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A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool
Annemarie Schiller1, Ting Zhang2, Ruliang Li3
1Research&Development Department, Cellular Technology Limited, Shaker Heights, OH 44122, USA. annemarie.schiller@student.uni-halle.de.
Abstract:
Testing of peripheral blood mononuclear cells (PBMC) for immune monitoring purposes requires verification of their functionality. This is of particular concern when the PBMC have been shipped or stored for prolonged periods of time. While the CEF (Cytomegalo-, Epstein-Barr and Flu-virus) peptide pool has become the gold standard for testing CD8 cell functionality, a positive control for CD4 cells is so far lacking. The latter ideally consists of proteins so as to control for the functionality of the antigen processing and presentation compartments, as well. Aiming to generate a positive control for CD4 cells, we first selected 12 protein antigens from infectious/environmental organisms that are ubiquitous: Varicella, Influenza, Parainfluenza, Mumps, Cytomegalovirus, Streptococcus, Mycoplasma, Lactobacillus, Neisseria, Candida, Rubella, and Measles. Of these antigens, three were found to elicited interferon (IFN)-γ-producing CD4 cells in the majority of human test subjects: inactivated cytomegalo-, parainfluenza-, and influenza virions (CPI). While individually none of these three antigens triggered a recall response in all donors, the pool of the three (the 'CPI pool'), did. One hundred percent of 245 human donors tested were found to be CPI positive, including Caucasians, Asians, and African-Americans. Therefore, the CPI pool appears to be suitable to serve as universal positive control for verifying the functionality of CD4 and of antigen presenting cells.
Insights
A new CPI (cytomegalovirus, parainfluenza, and influenza) viral pool effectively controls CD4 T-cell functionality. This universal positive control works across diverse populations, ensuring reliable immune monitoring of peripheral blood mononuclear cells (PBMC).
Area of Science:
- Immunology
- Cellular Immunology
- Immune Monitoring
Background:
- Peripheral blood mononuclear cells (PBMC) require functional verification for immune monitoring, especially after shipping or storage.
- A gold standard exists for CD8 T-cell functionality testing (CEF peptide pool), but a CD4 T-cell equivalent is lacking.
- A CD4 T-cell positive control should ideally use whole proteins to assess antigen processing and presentation.
Purpose of the Study:
- To develop and validate a universal positive control for assessing CD4 T-cell functionality.
- To identify protein antigens that elicit robust interferon-gamma (IFN-γ) production in CD4 cells.
Main Methods:
- Selected 12 ubiquitous protein antigens from infectious and environmental organisms.
- Screened antigens for their ability to induce IFN-γ-producing CD4 cells in human subjects.
- Evaluated a pool of three inactivated viral antigens (cytomegalovirus, parainfluenza, influenza - CPI) as a potential positive control.
Main Results:
- Inactivated cytomegalovirus, parainfluenza, and influenza virions (CPI) elicited IFN-γ-producing CD4 cells in most subjects.
- Individually, these antigens did not induce a recall response in all donors.
- The CPI pool demonstrated 100% positivity in 245 diverse human donors (Caucasians, Asians, African-Americans).
Conclusions:
- The CPI pool serves as a highly effective universal positive control for CD4 T-cell functionality.
- This control is suitable for verifying the functionality of both CD4 cells and antigen-presenting cells.
- The CPI pool ensures reliable immune monitoring across diverse populations.
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