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Updated: Aug 8, 2026
Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry
Published on: May 5, 2017
Extensive analysis of lymphocyte subsets in normal subjects by three-color immunofluorescence
F Vuillier1, D Scott-Algara, G Dighiero
1Service d'Immunohématologie et d'Immunopathologie, Institut Pasteur, Paris, France.
Insights
This study characterizes immune cell subsets in normal controls using monoclonal antibodies. Key findings include detailed B cell populations and T cell subset distributions, with some uncertainties remaining for CD8 subsets.
Area of Science:
- Immunology
- Cell Biology
- Flow Cytometry
Background:
- Accurate characterization of immune cell subsets is crucial for understanding immune function and disease.
- Defining specific cell surface markers aids in distinguishing various lymphocyte populations and their activation states.
Purpose of the Study:
- To define CD20, CD4, CD8, CD16, and CD56 cell subsets and their activation status in normal controls.
- To investigate the phenotypic characteristics of B cells, CD4+ T cells, CD8+ T cells, and Natural Killer (NK) cells.
Main Methods:
- Utilized flow cytometry with 11 combinations of 3 monoclonal antibodies.
- Studied 30 normal control individuals to establish baseline immune cell profiles.
Main Results:
- B cells predominantly expressed CD5 (approx. 92%), with a minor CD5+ fraction showing inter-individual variability.
- CD4+ T cell subsets included naive (CD45RA+CD29-) at 31.56%, memory (CD4+CD29+CD45RA-) at 46.54%, and transitional cells.
- NK cells coexpressed CD16 and CD56 (approx. 5%), with distinct populations expressing only CD16 or CD56.
Conclusions:
- Established reference ranges for major lymphocyte subsets in healthy individuals.
- Highlighted complexities in defining CD8 subsets, including cytotoxic T lymphocytes and suppressive cells.
- Confirmed the presence of activated lymphocytes (DR expression) and limited expression of CD25 across major subsets.
Abstract:
In the present work, 30 normal controls were studied with 11 combinations of 3 monoclonal antibodies in order to define CD20, CD4, CD8, CD16 and CD56 subsets as well as their activation status. Our results indicated that among B cells, CD5- cells predominated (about 92%), the minor CD5+ fraction showed variations between individuals but sometimes was as high as 20% in B cells; the CD45RA+CD29- subset (naive cells) accounted for 31.56 +/- 10.03% in CD4 cells, as compared to CD4+CD29+CD45RA- (memory cells) (46.54 +/- 10.35%), while CD4+CD29+CD45RA+ (transition cells between naive and memory state) represented 9.6 +/- 5.3% as compared to double-negatives (12.3 +/- 5.24%). Some uncertainties persist in defining CD8 subsets. NK CD8 cells accounted for about 20% of total CD8 according to CD16 and CD56 expression. Cytotoxic T lymphocytes could be delineated by S6F1 (76.22 +/- 11.28% within CD8 cells), though this marker appeared to be expressed by both cytotoxic and NK cells. Phenotypic definition of suppressive CD8 cells remains uncertain despite the fact that suppressor activity was found among CD8 lymphocytes expressing CD11b and probably CD57. NK cells coexpressed CD16 and CD56 markers in about 5% of all lymphocytes, while NK cells expressing CD16 alone accounted about 6%. Exclusive expression of CD56 was found in about 5%. Finally, while activated cells characterized by DR expression represented about 4% of CD4 and 9% of CD8 cells, CD25 appeared to be poorly represented among CD4, CD8 and CD20.(ABSTRACT TRUNCATED AT 250 WORDS)
