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CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays
Christian R Kreher1, Markus T Dittrich, Robert Guerkov
1Department of Pathology, School of Medicine, Case Western Reserve University, BRB 928, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
Insights
Cryopreservation does not affect antigen-specific T cell responses in human peripheral blood mononuclear cells (PBMC). This finding supports using frozen PBMC for reliable immune monitoring in clinical trials.
Area of Science:
- Immunology
- Cellular immunology
Background:
- Antigen-specific T cell responses, including cytokine profiles, indicate T cell immunity.
- Cytokine enzyme-linked immunospot (ELISPOT) assays on fresh peripheral blood mononuclear cells (PBMC) are valuable for monitoring vaccine and immune therapy efficacy.
- Clinical trials face challenges with ELISPOT assays due to the need for fresh cells.
Purpose of the Study:
- To investigate the impact of cryopreservation on antigen-specific T cell responses in human PBMC.
- To determine if cryopreserved PBMC can yield valid ELISPOT data for immune monitoring.
- To compare cryopreservation effects on human PBMC versus murine spleen cells.
Main Methods:
- Systematic study of antigen-specific T cell responses in fresh and cryopreserved human PBMC from four healthy donors.
- Analysis of CD4+ and CD8+ T cells responding to six recall antigens.
- Measurement of cytokine production (interferon-gamma, interleukin-2, -4, -5) to define T cell cytokine signatures.
Main Results:
- Cryopreservation did not alter the frequencies or the type 1/type 2 cytokine signatures of antigen-specific CD4+ and CD8+ T cells in human PBMC.
- Human PBMC maintained their T cell response profiles after freeze-thawing.
- Cryopreservation led to decreased cytokine secretion in murine spleen cells, contrasting with human PBMC findings.
Conclusions:
- Cryopreservation is a viable method for preserving human PBMC for ELISPOT analysis.
- Frozen human PBMC can be reliably used for ex vivo immune monitoring in clinical research.
- Cryopreservation effects differ between human and murine immune cells.
Abstract:
The frequency and the cytokine signature of antigen-specific T cells in the blood reflect the magnitude and the quality of T cell immunity in vivo. Recently, cytokine enzyme-linked immunospot (ELISPOT) assays performed on freshly isolated peripheral blood mononuclear cells (PBMC) emerged as a promising tool for monitoring these key parameters, providing direct feedback information on the efficacy of vaccinations and immune therapies. However, performing ELISPOT assays with freshly isolated cells is not readily feasible in the context of clinical trials. The ability to obtain valid ELISPOT data on cryopreserved samples would greatly enhance ex vivo immune monitoring capabilities. We have therefore systematically studied antigen-specific T cell responses in freshly isolated PBMC and after cryopreservation. Four healthy donors were selected that displayed T cell responses to six recall antigens. The antigen reactive T cells were defined as CD4 or CD8 cells, and their cytokine effector class was established measuring interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-5. The donors were bled at three different time points, and their PBMC were tested fresh and after freeze-thawing. The results showed that the frequencies and type 1/type 2 cytokine signatures of recall antigen-specific CD4 and CD8 cells are unaffected after cryopreservation. In contrast to these data obtained on human PBMC, cryopreservation of murine spleen cells causes a decrease in cytokine secretion.
