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In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function
Published on: October 16, 2013
Expression of CD69 after in vitro stimulation: a rapid method for quantitating impaired lymphocyte responses in
J F Krowka1, B Cuevas, D C Maron
1California Department of Health Services, Berkeley 94704, USA.
Insights
This study developed a CD69 assay to assess T cell immune responses in people with human immunodeficiency virus (HIV). The assay revealed reduced T cell activation in HIV+ individuals, highlighting potential immune defects.
Area of Science:
- Immunology
- Virology
- Cellular Biology
Background:
- Human immunodeficiency virus (HIV) infection impairs T cell function.
- Assessing T cell immune responses is crucial for understanding HIV pathogenesis and treatment.
- Existing methods for evaluating T cell activation may have limitations.
Purpose of the Study:
- To develop and validate a flow cytometric assay measuring CD69 expression on T cells.
- To analyze immunological responses in T cell subsets from HIV-infected (HIV+) and HIV-seronegative (HIV-) donors.
- To investigate the effect of HIV and gp120 on T cell activation.
Main Methods:
- Development of a flow cytometric assay using CD69 as an activation marker.
- Isolation and culture of peripheral blood lymphocyte (PBL) subsets (CD3+, CD4+, CD8+) from HIV+ and HIV- donors.
- In vitro stimulation of PBLs with antigens, polyclonal activators, and recombinant gp120.
- Measurement of CD69 expression and comparison with [3H]thymidine incorporation.
Main Results:
- Lower CD69 expression was observed on stimulated T cells from HIV+ donors compared to HIV- donors.
- Both CD69 expression assay and [3H]thymidine incorporation detected lymphocyte responses.
- Recombinant gp120 inhibited CD69 expression on phytohemagglutinin-stimulated T cells, suggesting an impact on T cell activation.
Conclusions:
- The CD69 expression assay is a useful tool for rapid assessment of immune response defects in HIV+ patients.
- The assay can evaluate phenotypically defined lymphocyte subsets.
- The findings suggest that gp120 may negatively modulate T cell activation in HIV infection.
Abstract:
A flow cytometric assay based on expression of the activation antigen CD69 was developed to analyze immunological responses of T cells from human immunodeficiency virus (HIV)-infected (HIV+) or HIV-seronegative (HIV-) donors after in vitro simulation by antigens and polyclonal activators. The levels of CD69 on freshly-isolated or unstimulated, cultured CD3+, CD4+, or CD8+ peripheral blood lymphocyte (PBL) subsets were low and did not differ greatly between HIV+ and HIV- donors. The frequencies of CD3+, CD4+, and CD8+ lymphocytes from HIV+ donors that expressed CD69 after culture with antigenic or mitogenic stimuli were significantly lower than in HIV- donors. Comparison of CD69 expression with [3H]thymidine incorporation revealed that both assays could detect lymphocyte responses to antigenic or mitogenic stimuli. The CD3+ PBL from HIV+ or HIV- donors did not show increased CD69 expression after culture with soluble or cross-linked recombinant envelope glycoprotein, gp120. The gp120, however, significantly inhibited CD69 expression in phytohemagglutinin-stimulated T cells in vitro and may also affect T-cell activation in vivo. These studies demonstrate the usefulness of this CD69 expression assay for the rapid assessment of defects in immune responses of phenotypically defined lymphocyte subsets in HIV+ patients and for testing the effects of agents that modulate immune activation.
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