The Phenotypic Markers of CD 4 + CD 25 + T Regulatory Lymphocytes

Regulatory T cells (Tregs) play a principal role in immune reactions including autoimmunity, transplantation tolerance, anti-infectious immunity and cancer. These cells represent about <5% of the peripheral CD4+ T cell population and play a crucial role in the maintenance of immune homeostasis against selfantigens [1, 2]. Numerical or functional deficit of Treg cells is linked to many autoimmune diseases such as rheumatoid arthritis [3], multiple sclerosis or type 1 diabetes. These Treg features result from their potent capacity to reduce the activation and expansion of conventional T cells [4] by suppressing their biological activities like proliferation and blocking the production of proinflammatory cytokines TNF alpha and IFNgamma, among others [5]. Abstract


Introduction
Regulatory T cells (Tregs) play a principal role in immune reactions including autoimmunity, transplantation tolerance, anti-infectious immunity and cancer.These cells represent about <5% of the peripheral CD4+ T cell population and play a crucial role in the maintenance of immune homeostasis against self-antigens [1,2].Numerical or The two main groups of Tregs represent natural Tregs (nTregs) and induced or adaptive Tregs (iTregs) [6].nTregs originate in thymus and express high levels of the IL-2 receptor α-chain (CD25) [7] and the forkhead-winged helix transcription factor FoxP3, which is inevitable for their development and function [8,9].FoxP3 + iTregs arise in periphery from conventional naive CD4 + FoxP3 − T cells.Under the influence of suppressive cytokines and antigen-specific activation they develop into FoxP3 + Tregs.[6].
Today the most widely accepted phenotype for Tregs is the coexpression of CD4, CD25 (α-chain of the IL-2 receptor), and of FoxP3 [10].

Mechanisms of Treg Function
Tregs can suppress a whole range of immune cells including B cells, NK cells, NKT cells, CD4 + or CD8 + T cells, and both monocytes and dendritic cells.The most important and extensively studied is the suppression of CD4 + CD25 -conventional T cells (TCONV).Main function of Tregs is to suppress the activation of naive TCONV, but they can also inhibit activated effector T cells and memory CD4 + [11] and CD8 + T cells [12].Tregs can suppress their proliferation directly [13] without the presence of antigen presenting cells (APCs).There are evidences for either contact-dependent suppression or suppression via immunosuppressive cytokines or other factors.One efficient way to supppress immune responses is direct killing of CD4 + effector cells, which was described by Grossman et al., [14].They have shown that upon CD3/CD46 activation human nTregs express the serine protease granzyme A and kill TCONV in a perforindependent manner without CD95-CD95L interaction.
Nevertheless, the main mechanism of suppression of Tregs consist in influencing the activation status of APCs.In this regard the key molecule is CTLA4, which competitively inhibits the binding of CD28 to its ligands CD80 and CD86 and thus inhibits co-stimulation of effector T cells.CTLA4 together with the adhesion molecule LFA-1 also downregulates the expression of CD80 and CD86 on APCs [15,16].Capturing of CD80/86 ligands by CTLA4 is a process called trans-endocytosis [17].Tregs do not only reduce antigen presenting activity of APCs, but also support an immunosuppressive cytokine milieu by reducing IL-6 while increasing IL-10 production by DCs [18].
Tregs can produce high amounts of membrane-bound and soluble TGF-ß, and blocking TGF-ß partially abrogated suppression of T cell proliferation in vitro using murine or human T cells [19,20] by inhibiting the TCR/CD3 pathway [21].An important role in Treg-mediated suppression plays cytokine IL-10, which exerts immunosuppressive effects on various cell types [22].Another way of suppression offers IL-35, the cytokine involved in Tregmediated suppression, which was shown to directly inhibit TCONV proliferation [23].
Hydrolysis of extracellular ATP to ADP or AMP by the ectoenzyme CD39, expressed by all murine Tregs and by about 50% of human Tregs, represents another Treg-mediated anti-inflammatory mechanism [24].
Treg cells express high levels of CD25 and consecutively deprive the environment of IL-2, which can also affect the survival of effector T cells.

Human and Mouse Differences
To describe all differences between human and mouse systems is not the topic of this rewiev.Shortly speaking, in mice most of

FoxP3
The transcription factor FoxP3 is regarded to be a lineage molecule for Tregs.It is neccessary for their thymic development, function and phenotype and is responsible for controlling the expression of a number of genes including suppresive cytokines and Treg surface molecules [8,10,26,27].However, FoxP3+ T cells are phenotypically and functionally heterogeneous and involve both suppressive and nonsuppressive T cells.Moreover, FoxP3 is an intracellular molecule, detection of which requires fixation and permeabilization of cells and the fixed cells cannot be used in studies of Treg function.Intracellular FoxP3 staining is hardly usable on a daily clinical routine basis in large series of samples (several steps of incubation, washing, long incubation times, use of isotype control).
FoxP3 has been broadly used for characterization of thymus derived naturally occurring Tregs, although it is well known that is also expressed in peripheral induced Treg [28] and even in human activated CD4 + CD25 − T effector cells without suppressive activity [29][30][31][32][33]. Therefore, to delineate Treg subset with maximal precision, the use of quantitative analysis and a combination of other markers rather than just qualitative assessment of Foxp3 and CD25 expression is necessary.Despite these facts CD4 + CD25 + FoxP3 + is still generally accepted as the most reliable phenotype of Treg cells [8,9,34].
Because of the absence of a specific Treg cell marker, it has not been possible to distinguish between Treg cells and conventional activated T cells [35].Later, epigenetic differences in DNA methylation within FoxP3 have been detected between Treg cells and non-regulatory T cells [36], which offered a promising option for the exact quantification of natural Treg cells.Tatura et al., [37] described the application of QAMA assay (quantitative analysis of methylated alleles), that is based on epigenetic differences within the FoxP3 Tregspecific demethylated region (TSDR) between Treg cells and all other major blood cells.

HELIOS
Helios is an Ikaros family transcription factor which was shown to play an important role in Treg function and which was at first believed to differentiate induced Treg cells from natural Treg cells [38,39].The aforesaid studies demonstrated the existence of two subsets of FoxP3 + Tregs which express Helios and which can discern nTregs (FoxP3 + Helios + ) from peripherally-induced (FoxP3 + Helios − ) iTregs [39].But recently Himmel et al., found, that a lack of Helios expression does not exclusively identify human iTregs, and, their data provide the first evidence for the coexistence of Helios + and Helios − nTregs in human peripheral blood [40].However, it has been reported very recently that Neuropilin 1 (Nrp1, CD304), highly expressed by most of nTregs, may be used to distinguish these two subsets [41,42].

CD25
Lot of studies indicate that CD25 (IL-2R -α) is a principal cell surface marker of Tregs [25,46], but some of them have showed that only the CD4 + T cell subset expressing the high levels of CD25 (termed CD25 high ) exerts in vitro suppressive activity and has the greatest regulatory potential [25,47].However, conventional/effector T cells [10,25,48] and a portion of CCR7 + central memory T lymphocytes start expressing CD25 upon TCR-mediated activation [49].Therefore, even highly pure CD4 + CD25 high Treg populations may contain a significant fraction of proinflammatory T effector cells [13].

CD26
CD26 is an extracellular serine protease with dipeptidyl peptidase IV (DPPIV) activity [50].High expression of CD26 has been traditionally used as an indicator of immune activation and effector functions in T cells.Activated and memory T cells display a CD26 high phenotype, and T cytokines like IL-12 raise the number of CD26 molecules on T lymphocytes.High surface levels of this protease indicate TH1 effector responses [50,51].
Recently interesting data has been published by Salgado et al., [52] Their flow cytometry outcomes demonstrated high percentage of CD26 within CD4 + CD25 − or CD4 + FoxP3 −/low effector T lymphocytes, but negative or low levels (CD26 −/low ) in Treg cells, which were gated as the CD4 + CD25 high or the CD4 + FoxP3 high phenotype.While the negative marker CD127 is down modulated in CD4 + T effector lymphocytes after their activation, CD26 molecule within these activated cells is upregulated and becomes a CD4 + CD25 +/high CD26 + phenotype.The upregulation of CD26 within Treg cells (CD4 + CD25 high CD26 −/low ) is only slight.The differences between Treg and activated effector T cells regarding CD26 levels seem to be stable, therefore assessment of CD26 molecule, in combination with currently used markers as CD25, FoxP3, CD194 and others may be useful in distinguishing Treg cells from activated T effector cells.This approach could help to quantify or isolate Tregs from samples of patients with autoimmune or inflammatory diseases.

CD39
Two studies highlighted the importance of CD39/NTPDase1 (ecto-nucleoside triphosphate diphosphohydrolase 1) as a useful sign for the identification of Tregs [24,53].Mandapathil et al., [54] have further focused on CD39 as a positive selection marker of Tregs.Their paper pointed out that CD39 molecule can be successfully used for routine characterization and isolation of functionally unharmed human T regulatory cells from the peripheral blood of patients or healthy donors.The ectoenzyme NTPDase1 catalyzes the generation of AMP from ATP, which is necessary to produce adenosine, an important mediator of active suppression [24,55].Some papers have also been published about the frequencies of CD39 + CD4 + T cells of patients infected with HTLV-1 [56]

CD45RA
The naïve T cell marker CD45RA and FoxP3 have been identified on the population of CD4 + CD25 + T cells from peripheral blood and these cells showed potent suppressive functions [62].Miyara et al., [27] described CD45RA + FoxP3 low T cells as a resting Tregs and Tregs with a CD45RA − FoxP3 high phenotype as activated Treg cells.While activated Tregs are terminally differentiated and rapidly die, resting Tregs show a high proliferative capacity and convert into their activated descendants.The subpopulation of CD45RA + Tregs can be used for cellular therapies, because it retains its suppressive activity also after in vitro expansion [63].

CD49d
Markus Kleinewietfeld et al., [64] have reported that the application of depleting antibodies for CD49d (alpha chain of VLA-4 integrin), a marker of effector T cells producing proinflammatory cytokines, will eliminate conventional T cells from Tregs and in combination with the CD127 antibody can give highly pure population of FoxP3 + Tregs.
CD49d molecule is expressed on most of IFNgamma or IL-17-producing proinflammatory T cells and is reduced on Tregs, even though later data from the same group [24] as well as other researchers [65] admitted, that some degree of CD49d is expressed also in some subsets of Treg cells.In healthy subjects, the results obtained by both methods were clearly positively correlated, while the correlation between both methods in septic patients was only poor [37].This finding revealed that quantification of Treg cells by QAMA detects CD4 + T cells with unmethylated FoxP3−TSDR, hidden in the CD25 med/low fraction of flow cytometry.

CD101
So that although CD127 was for a long period an efficient tool to determine the phenotype and functional activity of Treg [47, 78], there were increasing controversy in comparisons with CD4 + CD25 + FoxP3 + cells, particularly in the context of chronic infections [77].It became obvious, that the mere existence of an underlying disease (e.g., HIV infection) [76] or the in vitro activation [79] cause an intense CD127 down modulation on formerly CD127 + T effector cells.

FR4
The folate receptor (FR), is also known as the folic acid binding protein.There are four isoforms of the human FR: FR-α,-ß,-γ and-δ.They are expressed on epithelial cells, macrophages and malignant cells.Since FRs are overexpressed on approximately 40% of human cancers, folate conjugates have been used to deliver attached imaging and therapeutic agents sele ctively to malignant cells.Recently, FR-δ has been described also on human Tregs

Conclusion
Finding a specific cell surface marker of Treg cells which would allow their identification and isolation was the topic of a past decade.Presumably no other Treg-specific positive surface marker shall be identified in the future, but revealing the molecule CD26 as another negative marker in combination with other listed molecules could slowly amend our possibilities in distinguishing this important population from effector T cells and improve our diagnostic possibilities.
2013), Research in Immunology: An International Journal, DOI: 10.5171/2013.119348 The role of Runt-related transcription factor (RUNX) family proteins in relation to Treg development and function has been explored during last few years.It was shown that Foxp3 expression in nTregs is dependent on RUNX proteins and correlates with the binding of RUNX to Core-binding factor β (CBF-β).Heterodimers of RUNX and CBF-β have been shown to play an important role in maintenance and development of Tregs in both mice and humans, since they controll Foxp3 expression and also the expression of target genes [43].Complex of RUNX1 and _____________________________________________________________________________ ______________ Jan Lastovicka (2013), Research in Immunology: An International Journal, DOI: 10.5171/2013.119348 CBF-β seems to be indispensable for the suppressive function of human nTregs [44].Similar importance has the complex of CBF-β with RUNX1 and RUNX3 for the TGF-β mediated iTreg development and function [45].RUNX deficient mice produce autoimmune disorders resembling those occurring in Foxp3-mutants, although the symptoms are less severe [44].
2013), Research in Immunology: An International Journal, DOI: 10.5171/2013.119348 increases the proliferation and cytokine production of responder T cells.This unique cell subset produces a distinct repertoire of cytokines in comparison to the other CD4 + T cell subsets.It was proposed that this novel CD4 + T cell population counterbalances the suppressive activity of Tregs in periphery and molecule expressed on CD4 + T cells together with CD25 co-expression, is the marker of T cells with distinct effector (CD39 + CD25 − ) or regulatory (CD39 + CD25 + ) function.Moreover, further studies have shown, that activated T cells upregulate CD39 [60], and a new subset of human CD4 + CD39 + FoxP3 − T cells that produce IFN-gamma and IL-17 has been found [61].
or in HIV patients [57], regarding their correlation to Tregs.