Monitoring Immature B Cell Reconstitution

Peripheral blood mononuclear cells (PBMCs) were labeled with a combination of the following mAbs: CD3 (SP34–2), CD4 (L200), CD8 (SK1), CD21 (B-ly4), CD27 (M-T271), CD28 (CD28.2), CD95 (DX2), and IgM (G20–127) (BD Pharmingen, San Jose, CA), CD20 (2H7) (Biolegend, Inc., San Diego, CA) and FOXP3 (236A/E7) (eBioscience, Inc., San Diego, CA). Based on previous reports, which suggest that long-term allograft survival is associated with a predominance of immature B lymphocyte reconstitution,^{12 (link),13 (link)} CD3^-CD20⁺CD21⁺IgM⁺ or CD3^-CD20⁺CD21^-CD27⁺ B cells representing immature or mature B cell phenotypes, respectively, were monitored before and after conditioning. The fluorescence of the stained samples was analyzed using FACSverse (BD Biosciences, San Jose, CA) and Accuri flow cytometers (BD PharMingen), and FlowJo software (Tree Star, Inc., Ashland, OR).

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Oura T., Hotta K., Rosales I., Dehnadi A., Kawai K., Lee H., Cosimi A.B, & Kawai T. (2019). Addition of Anti-CD40 Monoclonal Antibody to Nonmyeloablative Conditioning with Belatacept Abrogated Allograft Tolerance Despite Induction of Mixed Chimerism. Transplantation, 103(1), 168-176.

Publication 2018

CD20 (2H7) FOXP3 (236A/E7) FACSverse Accuri flow cytometers

Allograft B cell Fluorescence Immature b lymphocyte Mabs Peripheral blood mononuclear cells Phenotypes Tree

Corresponding Organization : Massachusetts General Hospital

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Variable analysis

independent variables

Conditioning treatment

dependent variables

Frequency of CD3-CD20+CD21+IgM+ B cells (immature B cells)
Frequency of CD3-CD20+CD21-CD27+ B cells (mature B cells)

control variables

Peripheral blood mononuclear cells (PBMCs) from subjects
Labeling of PBMCs with the specified monoclonal antibodies (mAbs)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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