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Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs
Published on: December 27, 2016
Detection of proliferating cell nuclear antigens and interleukin-2 beta receptor molecules on mitogen- and
J Hesketh1, D Dobbelaere, J F Griffin
1Department of Microbiology, University of Otago, Dunedin, New Zealand.
Insights
Proliferating cell nuclear antigen (PCNA) is a more accurate marker for lymphocyte activation than interleukin-2 receptors (IL-2R) in deer. PCNA reliably identifies antigen-reactive cells in deer exposed to Mycobacterium bovis.
Area of Science:
- Veterinary Immunology
- Cellular Immunology
- Biomarker Discovery
Background:
- Lymphocyte activation is crucial for adaptive immunity.
- Identifying reliable markers for lymphocyte activation is essential for disease monitoring.
- Interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) are potential markers.
Purpose of the Study:
- To compare the efficacy of IL-2R and PCNA as markers of lymphocyte activation in deer.
- To evaluate their utility in identifying antigen-specific immune responses.
Main Methods:
- Deer lymphocytes were activated using mitogen (Concanavalin A) and antigen (Mycobacterium bovis-derived purified protein derivative - PPD).
- Expression of IL-2R and PCNA was detected using heterologous polyclonal and monoclonal antibodies, respectively.
- Lymphocyte transformation (LT) activity was used as a measure of activation.
Main Results:
- Both IL-2R and PCNA were co-expressed on mitogen-activated blast cells.
- PCNA expression more accurately reflected both antigen- and mitogen-induced lymphocyte activation compared to IL-2R.
- PCNA expression successfully identified antigen-reactive cells in M. bovis-exposed deer, with significantly higher levels in infected animals.
Conclusions:
- PCNA is a superior and more reliable biomarker for assessing lymphocyte activation and antigen-specific responses in deer.
- PCNA can be effectively used to detect cellular immune responses to M. bovis infection.
Abstract:
The expression of interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) were compared for their usefulness as markers of lymphocyte activation. Heterologous polyclonal (anti-bovine IL-2R) and monoclonal (anti-human PCNA) antibodies were used to detect the expression of these molecules on activated deer lymphocytes. Both molecules were co-expressed on blast cells which had been activated with mitogen [concanavalin A (Con A)]. There was detectable up-regulation of IL-2R expression in response to antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] stimulation while PCNA expression mimicked lymphocyte transformation (LT) reactivity. PCNA expression was found to more accurately reflect both antigen- and mitogen-activated lymphocyte activation, as estimated by LT activity. The expression of PCNA was used to identify antigen reactive cells from animals exposed to M. bovis. A very low percentage (1.1 +/- 0.4%) of peripheral blood lymphocytes from non-infected animals could be stimulated to express PCNA by in vitro culture with antigen (PPD). Within the infected group both diseased and healthy, 'in-contact', animals expressed significantly higher levels of PCNA upon antigen stimulation.
