A flow cytometry based assay for the enumeration of regulatory T cells in whole blood

Abstract

The analysis of regulatory T cells (T-reg(s)) is becoming an increasingly important consideration in the development of novel immunotherapeutic strategies. Accurate quantification of T-regs during treatment protocols is crucial, particularly where the therapeutic strategy is targeting T-regs. The TruCOUNT™ method has utility for enumerating different immune cells but has not been used to detect T-regs. We have utilized this technology to develop an assay to enumerate human T-regs in whole blood, based on CD127 expression. The mean number of CD4⁺CD25⁺CD127^lo T-regs per μl of whole blood was 48 ± 16.9 with a range of 18 – 79 (n = 22) and the average percentage was 6.1 ± 1.9% (range 2.2–10.4%). The percentages of CD4⁺CD25⁺CD127^lo T-regs were similar when detected in whole blood or density-gradient separated PBMC, and were comparable to those distinguished using the T-reg marker FoxP3. The assay was robust and reliable for enumeration of the lower frequency T-regs, with CV's for intra-assay repeatability and inter-assay precision of < 9% and < 35%, respectively. The CV's for the detection of total CD4⁺ T lymphocytes using this assay were < 2% for intra-assay repeatability and < 18% for inter-assay precision, providing further evidence for reproducibility. This assay has a number of advantages over current methods, including small sample volume, the ability to determine absolute cell counts, and no need for hematology cell analyzers. This assay will simplify clinical trial immune monitoring and can be used to provide crucial data on patient T-reg numbers before, during, and after therapeutic interventions.

Introduction

Naturally occurring regulatory T cells (T-reg(s)) are an important T cell subset involved in immune regulation. T-reg percentages have been shown to differ in numerous disease states, such as cancers and autoimmune disorders, compared to healthy controls (reviewed by (Allan et al., 2008)). For example, T-reg frequencies were increased in chronic lymphocytic leukaemia patients (Piper et al., 2011), and present in decreased numbers in gastric cancer patients (Szczepanik et al., 2011) and malignant glioma patients (Fecci et al., 2006), compared with healthy controls. It is widely accepted that the presence of T-regs may help or hinder immunotherapeutic approaches to treating cancer, autoimmunity, and transplantation. Therefore, documenting the number and role of T-regs during such treatment is important. This highlights the need for a reliable, practical assay that can accurately monitor T-regs in patients over the course of immunotherapeutic treatments using small amounts of blood or tissue samples.

The TruCOUNT™ assay, a flow cytometry based cell-counting method, allows the calculation of absolute cell numbers within a small sample of whole blood by reference to a known number of beads contained within the TruCOUNT™ tube. This optimized assay has been used as an alternative to traditional hematology cell analyzers to detect rare cell subsets, including blood dendritic cells, in both healthy donors and patient samples (Vuckovic et al., 2004). In addition, this assay accommodates the inclusion of multiple cell markers, allowing the identification and enumeration of several cell types within the same whole blood sample. Inclusion of the T-reg population in such counting protocols has been restricted due to the lack of appropriate and compatible cell markers.

The traditional T-reg marker forkhead box protein 3 (FoxP3) (Fontenot et al., 2003, Hori et al., 2003) is detected intracellularly following cell permeabilization. The requirement for permeabilization is not only time-consuming; it is also highly susceptible to cell loss, reduced fluorescence of antibody conjugates included in the staining mix, and therefore inaccurate quantification (Fazekas de St Groth et al., 2011). Moreover, in humans, FoxP3 is transiently upregulated in non-suppressive activated effector T cells, which can result in an overestimation of T-regs when using this marker (Walker et al., 2003, Wang et al., 2007). Together this suggests that FoxP3 expression is not sufficient in humans to identify suppressive T-regs, and that inclusion of additional or alternative markers for their phenotyping is essential.

It has now been demonstrated that human T-regs can be distinguished by low surface expression of CD127 (Liu et al., 2006, Seddiki et al., 2006, Hartigan-O'Connor et al., 2007, Fazekas de St Groth et al., 2011). Importantly, purified CD4⁺CD25⁺CD127^lo T-regs were confirmed to be functionally suppressive (Liu et al., 2006, Seddiki et al., 2006, Hartigan-O'Connor et al., 2007, Fazekas de St Groth et al., 2011). Furthermore, this marker has been used on clinical samples to measure T-reg frequencies, including graft versus host disease (GvHD) patients following stem cell transplantation (Bremm et al., 2011), in asthmatic patients on oral glucocorticoid therapy (Moniuszko et al., 2010), and in prostate cancer patients before and after vaccination (Vergati et al., 2011). We have designed a flow cytometry based assay, utilizing CD127/CD25 expression and TruCOUNT™ tubes that reliably and accurately enumerates T-regs from 50μl of whole blood. Use of such an assay could help understand how the number and frequency of T-regs relates to clinical immune responses during therapeutic intervention.