A modified flow cytometry method for objective estimation of human CD4⁺ regulatory T cells (CD4⁺ Tregs) in peripheral blood, via CD4/CD25/CD45RO/FoxP3 labeling

Abstract

Background: Several methods exist for flow-cytometric estimation of human peripheral blood CD4 + T regulatory cells (CD4 + Tregs). Methods:We report our experience with the estimation of human CD4 + Tregs via three different characterizations using flow cytometry (CD25 high FoxP3 + , CD25 high CD127 low/− FoxP3 + , and CD4 + CD25 high/int CD45ROFoxP3 + ) in normal subjects. We have used these methods on the control populations from two studies (32 and 36 subjects, respectively), the latter two methods retrospectively on the subjects of the first study. The six CD4 + T cell fractions obtained by the third method were differentially colored to ascertain the Abbreviations: CD45RA-RO, CD45RA or CD45RO; eTregs, effector Tregs (CD4 + CD25 high CD45RA − , or CD4 + CD25 high CD45RO + ), or active Tregs (aTregs); IPEX, immune deficiencypolyendocrinopathy-enteropathy-X-linked; iTregs, induced Tregs; mAb, monoclonal antibody; MFI, mean fluorescence intensity; nTregs, naïve Tregs (CD4 + CD25 int CD45RA + , or CD4 + CD25 int CD45RO − )

The KAG motif of HLA-DRB1 (β71, β74, β86) predicts seroconversion and development of type 1 diabetes

“…It is the overall immune response, together with other T1D susceptibility genes and unknown environmental conditions, that determines β-cell autoimmune response eventually evolving to clinical type 1 diabetes [ 48 , 49 ]. The convenient separation of naïve, effector and regulatory CD4 + T cells into various subpopulations by cytometry may demonstrate the relative distribution of epitope-specific T cells into the various subpopulations, with distinct pathogenic properties and roles [ 50 , 51 ].…”

Association of HLA-DQ Heterodimer Residues −18β and β57 With Progression From Islet Autoimmunity to Diabetes in the Diabetes Prevention Trial–Type 1

“…The comparatively more stable pDQ7, in no small part due to β57Asp, would be expected to lead to comparatively more Treg cells ( 48 ). The most strongly T1D-protective HLA-DQA1*01:02-B1*06:02 heterodimer appears to protect through at least two mechanisms: 1 ) by binding strongly to diabetogenic epitopes, “stealing,” in fact, such peptides from T1D-susceptible HLA class II heterodimers, and 2 ) by inducing Treg cells able to block diabetogenic effector T cells ( 49 , 50 ). Such studies should be extended to heterodimers such as HLA-DQ7, shown to confer protection from T1D.…”

Dawn of Monitoring Regulatory T Cells in (Pre-)clinical Studies: Their Relevance Is Slowly Recognised

“…The other CD4 + CD25 + T cells are activated effector T cells. According to the vision of several experts in the field, CD3, CD4, CD25, CD127, and FoxP3 are the minimally required markers to define human Treg cells in flow cytometric samples and addition of Ki67 and CD45RA/RO could provide information on the activation status of Tregs (173) or improve selection of pure Treg fractions (182). Such Treg panel would also allow for monitoring of different effector T cell subsets (naïve/memory state of both CD4 + and CD8 + T cells).…”

“…Identification of functional Tregs via marker gene analysis (e.g., FoxP3, CTLA-4, and IL-10) may also be a simple and quick method, although the level of mRNA expression does not necessarily reflect protein expression and this read-out is also considered surrogate for Treg functionality (201). Simply distinguishing between resting and activated Tregs and effector T cells can also provide information about the presence or absence of suppressive T cells in a sample (182,187). More considerations and technical challenges for Treg functionality assays can be found in the public domain (25,115,183,194,(202)(203)(204).…”