Toward standardization of Foxp3+ regulatory T‐cell measurement in clinical settings

Regulatory T-cells (T-regs) are a specialized subpopulation of T lymphocytes that act to suppress activation of other immune cells and to maintain immune system homeostasis, self tolerance, as well as to prevent excessive immune responses to foreign antigens (1). Over the last few years, evidence from both humans and animal models has accumulated that this cell subpopulation may play an important role in the pathogenesis of several diseases, suggesting that T-regs may be manipulated to treat those human diseases in the pathogenesis of which they are implicated (1). Several reports provided evidence that autologous stem cell transplantation for autoimmune diseases may induce immunologic self-tolerance by reprogramming autoreactive T cells, thus restoring the immune regulatory network (1,2). The recent demonstration that T-regs could separate graft versus host disease from graft versus tumor reactivity represent another field in which the assessment of FoxP3þ T-regs play a role, and suggests that their immunosuppressive potential might be manipulated (1). Inhibition and depletion of T-reg inhibition represent another field of intervention in cancer immunotherapy (3). Furthermore, several studies also indicate that a defective T regulatory cell compartment can be modulated by B cell-targeted therapy or by antithymocyte globulin treatment, thus opening new scenarios in the treatment of various hematological malignancies as well as autoimmune diseases (1,4). Here, Grant et al. propose a protocol and its validation for the assessment of Foxp3-expressing T-regs, and showed the results of their pilot study in type 1 diabetes, multiple sclerosis, and healthy controls (5). Prior to the identification of the forkhead family transcription factor FoxP3, surface expression of CD4 and CD25 was considered to be key markers for the identification and enumeration of T-regs. As FoxP3 is an intracellular antigen, its detection required standardization, as provided by Grant et al. (5). Ideally, to standardize T-reg measurement by flow cytometry, various monoclonal antibodies from different vendors, cell preparation protocols, fixation and permeabilization reagents, and cell isolation procedures should be carefully and comparatively evaluated. In the study by Grant et al., two antibodies from eBioscience and one from BioLegend were evaluated as probes for FoxP3 in a six-color flow cytometric assay. Clone 206D yielded optimal staining, while the fix and perm reagents from the two companies yielded similar results (5). Data were also similar when Ficoll-isolated and whole blood samples, and between fresh and frozen cells. The importance of these data is that they would permit the analysis of FoxP3 after different sample preparation protocols (5). However, it should be pointed out that in the study by Grant et al., washed samples instead of whole blood samples were used in some experiments, as the washing step was needed to eliminate background interference of protein in plasma with the assay results (5). This is a critical point that needs to be validated in further investigations. Specifically, it needs to be elucidated whether or not the proposed washing step would lead to loss of any T-cell subset. However, as far as the fixation reagent is concerned, I would believe that further studies are needed to assess the validity of various commercially available fix-perm reagents to detect FoxP3 staining in T-regulatory lymphocytes (6). The mainstay of Grant’s results have been obtained using Ficoll-isolated mononuclear cells. With respect to the staining procedure, Grant et al. make a clear case for the use of isotype controls for analyzing FoxP3 expression by T-regs. As recently discussed at the 2008 Annual Meeting of the Clinical Cytometry Society, isotype controls do have a place in intracellular stainings (7). From the scope of quantitative flow cytometry, it is still object of debate whether data on FoxP3-expressing T-regs should be expressed as percentage of positive cells or as mean intensity of fluorescence (MFI). Interestingly, conclusions based on % FoxP3 positivity and MFI as a function of disease status were by and large similar (see Figure 5 of reference 5). In the report by Grant et al., sodium heparin was chosen as anticoagulant; authors preferred heparin over EDTA as some previous data have indicated that heparin shows better stability of leukocyte subsets over 4–5 days, in comparison with EDTA (5). This aspect needs to be further investigated to ascertain the role played by the various anticoagulants for the assessment of FoxP3expressing T-regs.