Summary

CD4+CD25+ T cells arise during the normal process of maturation in the thymus and survive in the periphery as Treg cells. They constitute about 5-10% of  in the periphery and believed to be generated in the thymus. CD4+CD25+ T cells are potent inhibitors of polyclonal T cell activation. After activation via the TCR, they inhibit IL-2 production by the responding T cells. Unlike other regulatory T cells, which produce inhibitory cytokines, these cells suppress immune resposnes by a contact-dependent mechanism, at least in vitro.

Phenotype

It is not only the expression of CD25 on the surface o f CD4 T cells, but the actual level of CD25 expression that is critical in distinguishing between regulatory and non-regulatory CD4 T cells. Heterogeneity of isolated T regulatory cell populations may in fact explain contradictory findings as to the mode of action of T regs. For example, separation of CD4+ T cells into CD25neg, CD25med and CD25high subsets by FACS sorting reportedly resulted in the segregation of an IL-10, TGFB and CTLA4 independent but cell contact dependent suppression only with the CD25 high subset. Moreover, the CD25high subset was mainly CD62Lhigh (>95%), a hallmark for resting cells that have not been recently activated. In contrast, the CD25med population showed functional variability in vitro; they could enhance or suppress proliferation in cocultures and they contain the majority of recently activated CD62L lo/- CD4+ cells in the circualtion.

Most surface proteins that are expressed on Treg cells such as CD25, CTLA4, GITR, PD0L1, CD45RO and HLA-DR can also be found on activated T responder cells. However, it has been shown that 1-2 weeks after polyclonal activation in the presence of exogenous IL-2, bulk populations of Treg cells demonstrate sustained high expression of CD25 and CTLA4 on their surface while activated CD4+CD25- T responder cells are low or negative for CD25 and CTLA4 as they are only transiently expressed.

Natural Regulatory T cells are characterized by the surface expression of CD4 and CD25. Murine CD4+CD25+ T cells express little CD45RB and a significant proportion express cytolytic T lymphocyte associated antigen 4 (CTLA-4).

Pure populations of human CD25+CD4+ T cells contain both CD45RO+ and CD45RO- cells, but the suppressive activity is enriched in the CD45RO+ memory fraction. CD4+CD25+T cells isolated from human peripheral blood express CD45RO+ memory T cell markers and compared to CD4+CD25- T cells, highly express activation markers such as HLA-DR. Further, CD4+CD25+T cells constantly express CTLA-4 within the cells and the expression of CTLA-4 is enhanced by stimulation. CD4+CDE25+ T regs have ability to produce suppressor cytokines such as IL-10 and TGF B1 which is lower than that of mice. However proliferation inhibitory action against CD4+CD25+ T cells is not canceled by neutralizing antiboides against these cdytokines and the inhibitory action requires direct intercellular contact between CD4+CD25- T cells and CD4+CD25+ Tregs.

Express cytotoxic T-lymphocyte antigen 4 (CTLA4) constitutively and this may have significance in their functional role.

They show a partially anergic phenoytpe, in that they proliferate poorly upon TCR stimulation in vitro and their growth is dependent on IL-2. Some stimulations such as stimulation with anti-CD3 antibodies, stimulation with anti-CD3 antibodies and anti-CD28 antibodies, simulation with allogeneic mature DCs do not cause CD4+CD25+ regulatory T cells to synthesize DNA and to produce cytokines. Stimulation with cytokines such as IL-2, IL-4, IL-15, in addition to that with anti-CD3 and anti-CD28 antibodies enhance the ability of CD4+CD25+ regulatory T cells to synthesize DNA, but the ability is not comparable to that of CD4+CD25- T cells.

Although human Treg cells do exhibit features similar to those of repeatedly activated T cells in that they have short telmeres, contain low levels of Bcl-2, and express CD95 on their surface, theya re not particularly sensitive to activaiton induced cell death (AICD) and are sensitive to death induced by cytokine deprivation.

Regulatory CD4+ T cells express CCR4, CCR8 and CCR5. It is noteworthy that blood-borne M-DCs, in contrast to P-DCs, constitutively produce CCL17 and CCL22 ex vivo. This production of CCR4 ligands by im M-DCs could lead to the preferential attraction of CD25+Treg cells, a mechanism expected to favor tolerance induciton. The production of CCL22, a CCR4 lilgand, is markedly enhanced by 1,25(OH)2D3 in bood M-DC but not P-DCs. Besides maintaining periopheral immunological tolerance in homeostatic conditions, Treg cells could turn-off and limit ongoing inflammatory responses.

How they develop

CD4+CD25+ T cells seem to be members of a unique lineage of T cells that are selected during the process of T cell differentaition in the thymus. Once the natural TReg cells emerge from the thymus, co-stimulation through CD28 is required to maintain a stable pool of the cells in the periphery by promoting their self-renewal through homeostatic proliferation, and by supporting their survival.

TGF-ß reportedly can induce certain CD4+ T cell in the naive (CD45RA+RO-) fraction in human peripheral blood to develop powerful, contact-ependent suppressive activity that iis not antagonized by anti- TGF-ß or anti-IL-10 mAbs. The costimulatory effects of TGF-ß on niave CD4+ T cells up-regulated CD25 and CTLA-4 expression, inccreased their transition to the activated phenotype, but decreased activation induced apoptosis. These CD4+CD25+ regulatory cells prevented CD8+ T cells from proliferation in response to alloantigens and from becoming cytotoxic effector cells. The T regs exerted their suppressive activities in low numbers and once activated, their suppressive properties were Ag nonspecific. Although <1% of naive CD4+ T cells expressed CD25, depletion of this subset before priming with TGF-ß markedly decreased the generation of suppressive activity.

The amount of activation of APCs is critical to the development and induction of natural T regulatory cells. For example, mature DCs are more efficient at inducing the proliferation of transgenic natural T regulatory cells than are immature cells.

Activation/proliferation

In vitro CD4+CD25+ TR cells suppression requires activation of TR cells via their TCR. Although the activation of CD4+CD25+ T cells is antigen-specific, once activated, these cells inhibit both CD4+ and CD8+ T cell responses in an antigen-nonspecific manner. , Tregs require activation by their TCR to become suppressive, but once activated, their effector function is completely nonspecific.

Increasing natural Treg cell function or numbers could potentially be achieved by providing cytokines that favor natural Treg cell activity or survival  such as IL-2 or TGF-beta.

In mice, CD25+CD4+ T cells can proliferate in the absence of added cytokines in culture and in vivo when wtimulated by antigen-loaded DCs, especially mature DCs.

Functional Characteristics 

nTregs are potent suppressors of CD4+CD25- T cell activation in vitro. When CD4+CD25- T cells are stimulated with anti-CD3 antibodies or allogeneic mature DC in the presence of CD4+CD25+ Regulatory T cells, in comparison with that in the absence of CD4+CD25+ T regs, proliferation inhibitory action is observed in a CD4+CD25+ T reg number-dependent manner.

suppress both proliferation and IFNy production by CD8+ T cells induced either by polyclonal or Ag specific stimuli. Inhibit CD8+ responders by inhibiting both IL-2 production and upregulation of CD25 expression.

What can break their functional characteristics? 

High doses of IL-2 or anti-CD28 antibody reportedly abolish CD25+CD4+ T cell mediated suppression, either by abroating the suppressive activity of CD25+CD4+ T cells or by stimulating CD25- CD4+ T cells to overwhelm suppressive signals from CD25+CD4+ T cells, or both.

Removal of IL-2 or anti-CD28 antibody from culture milieu allows anergy-broken and non-suppressive CD25+CD4+ T cells to revert to their original anergic state and to reacquire their suppressive activity. This indicates that the anergic/suppressive state is the basal default condition of CD25+CD4+ T cells.

Mechanisms of Immunosuppression 

A number of possible mechanisms for CD25+CD4+ T cell mediated suppression have been proposed. These include (1) on APC, CD25+CD4+ T cells may directly deliver to CD25-CD25+ T cells a negative signal for activation/proliferation; (2) CD25+CD4+ T cells may express various adhesion molecules (such as ICAM-1, LFA-1 and CD2) at higher levels then CD25-CD4+ T cells, (3) may physically interfere with the interaction of the latter with APC (e.g., by competing for co-stimulatory molecules sucha s CD80/86 or (4) CD25+CD4+ T cells may chemically modify functions of cell surface moelculesw already expressed on APC and thereby interfere wtih activation/prolifesration of CD25-CD4+ T cells on APC.

Role of costimulatory molecules: The block of T regulatory cell mediated suppression is independent of expression of costimulatory molecules on DCs. Thus when MyD88 deficient DCs are stimulated with LPS (MyD88 independent pathway is sufficient for the upregulation of MHC class II and costimulatory molecules in DCs), they upregulate normal levels of costimulatory molecules. When these same DCs are used as APCs, LPS can not interfere with T regulatory cell mediated suppression despite normal upregulation of CD80, CD86 and CD40.

Role of cytokines: Cytokines secreted by DCs are required for overcoming T regulatory cell mediated suppression.

The role of cytokines in the suppressive effects mediated by CD25+CD4+ Tr cells, and thus their relationship to Tr1 and Th3 cells, is highly controversial. Some studies failed to detect production of IL-10 and TGF-B by human CD25+CD4+ T cells, whereas other clearly showed secretion of these cytokines in response to a variety of stimuli. Interestingly, when activated by alloantigens, CD25+CD4+ T cells produced IL10, TGF-B, low levels of IFNy, but no IL-4 or IL-2, a profile of cytokine production which was remarkably similar to that of Tr1 cells. Nevertheless, neither IL-10 nor TGF-B seemed to be directly required for their in vitro suppressive effects. Suppression does not involve killing of the responder cells and is mediated through a cell contact-dependent mechanism independent of IL-4, IL-10 and TGF –?.

Neutralization of Il-4, IL-10 and/or TGF-B neutralization with specific antibodies did not exhibited any significant effects on CD25+CD4+ T cells mediated suppression in one study. However, CD25+ T regs cells that control intestinal inflammation have reportedly been dependent on TGF-ß.

In one study, IL-4 was able to reduce suppressive effectsw by both CD4+ and CD8+ Treg cells from human thymus with respect to Th2 cells but not Th1 cells. The suppressive activity on Th2 clones was also completely inhibited by the addition of IL-7, IL-9 and IL-15. Th1 clones, however, were only reverted by addition of IL-15. In addition, Th2 cells had lower susceptibility to suppressive activity of the human CD25+ Tregs even with addition or these cytokines.

CD4+CD25+ T reg cells have reported to cause CD25- responder type T cells to become suppressive themselves, however, by the production of the inhbitory cytokines TGF-B and IL-10. This mechanism is referred to as “infectious tolerance”.

IL-6 has reportedly contributed to the loss of CD4+CD25+ suppression. Induction of IL-6 by microbial products is required to induce T cell activaiton in the presence of T regulatory cells. For example, stimulation of mouse spleenic cells with LPS reverses T reg cell-mediated suppression, restoring T cell proliferation to near normal levels. This reveral is independent of expression of costimulatory molecules on DCs but dependent on MyD88 since when MyD88 deficient DCs are stimulated with LPS suppression is not reversed despite normal up-regulation of CD80, CD86 and CD40. (MyD88 independent pathway is sufficient for upregulation of MHC class II and costimulatory molecuels in DCs). Neutralization of IL-6 almost completely blocked the ability of spleenic mouse DC culture medium to bock suppression.

Although IL-6 can act systemically, production of high levels of IL-6 during infection will not normally result in a nonspecific block of suppression, because activation of antigen-specific T cells still requires the costimulatory signals provided in a cognate T cell-APC interactions. However, possibly during chronic infections, conditions may arise that lead to IL-6-mediated release of suppression of self-reactive T cells, which may explain the link between infection and some autoimmune diseases.

Chemokines: Although several surface molecules expressed by CD25+TCs cells have been suggested to provide key molecular signals for immunosuppression, multiple mechanisms are probably operative. CCR4, CCR5 and CCR8, a pattern of selectively expressed by CD25+T cells could guide them to their cellular targets and control their interaction with APCs and T cells.

Cell-Contact: Suppression appears to be mediated by T-T interaction as nTregs suppress responses of TCR transgenic CD8+ T cells stimulated with soluble peptide MHC class I tetramers in the complete absence of APC.

Negative signals: The supressive activity of CD25+CD4+ T cells may involve signals through CTLA-4 and/or glucocorticoid-induced TNF receptor (GITR). Stimulation of GITR reportedly abrogates CD25+CD4+ T cell mediated suppression. In addition removal of GITR expressing T cells or administration of monoclonal antibody to GITR produced rogan specific autoimmune disease in otherwise normal mice. One theory behind the function of CTLA-4 is that ligation of CTLA-4 uncouples TCR ligation from the downstream signaling events that lead to full T cell activation. However, ligation of CTLA-4 has also been shown to induce TGF-ß secretion by both naive and effector CD4+ T cells, suggesting that it may mediate positive signaling for secretion of immune-suppressive cytokines.

In vitro measures of suppression by human CD4+CD25+ cells

The widely utilized method to examine regulatory cell function is the in vitro co-culture supression assay in which the potential Treg cells are added, often in decreasing numbers, to target cell CD4+CD25- responder T cells in the presence of polyclonal stimulation.

In order to classify CD25+ subset of CD4 cells as CD4+CD25+ Treg as opposed to Tr1 or Tr3 Treg cells, the suppressive ability of the isolated population must be shown to depend on cell contact.

Effect of T regulatory Cells on DCs

Whereas activated CD4+CD25- T cells are potent stimulators of imDC which in turn stimulate allogeniec CD4+ T cells in MLR, CD4+CD25+ T cells do not stimulate DCs. In fact, there is up to a 3 fold reduction in the capacity of DC cocultured with CD4+CD25+ T cells to stimulate alloeneic T cells compared with DC cocultured with CD4+CD25- T cells. This was shown by cocultruing human DCs with CD4+CD25+ and CD4+CD25- T cells. DCs were then separated form the T cells and seeded with responder T cells. Prolfieration was measured by thymidine incorporation.

Tregs and HIV 

Accumulating evidence indicates that immunity to HIV infection may be controlled by natural Treg cells. AIDS is associated with the loss of CD4+ T cells. Removal of Treg cells from PBMC populations results in increased anti-HIV CD4+ T cell responses. In addition, the in vitro HIV specific CD4 and CD8 T cell responses in most HIV infected people is substantially abrogated by Treg cells. Such suppression is cell contact dependent and cytokine independent, supporting the idea of involvement of natural Treg cells.

Depletion of CD25+ T cells:

Treatment of mice with multiple injections of monoclonal antibodey PC61 depletes mice of CD25+ T cells.