TY - JOUR
T1 - Optimized Protocols for In-Vitro T-Cell-Dependent and T-Cell-Independent Activation for B-Cell Differentiation Studies Using Limited Cells
AU - Marsman, Casper
AU - Verhoeven, Dorit
AU - Koers, Jana
AU - Rispens, Theo
AU - ten Brinke, Anja
AU - the T2B Consortium
AU - van Ham, S. Marieke
AU - Kuijpers, Taco W.
AU - T2B Consortium
N1 - Funding Information: We acknowledge the support of patient partners, private partners, and active colleagues of the T2B Consortium; see Appendix 1 and website: www.target-to-b.nl. We thank Simon Tol, Erik Mul, Mark Hoogenboezem, and Tom Ebbes of the Sanquin Central Facility for the maintenance and calibration of the FACS machines. Funding Information: This collaboration project is financed by the PPP Allowance made available by Top Sector Life Sciences & Health to Samenwerkende Gezondheidsfondsen (SGF) under project number LSHM18055-SGF to stimulate public–private partnerships and co-financed by health foundations that are part of the SGF. This project was also funded by the Landsteiner Foundation for Blood Transfusion Research (project grant number: LSBR 1609) and Sanquin Product and Process Development Call 2020. Publisher Copyright: Copyright © 2022 Marsman, Verhoeven, Koers, Rispens, ten Brinke, van Ham and Kuijpers.
PY - 2022/6/29
Y1 - 2022/6/29
N2 - Background/Methods: For mechanistic studies, in-vitro human B-cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T-cell-dependent (TD) and T-cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols make the interpretation of results challenging. The aim of the present study was to achieve the most optimal B-cell differentiation conditions using isolated CD19+ B cells and peripheral blood mononuclear cell (PBMC) cultures. We addressed multiple seeding densities, different durations of culturing, and various combinations of TD and TI stimuli including B-cell receptor (BCR) triggering. B-cell expansion, proliferation, and differentiation were analyzed after 6 and 9 days by measuring B-cell proliferation and expansion, plasmablast and plasma cell formation, and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B-cells and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2,500 and 25,000 B–cells per culture well for the TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B-cell cultures, which allows dismissal of additional B-cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed a little effect on phenotypic B-cell differentiation; however, it interferes with Ig secretion measurements. The addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B-cell differentiation and Ig secretion in B-cell but not in PBMC cultures. With this approach, efficient B-cell differentiation and Ig secretion were accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more in-depth analysis of B-cell differentiation in primary human B-cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B-cell differentiation of patient samples from different cohorts of B-cell-mediated diseases.
AB - Background/Methods: For mechanistic studies, in-vitro human B-cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T-cell-dependent (TD) and T-cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols make the interpretation of results challenging. The aim of the present study was to achieve the most optimal B-cell differentiation conditions using isolated CD19+ B cells and peripheral blood mononuclear cell (PBMC) cultures. We addressed multiple seeding densities, different durations of culturing, and various combinations of TD and TI stimuli including B-cell receptor (BCR) triggering. B-cell expansion, proliferation, and differentiation were analyzed after 6 and 9 days by measuring B-cell proliferation and expansion, plasmablast and plasma cell formation, and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B-cells and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2,500 and 25,000 B–cells per culture well for the TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B-cell cultures, which allows dismissal of additional B-cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed a little effect on phenotypic B-cell differentiation; however, it interferes with Ig secretion measurements. The addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B-cell differentiation and Ig secretion in B-cell but not in PBMC cultures. With this approach, efficient B-cell differentiation and Ig secretion were accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more in-depth analysis of B-cell differentiation in primary human B-cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B-cell differentiation of patient samples from different cohorts of B-cell-mediated diseases.
KW - B-Lymphocytes
KW - B-cell activation
KW - B-cell differentiation
KW - CD40L
KW - Cell Differentiation
KW - CpG
KW - Humans
KW - IL-2
KW - IL-21
KW - Leukocytes, Mononuclear
KW - Lymphocyte Activation
KW - T-Lymphocytes
KW - plasma cells
UR - http://www.scopus.com/inward/record.url?scp=85134205407&partnerID=8YFLogxK
UR - https://pure.uva.nl/ws/files/127351057/Supplementary_Material_Optimized_Protocols_for_In_Vitro_Table_1.XLSX
UR - https://pure.uva.nl/ws/files/127351059/Supplementary_Material_Optimized_Protocols_for_In_Vitro_Table_2.XLSX
UR - https://pure.uva.nl/ws/files/127351061/Supplementary_Material_Optimized_Protocols_for_In_Vitro_Data_Sheet_1.pdf
UR - https://pure.uva.nl/ws/files/127351063/Supplementary_Material_Optimized_Protocols_for_In_Vitro_Data_Sheet_2.pdf
U2 - https://doi.org/10.3389/fimmu.2022.815449
DO - https://doi.org/10.3389/fimmu.2022.815449
M3 - Article
C2 - 35844625
SN - 1664-3224
VL - 13
JO - Frontiers in immunology
JF - Frontiers in immunology
M1 - 815449
ER -