Technical noteImmuno-monitoring of CD8+ T cells in whole blood versus PBMC samples
Introduction
Increasingly complex manipulations of the immune system to boost T cell responses are now being tested for the development of effective immunotherapies against infectious diseases or cancer in humans (e.g. vaccination or adoptive cell transfer strategies). This requires more and more precise analysis for careful immuno-monitoring of patients in order to determine the effects of these new strategies, in an effort to optimise and accelerate the development of successful strategies. The detection of T cell activity is often no longer sufficient, as precise characterisation of T cells is needed. In addition, the analysis of samples that have undergone manipulation such as culture of cells yields mostly subjective information. With improved tools available for analysis of cellular immune responses, it is now possible to perform detailed ex vivo analysis of natural immune responses as well as responses to immunotherapy. Ex vivo phenotypic analysis can provide information on the activation status of T cells, their differentiation status, and functional analysis on their effector mechanisms. For this purpose, direct analysis of fresh whole blood samples appears to be the method of choice as cells remain largely unmanipulated (Appay and Rowland-Jones, 2002).
Nonetheless, for practical reasons, studies are often performed using cryopreserved purified PBMC samples. Several T cell analyses comparing whole blood and purified PBMC procedures have shown that these two procedures can yield noticeably different results. For instance, variations in CD4 / CD8 ratios have been reported, and it has been suggested that the whole blood procedure may be more accurate (De Paoli et al., 1984, Renzi and Ginns, 1987, Ashmore et al., 1989, Romeu et al., 1992). These studies were usually performed on whole lymphocyte populations (e.g. CD4, CD8, NK), but did not include subpopulations (e.g. naïve and antigen experienced cells). A more recent study demonstrated that assessment of chemokine receptor expression could also vary from one procedure to another (Berhanu et al., 2003).
We report here that the simple step of Ficoll purification of PBMC can induce a bias in subpopulation distribution, in particular of CD8+ T cells, and lead to inaccurate measurement of antigen specific CD8+ T cell responses. The main obstacle for consistent use of whole blood is in performing longitudinal analysis, due to the need for comparative data that may be best obtained by including samples from different dates in the same assay. However, our experience in the context of longitudinal immuno-monitoring of T cell based therapy suggests that immediate monitoring on fresh whole blood is entirely feasible.
Section snippets
Whole blood samples
Samples from HLA-A2+ healthy or vaccinated melanoma patients were obtained from volunteers attending the clinic. The relevant local Institutional Review Boards and Ethics Committees approved the study. Heparinised blood samples were used fresh within 4 h, and peripheral blood mononuclear cells (PBMCs) were separated from blood using Ficoll–Hypaque™ or NycoPrep™ according to the manufacturer's recommendations.
Reagents
HLA-peptide tetrameric complexes (“tetramers”) were produced as previously described (
Bias towards over-representation of the naïve CD8+ T cell subset in purified PBMC versus whole blood
In the periphery, CD8+ T cells exist in multiple subsets endowed with distinct characteristics and functions in the immune responses. Naïve cells are unprimed and have no effector function but constitute a diverse precursor pool, waiting to encounter foreign antigens, to become memory/effector T cells. In contrast, antigen primed CD8+ T cells constitute a heterogeneous population with various differentiated subsets that can be distinguished according to the expression of various cell surface
Acknowledgements
We thank the donors for study participation and blood donation. This work was sponsored by Fond'action Contre le Cancer, the Ludwig Institute for Cancer Research, the Nelia et Amadeo Barletta Foundation, and by the National Center of Competence in Research (NCCR) Molecular Oncology.
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