Technical note
Immuno-monitoring of CD8+ T cells in whole blood versus PBMC samples

https://doi.org/10.1016/j.jim.2005.11.007Get rights and content

Abstract

The study of natural T cell responses against pathogens or tumors, as well as the assessment of new immunotherapy strategies aimed at boosting these responses, requires increasingly precise ex vivo analysis of blood samples. For practical reasons, studies are often performed using purified PBMC samples, usually cryopreserved. Here, we report on FACS analyses of peripheral blood T cells, performed by direct antibody staining of non-purified total blood. For comparison, fresh PBMC, purified by Ficoll, were analysed. Our results show that the latter method can induce a bias in subpopulation distribution, in particular of CD8+ T cells, and sometimes lead to inaccurate measurement of antigen specific CD8+ T cell responses. Direct analysis of total blood can be applied to longitudinal immuno-monitoring of T cell-based therapy. While the need to purify and cryopreserve PBMC for subsequent studies is obvious, the use of whole blood has the advantage of providing unbiased results and only small amounts of blood are used.

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.

Cited by (44)

  • Protocol to quantify and phenotype SARS-CoV-2-specific T cell response using a rapid flow-cytometry-based whole blood assay

    2022, STAR Protocols
    Citation Excerpt :

    Our data show that the overall frequencies of CD4+ and CD8+ T cells and their activation profile were similar between the two assays. the proportion of naïve T cells was slightly lower in the whole blood assay compared to the ICS on PBMC (median: 45% vs 48% for the CD4 compartment and 29.9% vs 33.2% for the CD8 compartment, respectively) as previously reported by Appay et al. (Appay et al., 2006). We then compared the frequency of SARS-CoV-2 spike-specific CD4 T-cell response (producing any measured cytokine: IFN-γ, TNF-α or IL-2) between the whole blood assay and PBMC (Figure 6B).

  • Monitoring inflammation in psychiatry: Caveats and advice

    2022, European Neuropsychopharmacology
    Citation Excerpt :

    Several studies have contributed to the challenge of harmonizing methods in academic research laboratories. Particular attention has been given to sample type with comparative assessments of fresh or frozen purified Peripheral Blood Mononuclear Cells (PBMCs) and whole blood (Hoffmeister, Bunde et al. 2003, Appay, Reynard et al. 2006). Additional parameters that have been considered include the choice of mAbs (Maecker, McCoy et al. 2012, Finak, Langweiler et al. 2016), the use of liquid, lyophilized or freeze-dried reagents (Maecker, McCoy et al. 2012) and the calibration and settings of the optical bench of multi-laser cytometers for longitudinal, multi-user and inter-laboratory standardization (Kalina, Flores-Montero et al. 2012).

View all citing articles on Scopus
View full text