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Phenotypic analysis of human peripheral blood lymphocytes by automatic sampling flow cytometry after stimulation with
L S Azzolina1, G Stevanoni, M Tommasi
1Istituto di Scienze Immunologiche, Università degli Studi di Verona.
Insights
This study details how different mitogens like PHA, ConA, and PWM affect human peripheral blood lymphocyte (PBL) phenotypes. Automated flow cytometry provides accurate data for monitoring patients in transplantation and immunopathology.
Area of Science:
- Immunology
- Cell Biology
- Flow Cytometry
Background:
- Peripheral blood lymphocyte (PBL) phenotyping is crucial for understanding immune responses.
- Standardized methods are needed for accurate analysis of PBL subsets.
- Assessing PBL responses to various mitogens and in mixed lymphocyte culture (MLC) is important for clinical applications.
Purpose of the Study:
- To analyze human peripheral blood lymphocyte (PBL) phenotypes after stimulation with phytohemagglutinin (PHA), concanavalin A (ConA), and pokeweed mitogen (PWM).
- To evaluate PBL subset changes in mixed lymphocyte culture (MLC).
- To establish reference phenotypic ranges for fresh and control PBL cultures.
Main Methods:
- Human PBLs were stimulated with PHA, ConA, and PWM for 3 days, and in MLC for 7 days.
- PBLs were labeled with 10 fluorescent monoclonal antibodies and analyzed using automated flow cytometry.
- A microsample delivery system was used for sampling from 96-well microtiter plates.
Main Results:
- PHA primarily stimulated T PBLs expressing CD3, CD5, CD7, CD8, and CD25, with minimal effects on natural killer (NK) cells, B cells (CD19), and HLA-DR+ PBLs.
- ConA showed differential mitogenic effects, stimulating CD4+ T cells more than CD8+ T cells, with incomplete overlap with PHA stimulation.
- PWM stimulated T cell subsets (CD3, CD5, CD7, CD4, CD8, CD25) but not NK, HLA-DR, or B cells.
- MLC stimulation increased all tested T, B, and NK PBL subsets, with significant proliferation observed in cells expressing CD1, CD4, CD5, CD7, CD25, NK, CD19, and HLA-DR.
Conclusions:
- The study established reference phenotypic ranges for human PBLs under various stimulation conditions.
- Automated flow cytometry offers a comprehensive and accurate method for PBL phenotyping.
- This approach can enhance patient monitoring and follow-up in transplantation and immunopathology.
Abstract:
Human peripheral blood lymphocyte (PBL) phenotypes have been analyzed before and after stimulation with phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) for 3 days and in mixed lymphocyte culture (MLC) for 7 days. PBL labeled with each of 10 fluorescent monoclonal antibodies were automatically sampled for flow cytometry from 96-well microtiter plates using a microsample delivery system. The reference phenotypic ranges were determined in fresh cells and control cultures. PHA was mostly mitogenic for T PBL bearing the CD3, CD5, CD7, CD8 and CD25 differentiation clusters, and a low density of CD1 and CD4 had a small effect on human natural killer cells (HNK) and also did not stimulate B (CD19) and HLA-DR+ PBL. There was an incomplete phenotypic overlapping between PHA- and ConA-stimulated cultures, ConA being more mitogenic for CD4 and less mitogenic for CD8 PBL. The mitogenic effect of PWM was evident on CD3, CD5, CD7, CD4, CD25 and CD8, but not on HNK, HLA-DR and CD19 B PBL, which presumably had already differentiated into antibody-secreting cells. After MLC stimulation all T, B and HNK PBL subsets tested were increased, but the cells bearing CD1, CD4, CD5, CD7, CD25, HNK, CD19 and HLA-DR had the greatest proliferation with respect to the unmixed control PBL. The present approach to the phenotyping of PBL subsets could offer more complete and accurate data for monitoring and follow-up of patients in transplantation and immunopathology hospital wards.