The concept of immune regulation by suppressor T cells was proposed more than 0 years ago, but at that moment, the necessary technologies to investi-gate these cells were still missing. How-ever, in the last decade, a solid basis for the role of Treg cells in immune regula-tion was confirmed in both animal mod-els and humans. The major functions of effector T cells are mediated by
dis-tinct subpopulations of T cells (Th1 and Th), which have a different cytokine profile. Recently, Treg cells were shown to play a significant role in the control of these immune responses, elicited by both self- and non-self antigens.
Sakaguchi et al. were the first to charac-terize the Treg cells as a minor popula-tion of CD4+ T cells that expresses the interleukin--receptor (IL-R) α-chain (CD5) and that is crucial for the control of autoreactive T cells in vivo (190). For instance, neonatal thymectomy of BALB/
c mice (but not C57BL/6 mice or rats) results in severe auto-immune disease targeting several organs, such as the heart, the gut or endocrine and exocrine glands (figure 17). Two main conclusions were reached from these observations.
First, CD4+CD5+ T cells are important in immune regulation, and second, the thymus is a major producer of Treg cells.
A little later, the suppressive activity of CD4+CD5+ T cells was consistently dem-onstrated in vitro (191). The thymic ori-gin of Treg cells was further confirmed in 001 by Jordan and co-workers. They demonstrated that naturally occurring Treg cells are generated during differen-tiation from the double-positive to the single-positive stage after self-antigen recognition in the thymus (19).
A parallel between CD4+CD5high Treg cells from mice and from humans was rapidly established (19). Human Treg cells have similar characteristics, such as hyporesponsiveness to polyclonal stimulation and suppressive effects that depend on cell-to-cell contact and/or immunomodulatory cytokines (figure 18). Strikingly, most Treg cells express activation/memory markers like CD45RO and MHC class II, and have
critically short telomeres (194). Hence, it was proposed that circulating Treg cells have already encountered their cognate antigen and have subsequently divided and matured (194). Soon after that, it was demonstrated that the transcription factor FOXP was selectively expressed in CD4+CD5+ T cells (see below) (195).
Classification of regulatory CD4+ T cells: naturally occurring or induced
Treg cells are currently classified in ma-jor categories. On one hand, the natural-ly occurring CD4+CD5+ Treg cells devel-op in the thymus during early, neonatal development with subsequent recir-culation in the periphery. On the other hand, there is increasing evidence for an additional subset of Treg cells, which is induced in the periphery. Distinct sub-types of these “induced” or “adaptive”
Treg cells have been described (196):
- Tr1 Treg cells, which are induced in vitro in the presence of select-ed cytokines, especially IL-10, - Th Treg cells, which are found
in mucosa after antigenic chal-lenge and for which TGF-ß is im-portant and finally,
- Natural-like Treg cells, which have the same phenotype as naturally occurring Treg cells.
It should be mentioned that sup-pressor activity is not confined to the CD4+CD5+ T cells. Some CD4+CD5- T cells, CD8+ T cells and even NK T cells or γδ T cells also exert immunosuppressive functions (196).
Several unanswered questions remain about the precise mechanisms of pe-ripheral induction of Treg cells.
There-Normal phenotype AUTOIMMUNITY Thymus
-2 -1 B 1 2 3 4 5 6 7 Autoimmune T cells production
Thymus
-2 -1 B 1 2 3 4 5 6 7 Thymectomy Regulatory T cells production
Wild-type BALB/c mouse
autoimmune T cells regulatory T cells Age (day)
Figure17.DemonstrationoftheroleofthethymusinthegenerationofregulatoryTcells.Neonatal thymectomy of BALB/c mice results in a severe autoimmune disease characterized by diffuse inflammation of several organs (heart, gut, endocrine and exocrine glands). The conclusions of this pioneer experiment were that suppressor T cells are generated in the thymus early in life and that these suppressor T cells control autoreactive cells in the periphery. B = Birth. Adapted from : Sakagushi S., History of regulatory T cells. Regulatory T cells. Vancouver, British Columbia, Canada ; 1-6 feb 007
CD4+CD25+ Treg cells : key concepts
poor proliferation after polyclonal or antigen-specific stimulus suppress proliferation and IL-2 production by naïve cells suppress both CD4+ and CD8+ T cells
exogenous IL-2 & additionnal costimulatory signals (i.e. CD28:B7) abolish the suppressive capability of Treg cells and force proliferation have to be activated in antigen-specific manner to be suppressive once activated, suppress in an antigen non-specific manner suppressive by both cell-cell contacts and inhibitory cytokines
Figure18.KeyconceptsofCD4+CD25+regulatoryTcells. See text. Adapted from Lohr J, Knoechel B, Abbas AK.
Regulatory T cells in periphery. Immunol Rev 006;1:149-16.
fore, different hypotheses are proposed in reference to the models for memory cell generation (197, 198) (figure 19). The linear model postulates that peripheral Treg cells are derived from effector T cells. In the second model (which is called the parallel model), the branch-point between Treg and effector T cell development occurs at the stage of ac-tivated T cells. Particular microenviron-ments, but also the presence of specific subsets of DC, may influence the lineage choice (198).
Major phenotypic markers of regula-tory T cells
The phenotypic characterization of Treg cells remains complex because in hu-mans, all markers proposed to precisely identify Treg cells are insufficient when used alone, as they are also expressed
on other cell types (e.g. CD5, FOXP, CD17, CTLA-4, GITR, OX-40, CD6L). For instance, these markers are expressed by recently activated T cells, although the intensity or the duration of expres-sion may be different.
The presence of inflammation may also cause interference, as the expression of “Treg” markers by activated T cells is boosted. At the moment, phenotypic characterization of Treg cells is based on the combination of the following markers: CD, CD4, CD5, +/- CD17 -/+
FOXP. FOXP is the most specific mark-er of Treg cells (see below). Howevmark-er, its intranuclear localization requires fixa-tion and subsequent permeabilizafixa-tion of the target cells for identification of its expression. Therefore, these cells can not be used for functional characteri-zations. Hence, the selection of viable
Antigen
Antigen IL-2
IL-2
IL-2 Linear model IL-2
Parallel model
Regulatory T cell
Regulatory T cell Naive
T cell
Activated T cell
Naive T cell
Activated T cell
Effector T cell
Effector T cell
IL-10, TGF-β, IFN-α immature DC low-dose antigen
etc...
IL-12, IFN-γ,...
fully matured DC high-dose antigen
etc...
Figure 19. Models proposed for regulatory T cells generation in the periphery.Two different hypotheses depict the putative mechanisms of peripheral regulatory T cells differentiation. The linear model proposes a final differentiation following the stage of effector T cell. The parallel model postulates that regulatory T cell differentiation is an alternative pathway of differentiation of activated T cells. In this latter model, the role of the microenvironment is of importance (see text). TGF-ß: Transforming-Growth-Factor-Beta; IL: interleukin; IFN: interferon; DC: dendritic cell. Adapted from : Shevach EM. CD4+CD5+ suppressor T cells : more questions than answers. Nat Rev Immunol 00;:89-400 and Lohr J, Knoechel B, Abbas AK. Regulatory T cells in periphery. Immunol Rev 006;1:149-16
Treg cells relies on surface markers such as CD5, possibly combined with other markers (i.e. CD17).
IL-2 receptor:the α-chain (CD5): is an important cytokine involved in the stimulation of T cell expansion. This is the first cytokine produced by CD4+
T cells after activation. Its high-affin-ity receptor, a three-chains molecule, is highly expressed on activated T cells and regulatory T cells, cell types that strongly depend on IL- (figures 20 and 21) (199). Naive T cells express the β (CD1) and the γ (CD1) signalling chains of this receptor but lack the high affinity binding α chain for IL- (CD5).
Within hours after activation, T cells pro-duce high amounts of the α chain and subsequently express the trimeric high-affinity IL- receptor. IL- binding sub-sequently promotes T-cell survival (e.g.
by promoting expression of the anti-ap-optotic factor bcl-) and expression of genes involved in cell cycle regulation.
IL- is strongly required for the genera-tion and maintenance of Treg cells in the periphery. This crucial function was illustrated in IL--deficient mice, which develop severe autoimmune diseases
and have a reduced frequency of circu-lating Treg cells (00, 01). In contrast, IL- and CD5 are not required for the thymic generation of FOXP+ naturally occurring Treg cells (0). However, mu-tations of the γ chain (CD1) resulted in the absence of functional Treg cells.
As CD1 is shared with other cytokine receptors such as IL-4, IL-7, IL-9, IL-15, this indicates that some of these cytokines are crucial during the generation and/or maintenance of functional Treg cells in the periphery (0).
In humans, CD4+CD5high T cells repre-sent about 5 to 10 % of the circulating CD4+ T cells. However, only the 1- % of the cells that express the highest levels of CD5 shows strong regulatory func-tion (0). Even if CD4+ Treg cells express high levels of CD5, this phenotypic marker alone does not allow for a pre-cise identification of Treg cells because of the CD5 expression, albeit at lower levels, by activated T cells, especially during inflammatory conditions. De-spite these problems, positive selection based on the CD5 expression, allowed for the demonstration of their suppres-sive function in both mice and humans.
CD25 synthesis
Growth Survival Transcriptionnal regulation
Effector differentiation Low affinity IL-2R
(Kd +/- 10-9M)
High affinity IL-2R (Kd +/- 10-11M)
JAK3
JAK1 IL-2Rβ
(CD122)
γC (CD132)
IL-2Rβ (CD122)
γC (CD132) IL-2Rα (CD25) IL-2
IL-2 IL-2
Figure20.ConstitutionofthehighaffinityIL-2Raftercellactivation. Interactions of interleukin- (IL-) with its high affinity trimeric receptor will notably promote T cell growth, survival and development of effector functions (see text). CD: cluster of differenciation; JAK: Janus Kinase. Adapted from: Malek TR, Bayer AL.
Tolerance, not immnunity, crucially depends on IL-. Nature Reviews 004;4:665-674.
Forkhead box Protein 3 (FOXP3):
FOXP3 is an X-chromosome-linked transcription factor of 41 amino-acids.
It belongs to the winged helix/forkhead family. This family is characterized by a 100-amino-acid monomeric DNA-bind-ing domain, called the fork-head do-main. This DNA-binding domain con-sists of three α helices and two large loops or “wings”, after which the family is named (04, 05). FOXP differs from other FOX proteins in that its forkhead domain is very close to the C-terminal part of the protein.
In mice, FOXP is selectively expressed in CD4+CD5+ T cells and expression of FOXP and CD5 is strongly correlated.
The absence of CD4+CD5+FOXP+ T cells leads to the development of multi-organ autoimmune diseases as illustrat-ed with scurfy disease in mice. Scurfy disease was first observed in 1949: as the disease is X-linked, only male mice develop severe eczema and a fatal evo-lution within weeks (06). Strikingly, the lesions resembled those of graft-versus-host disease, with huge amounts of infiltrating lymphocytes. The scurfy CTLA-4
high affinity receptor for B7 molecules constitutively expressed by Treg cells acquired expression on activated T cells mediates negative costimulatory signals
keep effector T cells in check (physiological terminator of activation) could be involved in suppressive mechanisms used by Treg cells mutations of CTLA-4 gene are associated with autoimmunity
CD25
α-chain of the IL-2 receptor
expression not specific for Treg cells (activation) oldest marker of Treg cells in use
human PBMC : high expression by 5-10 % of the CD4+ only the top 1-2 % of CD4+ that express CD25 are suppressive high CD25 density reflects high affinity of Treg cells for IL-2 allows positive selection of functionnal Treg cells
FOXP3
transcriptionnal regulator
expression is sufficient to convert naïve T cell into Treg cell predominantly expressed by CD4+CD25+ T cells the most specific marker for Treg cells (human, mouse) regulation of expression is different in rodents and humans sustained expression is required for suppressive activity not expressed in all suppressor T cells (i.e. Tr1 cells)
GITR
receptor of the TNFR superfamily not specific for Treg cell present in CD25+ and also in CD25- suppressor cells expression controlled by FOXP3 in suppressor cells not expressed in all suppressor T cells not necessary for suppressor T cell activity
inhibits Treg cell functions but increases Treg cell proliferation modulates Treg cell survival
CD127
α-chain of the IL-7 receptor
low expression on CD4+CD25+FOXP3+ Treg cells high expression on activated T cells promoter expression repressed by FOXP3 allows more accurate selection of functionnal Treg cells
OX-40
receptor of the TNFR superfamily not specific for Treg cell not specific ; also expressed by activated T cells involved in the generation of memory T cells could be involved in survival and functions of Treg cells OX-40 deficient mice : lowered frequency of Treg cells
Figure 22. Main phenotypic markers of regulatoryT cells.Principal markers are the CD5, FOXP, CTLA-4 (CD15) and CD17. Additional markers include GITR and OX-40 (CD14). Tables listed above summarize their major characteristics and special interests for regulatory T (Treg) cells characterization and isolation.
phenotype was also associated with over-expression of cytokine genes (07).
In 001, the association between scurfy disease and genetic mutations of FOXP was finally demonstrated (08). With this mouse model, Fontenot and co-workers demonstrated that Foxp expression was not induced upon acute infection in vivo (09). In agreement with previous work of Sagakuchi et al., this was sug-gestive of thymic development of Treg cells and absence of Treg cell generation in the periphery during the antigenic challenge in mice. Consistent with these observations, Foxp expression is not upregulated in CD4+CD5-FOXP- T cells following TCR stimulation in mice (196).
Finally, Foxp gene transfer converts na-ive CD4+CD5- T cells into a functional regulatory population, demonstrating the pivotal regulatory role of FOXP in the biology of the Treg cell (195, 10).
Situation in humans is more complex.
Like in rodents, mutations of FOXP are associated with Treg cell deficiency and subsequent auto-immune diseases, such as IPEX syndrome (11-1). How-ever, in humans, nearly all CD4+CD5high T cells express FOXP and are suppres-sive. Moreover, some CD4+T cells with intermediate CD5 expression also ex-press FOXP. These cells could be either precursors of Treg cells or transiently activated T cells (14). In contrast to mice, polyclonal or specific antigenic TCR stimulation was associated with long-lasting FOXP expression (14). In agreement with this, Wang et al. dem-onstrated the need for sustained FOXP expression to mediate long-lasting reg-ulatory activity (15). Transient FOXP expression is not associated with sup-pression. Considering these differences
FOXP3, Foxp3, FoxP3 or FOXP3?
Individual subclasses of FOX pro-teins are based on a phylogenetic analysis. A letter and an Arabic numeral designate subclasses.
Therefore, the actual name of any FOX protein is “Fox, subclass N, member X”, or for example, Foxp3. Genes that encode hu-man transcription factors are de-noted by uppercases letters (that is, FOXP3). Only the first letter is capitalized for the genes that en-code mouse transcription factors (Foxp3). Finally, the first and sub-class letter are capitalized for all other chordates (FoxP3). Both hu-man and mouse proteins are de-noted by uppercase letters (that is, FOXP3) (204).
IPEX syndrome: IPEX is the ac-ronym for Immune-Dysregula- tion-Polyendocrinopathy-Enter-opathy-X-linked. IPEX is the most famous syndrom associated with FOXP3 mutations. It is a X-linked disorder characterized by early presentation of severe endocrine autoimmunity (e.g. diabetes mel-litus, thyroiditis), watery diar-rhoea, eczema, failure to thrive and serious infections (e.g. men-ingitis, pneumonia). It is also associated with autoimmune haemolytic anemia, immune thrombocytopenia, neutropenia, eosinophilia and splenomegaly.
Multiple autoantibodies are found. This clinical phenotype is also observed in patients without mutations in FOXP3, indicating that other genes may be involved.
Treatment is essentially based on early bone-marrow transplanta-tion and haematopoietic stem cell transplantation.
with rodents, it seems crucial to always assess the suppressive functions of the human CD4+CD5+FOXP+ T cells in or-der to conclude that these cells truly are Treg cells, and not transiently activated effector T cells.
The development of both anti-FOXP monoclonal antibodies and Foxp knock-in mouse strains were major ad-vances for further characterization of Treg cells. Flow cytometry revealed that human FOXP is predominantly, but not exclusively, expressed in CD4+CD5+T cells. For instance, small proportions of CD5low/mediumFOXP+ and of CD8+FOXP+ were also identified. The CD5-/lo FOX-PGFP+ T cells from FoxpGFP knock-in mouse were also suppressive. In con-trast, cells negative for Foxp expres-sion lacked regulatory function (09).
Taken together, FOXP expression is the current gold standard for the identifi-cation of naturally occurring Treg cells, and this expression is related to the dif-ferentiation into T cells with a regulatory function.
CTLA-4 (CD152): CTLA-4 is a high-af-finity receptor for B7 molecules (CD80/
CD86) which are expressed at the sur-face of APC. Only Treg cells constitu-tively express CTLA-4 in both mice and humans. However, activated T cells also express CTLA-4 after only or 4 days of activation. As a consequence, CTLA-4 expression is not specific for Treg cells.
CTLA-4/B7 interaction on activated T cells induces inhibitory signals and hence, could act as a physiological ter-minator of their activation. This explains why CTLA-4 deficiency results in wide-spread autoimmunity against the host tissue (fatal lymphoproliferative disease with myocarditis, pancreatitis) (16).
The CTLA-4 pathway is thus important to keep T cells in check. In order to de-termine whether CTLA-4 is required for suppressive activity of Treg, several au-thors blocked CTLA-4 with specific an-tibodies. This neutralization abrogated the suppression in experiments where CD4+CD5- and CD4+CD5+ T cells from wild-type mice were mixed (16, 17).
In contrast, in humans, the blockade of CTLA-4 did not alter the suppressive activity mediated by CD4+CD5+ T cells (0, 18). These apparently contradic-tory observations probably reflect the existence of compensatory suppressive mechanisms that could involve TGF-ß (16).
GITR: The Glucocorticoid-induced TNFR-related protein (GITR ; TNFRSF18) is a re-ceptor that belongs to the TNF rere-ceptor superfamily (TNFRSF). Other members of this superfamily are 4-1BB (CD17), OX-40 (CD14) and CD95 (Fas). GITR is ac-tivated by its ligand, GITRL, The ligand is expressed predominantly by APC, even though other cells like endothelial cells can also express GITRL. Only Treg cells are GITRhigh at a resting stage. During activation, several other cell types, such as T cells, NK cells or APC, also express GITR. FOXP positively regulates the expression of GITR. Moreover, GITR ex-pression was associated with suppres-sive functions, even in the absence of CD5 expression. In contrast to these observations, other studies show that CD4+CD5+GITRhigh T cells are not sup-pressive and that Treg cells from mice that lack GITR are fully capable of sup-pression. The triggering of this receptor induces both pro- and anti-apoptotic effects (e.g. during negative selection
in the thymus), costimulates effector T cells and stimulates proliferation of Treg cells. Strikingly, GITR costimulation ab-rogates the suppressive activity of Treg cells and renders CD5- T cells resistant to Treg cell-mediated regulation. At the moment, GITR costimulation is consid-ered to be an inducer of the Treg cell expansion. However, GITR-induced ex-pansion is associated with transient loss of the suppressive activity (19).
OX-40 (CD134): Like GITR, OX-40 is a member of the TNFR superfamily. Al-though the best known costimulation is the interaction between the CD8 to CD80/86, other costimulatory mol-ecules, such as receptors belonging to the TNFRSF, can effectively potentiate the activation of T cells. The OX-40/OX-40L interaction is essential for optimal CD4+ T cell functions and critically con-tributes to the generation of memory
OX-40 (CD134): Like GITR, OX-40 is a member of the TNFR superfamily. Al-though the best known costimulation is the interaction between the CD8 to CD80/86, other costimulatory mol-ecules, such as receptors belonging to the TNFRSF, can effectively potentiate the activation of T cells. The OX-40/OX-40L interaction is essential for optimal CD4+ T cell functions and critically con-tributes to the generation of memory