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An in Vitro Model of Differentiation of Memory B Cells Into Plasmablasts and Plasma Cells Including Detailed Phenotypic and Molecular Characterization

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An in Vitro Model of Differentiation of Memory B Cells Into Plasmablasts and Plasma Cells Including Detailed Phenotypic and Molecular Characterization An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization. Michel Jourdan, Anouk Caraux, John de Vos, Geneviève Fiol, Marion Larroque, Chantal Cognot, Caroline Bret, Christophe Duperray, Dirk Hose, Bernard Klein To cite this version: Michel Jourdan, Anouk Caraux, John de Vos, Geneviève Fiol, Marion Larroque, et al.. An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phe- notypic and molecular characterization.. Blood, American Society of Hematology, 2009, 114 (25), pp.5173-81. 10.1182/blood-2009-07-235960. inserm-00446133 HAL Id: inserm-00446133 https://www.hal.inserm.fr/inserm-00446133 Submitted on 25 Jan 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Figure S1 Secretory Pathway Transcriptions factors Targeting and Translocation Folding of ER proteins Degradation of misfolded ER proteins by the ERAD pathway Protein glycosylation ER-Golgi vesicular trafficking Golgi proteins Figure S1. Biological plasma cell networks Biological pathways encoded by the 202 unique plasma cell genes overexpressed in D7 PBs+D10 PCs+BMPCs compared to D0 MBCs+D4 actBCs were analyzed with Ingenuity software. Data are those of the highest score (67) network with 33 PC genes out of the 50 in the network. This network mainly comprises genes coding for proteins inducing XBP1 expression or genes activated by XBP1. The networks are shown in supplemental Table 5. Figure S2 AB CD20 HLA-DRB1 CIITA CCR10 CD22 CCR7 CXCR5 CXCR4 # * # * * * * * * *** ******* ** * # * # * * ** ** *** *** *** * *** *** ** ** * *** *** CD62L * CD19 CD45 CD24 IGH * CCR2 # ITGA4 * ITGB1*** *** * ***** * *** * * ** *** ** # * # *** * # * * # * ** *** ** # ** * *** *** *** *** * * ** CD38 * PECAM1 CD138 # CD27 ICAM2 * EDG1 ITGAL * * ITGB2 ** *** # * * ** *** *** /CD31 *** *** ** ** *** # /S1PR1 ** # *** *** *** * ** *** *** *** *** * *** CD9 # CD40 # CD95 CD23 CXCL12 # VCAM1 # SELP/CD62 # /SDF1 # * *** * * * AICDA MKi67 /AID * * s s s s C C B C s B B P P C ct 7 0 P M a D 1 M 0 4 D B D D Figure S2. Visualization of gene expression using Amazonia “B cell to plasma cell Atlas”. Data are the expression of genes coding for main B and PC markers (A) and homing molecules (B). * indicates that the mean expression is different from that in D0 MBCs, ** from that in D4 actBCs, *** from that in D7 PBs and # between D10PCs and BMPCs. HSPA5 Figure S3 ERN1 # *** *** * ***** * Unfolded protein response DNAJB9 DNAJC3 * DNAJB11 * STCH * * SEL1L * *** # *** *** ***** ** ** # ***** # * *** * ** * * ** * * TXNDC5 * TXNDC11 * * ERO1L C2orf30 * ** *** *** ** Misfolded protein * *** * # ***** degradation * # * Protein folding SRP54 ** RPN1 SSR1 SSR3 * SSR4 *** *** *** ** *** * *** ** * * **** * # # # * * SEC61A TRAM1 SPCS2 SEC11L3 * *** *** * # # * * # * ***** * # ** ** * * * *** Targeting and translocation across ER membrane SEC23B # ARCN1* * ARFGAP3 LMAN2 KDELR2 ** * * * * # * *** ** ** ** ** * # # * * * * ** ** * * Vesicular trafficking and transport SLC33A1 * * *** FUT8 ** *** MGAT2 * ALG2 *** *** ** *** * ** # # # ** * * * * DAD1 * RPN2 * DDOST * GOLPH3 ** ** * * *** # *** ** * * * * * * Protein glycosylation Golgi protein Activation sCD40L + ODN sCD40L ODN sCD40L sCD40L Cytokines IL-2+IL-10 IL-2+IL- IL-2+IL- IL-2+IL-4 IL-2+IL-4+ +IL-15 10+IL-15 10+IL-15 IL-10+IL-12 Mean cell amplification 6.1 ± 1.8 3.3 ± 0.8* 2.0 ± 0.5* 0.8 ± 0.1 3.4 ± 1.2* n=19 n=4 n=10 n=2 n=6 Cell viability 94% 95% 87% 63% 93% CD20+CD38- activated 42.3 ± 12.2 35.0 ± 5.9 37.7 ± 9.2 45.1 ± 12.6 23.1 ± 7.2 B cells (%) n=13 n=4 n=3 n=2 n=6 CD20+CD38+ 16.4 ± 12.5 18.3 ± 9.3 36.3 ± 8.6 47.2 ± 19.8 27.4 ± 11.9 intermediate cells (%) n=13 n=4 n=3 n=2 n=6 CD20-CD38++ plasmablasts/plasma 19.5 ± 5.5 22.8 ± 6.3 15.4 ± 3.7 6.3 ± 6.2 34.6 ± 6.6 cells (%) n=13 n=4 n=3 n=2 n=6 CD20-CD38+CD138+ 2.1 ± 0,9 2.2 ± 1.0 2.2 ± 0.7 4.3 ± 0.5 4.4 ± 2.0 plasma cells (%) n=13 n=4 n=3 n=2 n=6 Table S1: B cell amplification and differentiation. Day 0-4. Step 1. Purified memory B cells were cultured for 4 days using either sCD40L and/or ODN activation and various cytokine cocktails. At day 4, cell counts and viability were determined and cell phenotype was assayed with fluorochrome-conjugated anti- CD20, CD38, or CD138 mAb, or isotype-matched control mAb. Flow cytometry was performed with a FACScan device. Results are shown as the mean ± SD of n experiments.* indicates that the data are significantly different from those obtained in the sCD40L+ODN+IL-2+IL-10+IL-15 group (P ≤ .008). Step 1 sCD40L + ODN ODN sCD40L+ + IL-2+IL-10+IL-15 + IL-2+IL-10+IL-15 IL-2+IL-10+IL-15 Step 2 IL-2 +IL-6+IL-10 IL-2+IL-6+IL-10 IL-2+IL-6 Culture conditions +IL-15 +IL-15 +IL-10+IL-12 Mean cell amplification 3.7 ± 1.3 1.6 ± 0.6 2.5 ± 0.7 in step2 n=18 n=9* n=6* Cell viability 81% 79% 72% Activated B cells 11.7± 5.1 13.7 ± 7.2 1.5 ± 1.8 CD20+CD38- (%) n=13 n=4 n=6 Intermediate cells 20.1 ± 8.8 37.2 ± 10.9 13.5 ± 10.8 CD20+CD38+ (%) n=13 n=4 n=6 Plasmablasts / 56.6 ± 7.7 41.6 ± 3.4 81.1 ± 9.5 plasma cells n=13 n=4 n=6 CD20-CD38++ (%) Plasma cells 15.9 ± 6;2 24.0 ± 12.4 30.1 ± 11.0 CD138+CD20-CD38++ n=13 n=4 n=6 (%) Yield of plasmablast/plasma cell 12.3 ± 6.1 1.6 ± 0.8 6.2 ± 2.8 generation for one n=13 n=4 n=6 starting memory B cells Table S2: Plasmablastic differentiation. Days 4-7. Step 2. Purified memory B cells were cultured for 4 days in the various culture conditions as described in supplemental Table 1 and further cultured for 3 days with various cytokine combinations as indicated. At day 3 (day 7 of the whole culture), cell counts and viability were determined and the cell phenotype was assayed with fluorochrome-conjugated anti-CD20, CD38, or CD138 mAb, or isotype-matched control mAb. Flow cytometry was performed with a FACScan device. Results are shown as the mean ± SD of n experiments. * Indicates that the data are significantly different (P ≤ .04) from those obtained in the sCD40L+ODN+IL-2+IL-6+IL-10+IL-15 group. Step 1 sCD40L + ODN ODN sCD40L +IL-2+IL-10+IL-15 +IL-2+IL-10+IL-15 +IL-2+IL-10+IL-15 Step 3 Culture conditions IL-6+IL-15+IFN-α IL-6+IL-15+IFN-α IL-6+IL-15+IFN-α Mean cell amplification 0.51 ± 0.09 0.52 ± 0.14 0.33 ± 0.13 in step 3 n=17 n=8 n=4 Cell viability 38% 43% 24% Activated B cells 2.2 ± 1.2 2.7 ± 0.7 1.2 ± 0.8 CD20+CD38- (%) n=13 n=5 n=4 Intermediate cells 13.6 ± 8.5 25.3 ± 3.0 8.4 ± 2.8 CD20+CD38+ (%) n=13 n=5 n=4 Plasmablasts / Plasma cells 79.0 ± 8.8 68.2 ± 3.5 86.3 ± 4.5 CD20-CD38++ (%) n=13 n=5 n=4 Plasma cells 54.8 ± 8.7 49.4 ± 5.0 65.0 ± 11.2 CD138+CD20-CD38++ n=13 n=5 n=4 (%) Yield of mature plasma cell generation for one 6.3 ± 3.3 0.86 ± 0.45 2.4 ± 1.3 starting B cells n=13 n=7 n=3 Table S3: Plasma cell differentiation. Day 7-10. Step 3. Purified memory B cells were cultured for 4 days in the various culture conditions as described in supplemental Table 1 and then for 3 days as described in supplemental Table 2. At day 7, cells were cultured with IL-6+IL-15+IFN-α for 3 days and cell count and viability were determined. The cell phenotype was assayed with fluorochrome- conjugated anti-CD20, CD38, or CD138 mAb, or isotype-matched control mAb. Flow cytometry was performed with a FACScan device. Results are shown as the mean ± SD of n experiments. Table S4. B cell and plasma cell genes. B cell genes (257) Plasma cell genes (202) Gene ID Gene Name Fold Change Score(d) Gene ID Gene Name Fold Change Score(d) 206126_at BLR1 0.02 -2.08 203153_at IFIT1 196.72 2.08 228592_at MS4A1 0.04 -2.08 201656_at ITGA6 176.21 2.08 207339_s_at LTB 0.04 -2.08 205569_at LAMP3 100.81 2.00 204440_at CD83 0.05 -1.94 206121_at AMPD1 99.61 1.86 209118_s_at TUBA3 0.05 -2.08 203868_s_at VCAM1 91.25 1.50 1554036_at ZBTB24 0.07 -1.30 205692_s_at CD38 66.37 2.08 210982_s_at HLA-DRA 0.07 -2.08 201287_s_at SDC1 52.81 2.08 214097_at RPS21 0.07 -2.08 213797_at RSAD2 46.56 1.78 214369_s_at RASGRP2 0.08 -1.53 212097_at CAV1 31.27 2.08 203932_at HLA-DMB 0.08 -1.94 226702_at LOC129607 30.88 1.32 213758_at COX4I1 0.08 -1.72 228486_at SLC44A1 25.70 2.08 200934_at DEK 0.09 -2.08 204415_at G1P3 24.14 1.69 218032_at SNN 0.10 -1.39 219159_s_at SLAMF7 22.11 2.08 217234_s_at VIL2 0.10 -2.02 214768_x_at IGKV1-5 18.99 2.08 206255_at BLK 0.10 -1.89 203675_at NUCB2 17.05 2.08 209795_at CD69 0.10 -1.72 205483_s_at G1P2 15.86 1.44 224838_at FOXP1 0.10 -2.08 214152_at CCPG1 15.63 2.08 221059_s_at COTL1 0.10 -2.08 214973_x_at IGHD 15.37 2.08 204057_at IRF8 0.11 -2.08 202688_at TNFSF10 14.66 2.02 207957_s_at PRKCB1 0.11 -2.08 216557_x_at IGHA1 /// IGHG1 13.25 2.08 203879_at PIK3CD 0.11 -2.08 219551_at EAF2 11.82 2.08 212543_at AIM1 0.11 -2.08 225032_at FNDC3B 11.63 2.08 222624_s_at ZNF639 0.12 -1.86 200983_x_at CD59 11.29 2.08 219667_s_at BANK1 0.12 -2.08 216542_x_at IGHG1 /// MGC2 10.69 2.08 218329_at PRDM4 0.13 -1.80 396_f_at EPOR 10.37 2.08 211656_x_at HLA-DQB1 0.13 -2.08 205352_at SERPINI1 9.30 1.89 219681_s_at RAB11FIP1 0.13 -2.05 224404_s_at FCRL5 8.91 2.08 203320_at LNK 0.13 -1.50 219505_at CECR1 8.90 2.08 206571_s_at MAP4K4 0.14 -2.08 202086_at MX1 8.90 1.44
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