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Updated: Aug 8, 2026

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry
Published on: March 5, 2019
Evidence for differential responsiveness of human CD5+ and CD5- B cell subsets to T cell-independent mitogens
1Istituto Nazionale per la Ricerca sul Cancro, I.S.T., Servizio di Immunologia Clinica, Genova, Italy.
Insights
CD5+ tonsillar B cells show a robust response to thymus-independent stimuli, producing antibodies and autoantibodies, unlike CD5- cells. Interleukin-2 enhances CD5+ cell proliferation and antibody production, suggesting distinct roles in immune responses and autoimmunity.
Area of Science:
- Immunology
- Cell Biology
Background:
- Tonsillar B cells are crucial for immune responses.
- Understanding B cell subsets, like CD5+ and CD5-, is key to immune regulation.
- Autoimmune diseases involve dysregulated B cell activity.
Purpose of the Study:
- To investigate the differential responses of CD5+ and CD5- tonsillar B cell subsets to thymus-independent stimuli.
- To explore the role of interleukin-2 (IL-2) in modulating these responses.
- To assess the potential implications for autoimmune disease pathogenesis.
Main Methods:
- Separation of tonsillar B cells into CD5+ and CD5- subsets.
- Stimulation with Staphylococcus aureus Cowan strain I (SAC) and anti-mu antibodies.
- Assessment of [3H]thymidine incorporation for proliferation.
- Measurement of IgM and IgG production.
- Analysis of autoantibody secretion (rheumatoid factor, anti-DNA).
- Flow cytometry for activation markers and cell cycle analysis.
Main Results:
- CD5+ cells showed significantly higher [3H]thymidine incorporation with SAC stimulation, augmented by IL-2.
- IL-2 enabled CD5+ cells to proliferate in response to anti-mu antibodies and produce IgM and IgG.
- CD5+ cells secreted autoantibodies, including rheumatoid factor and anti-single-stranded DNA.
- CD5- cells exhibited minimal proliferation to either stimulus, despite SAC-induced activation marker expression.
- IL-2 did not enhance CD5- cell responsiveness.
Conclusions:
- CD5+ and CD5- tonsillar B cells exhibit distinct responses to thymus-independent activation.
- CD5+ cells are more responsive and capable of antibody and autoantibody production, especially with IL-2 support.
- CD5- cells are activated by SAC but fail to enter the cell cycle, indicating differential activation pathways.
- These findings contribute to understanding B cell clonal expansion and autoimmune disease mechanisms.
Abstract:
Tonsillar resting B cells were separated into CD5+ and CD5- cell subsets and stimulated with the thymus-independent mitogens, Staphylococcus aureus Cowan strain I (SAC) or insolubilized anti-mu monoclonal antibodies (a mu Ab). CD5+ cells incorporated [3H]thymidine more efficiently than unfractionated cells when stimulated with SAC and their response was augmented by the addition of interleukin (IL) 2 to the cultures. CD5+ cells also proliferated in response to a mu Ab provided that IL 2 was present, SAC-, but not a mu Ab-stimulated CD5+ cells produced IgM and IgG molecules when IL 2 was added to the cultures and also secreted autoantibodies with rheumatoid factor activity and sometimes also with anti-single-stranded, but not double-stranded, DNA activity. The efficient response of CD5+ cells was not explained by the fact that they contained cells already activated in vivo. Thus, they did not express the CD23, CD69, CD71 and CD39 activation markers, failed to incorporated [3H]thymidine and to secrete Ig spontaneously or in response to IL 2 and were found to be in a quiescent state by cell cycle flow cytometric analysis. In contrast to CD5+ cells, CD5- cells displayed very little or no [3H]thymidine incorporation in response to SAC or to a mu Ab and their poor responsiveness was not altered by changing either the doses of the stimulants, the timing of the cultures, by co-culturing the cells together with CD5+ cells, or by adding IL 2 or IL 4. Immunofluorescence studies showed that freshly prepared CD5- cells did not have surface activation markers but that they expressed them following SAC stimulation. Thus, unlike that observed for CD5+ cells, SAC seems to be capable of activating CD5- cells but does not appear to be a sufficient stimulus for driving the cells into the subsequent phases of the cell cycle. The above findings, that demonstrate marked differences in the response to CD5+ and CD5- cells to thymus-independent stimuli, may bear relevance for the understanding of the normal clonal expansion of CD5+ cells as well as for the pathogenesis of autoimmune diseases.
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