1 week after TetDiph vaccination (#5 to #8) and 1 week after Pandemrix vaccination (#9 to #12). Henrik E. Mei et al. Blood 2015;125:1739-1748 ©2015 by American Society of Hematology

CD19− PC are detectable at sites of chronic inflammation and can secrete autoantibodies. CD19− PC are detectable at sites of chronic inflammation and can secrete autoantibodies. (A) Synovial biopsies from 2 patients with rheumatoid arthritis were analyzed by immunofluorescence histology for the presence of CD138+DAPI+CD19+ and CD138+DAPI+CD19− PC (1 out of 2 cases is shown). Upper and lower panels originate from the identical staining and section. (B) Kidney tissue from 2 patients after humoral rejection was digested by collagenase V, and mononuclear cells were analyzed by flow cytometry for the presence of CD19+ and CD19− PC. PC were identified using high expression of CD38 within a wide lymphocyte gate and electronic depletion of CD3+ cells, CD14+ cells, and dead cells. In 1 of the 2 cases, CD19 expression by PC was controlled using an isotype-matched control antibody, and the PC identity was confirmed by the detection of intracellular immunoglobulin (cyt κ and λ Ab light chains). Numbers denote mean fluorescence intensities. (C) CD19+ and CD19− BMPC from 1 SLE patient (serum anti-dsDNA antibodies, 84 U/mL) were isolated and subjected to an Elispot assay detecting dsDNA-specific IgG-secreting cells among 24 000 CD19+ BMPC/well and 27 000 CD19− BMPC/well. Representative wells (from 14 dsDNA-coated replicate wells and 7 bovine serum albumin-coated replicate wells per PC subset to determine assay background) and assay controls are shown. After background subtraction, frequencies of dsDNA-specific PC were 0.8 and 1.3 PC per 10 000 total CD19+ and CD19− BMPC, respectively. Henrik E. Mei et al. Blood 2015;125:1739-1748 ©2015 by American Society of Hematology