CD4+CD25+ T cells as key regulators of immune responses

ARTICLE

he past decade has seen strengthening support for the idea that suppressor T cells are key actors in maintaining peripheral tolerance and controlling inflammatory responses. Initially introduced in the 1950s, the concept of active suppression and regulatory cells was abandoned for a time due to its complexity and the lack of a clear regulatory mechanism. Regulatory cells endowed with the capacity to inhibit the development of a potentially dangerous immune response have been described in many models. The current complexity is in part due to the diversity of models used, which have enabled the identification of a regulatory component in almost every T cell subset and which have brought into evidence many much-debated regulatory mechanisms.

One group of naturally occurring CD4⁺ regulatory T cells expressing CD25 stands out and seems to be implicated in different experimental systems. Identified in rodents and in man, CD4⁺CD25⁺ regulatory cells derive from thymic precursors, are found in significant numbers in healthy individuals and prevent untoward reactions against potentially harmful self or exogenous antigens throughout life. Much of our knowledge of their mechanisms of action has been learnt from studies using lymphopenic animals, transgenic or cloned T cells and further progress will critically depend on the development of models aimed at understanding the role of CD4⁺CD25⁺ cells in "physiological" non-manipulated normal animals.

CD4⁺CD25⁺ cells and the control of multiple patho-physiological processes

CD4⁺CD25⁺ cells have been identified in mice, rats and in man and represent 5 to 15 % of the CD4⁺ peripheral T cell contingent in healthy individuals. These cells constitutively express the alpha chain of the IL-2 receptor (CD25), present a memory T cell phenotype and are produced continuously in the thymus. Their role in peripheral tolerance has been clearly demonstrated by the observation that the transfer of T cells lacking CD25⁺ regulatory cells leads to the appearance of multiple auto-immune disorders [1]. These results confirm the role of the thymus and CD4⁺CD25⁺ T cells in the maintenance of peripheral tolerance, as shown by pioneer studies of early thymectomy experiments in newborn mice. Since then a large number of reports have shown that CD4⁺CD25⁺ regulatory cells play a major role in controlling the appearance of other auto-immune diseases like type 1 diabetes, experimental auto-immune encephalomyelitis, auto-immune gastritis (AIG), thyroiditis and chronic inflammatory bowel diseases (IBD). These cells are also implicated in the inhibition of transplant rejection [2] and more generally in the control of T cell activation and the size of the effector T cell pool [3]. Clinically, the manipulation of CD4⁺CD25⁺ regulatory T cells might be a valuable approach to treat auto-immune diseases and to potentiate vaccine efficacy. Indeed, restoration or enhancement of CD4⁺CD25⁺ T cell suppressive activity may be beneficial for the treatment of auto-immune diseases; alternatively, the dampening of their function could potentiate the efficacy of anti-tumoral or anti-viral vaccines. In this respect, it has recently been shown that anti-tumoral immunity better develops in animals who have been made CD4⁺CD25⁺ deficient [4-6].

Activation/expansion of CD4⁺CD25⁺ cells

CD4⁺CD25⁺ cells are characterized by a weak sensitivity to stimulation via the antigen receptor (TCR) and inability
to produce IL-2, which correlates with their suppressive function. This anergy can be partly reversed in vitro when stimulation via the TCR is combined with IL-2 or a signal via CD28, which leads to the expansion of CD4⁺CD25⁺ cells with the maintenance or even increase in their suppressive function [7, 8]. CD4⁺CD25⁺ T cells are hypo-responsive to antigenic stimuli in vivo but can expand in response to lymphopenia [9]. This homeostatic expansion was demonstrated to strongly potentiate their regulatory capacity [9].

Although CD4⁺CD25⁺ cells are naturally occurring regulatory T cells produced throughout life within the thymus, it is probable that certain immunization conditions result in expansion of the CD4⁺CD25⁺ cell pool, or even in the stimulation of their suppressive function, leading to down-regulation of the immune response. Recent studies using TCR transgenic animals have shown that certain routes of antigen delivery ¯ the intravenous and oral routes in particular ¯ favored the expansion of CD4⁺CD25⁺ cells and stimulated their suppressive activity [10, 11]. This expansion could be due either to proliferation of CD4⁺CD25⁺ cells and/or to the differentiation of CD4⁺CD25 ^-  cells into regulatory cells. This latter mechanism has been recently demonstrated by transfer experiments of TCR transgenic naive CD4⁺ T cells into irradiated transgenic RAG2 ^- / -  recipients expressing the relevant antigen [12]. Results from this study clearly demonstrated that regulatory T cells can develop in the periphery from mature naive T cells in the absence of already preformed Treg cells.

The cellular and molecular parameters involved in expansion/activation of regulatory cells remain to be defined, but the nature of the antigen-presenting cell and the cytokine micro-environment seem to be determining factors. In particular, IL-10 promote the differentiation of Tr1 cells [13] and TGF-beta induce both CD4⁺CD25 ^-  and CD4⁺CD25⁺ T cells to differentiate into CD4⁺CD25⁺ regulatory T cells [14, 15]. Although not formally demonstrated for CD4⁺CD25⁺ cells, the repeated stimulation of CD4⁺ T cells by immature dendritic cells leads to the emergence of regulatory cells, producing IL-10 and related to Tr1 cells [16], a subset of regulatory CD4⁺ T cell clones previously described by Groux et al. [13]. The relationship between CD4⁺CD25⁺ T cells and Tr1 cells is unclear at present. The induction of Treg cells by immature DC might be particularly relevant in vivo since secondary lymphoid organs contain previously unsuspected significant numbers of immature dendritic cells such as Langerhans cells [17]. Moreover, it has been shown in vivo that tolerance induced by antigen administration through the respiratory tract is dependent on pulmonary dendritic cells, which induce the differentiation of Tr1 cells [18]. These dendritic cells were characterized by a mature phenotype and the capacity to produce IL-10. In the model of contact sensitivity to haptens in mice, it has been shown that mature Langerhans cells were capable of inducing the development of CD4⁺ T cells which control the intensity and duration of the inflammation [19]. The mechanism involved in the induction of regulatory cells by dendritic cells does not therefore correlate with their level of maturity and could implicate the recruitment of sub-populations of particular DCs and/or a functional conditioning of the DCs by the micro environment at the site of antigen penetration.
Several mechanisms of regulation

CD4⁺CD25⁺ cells are able to regulate the proliferation and differentiation of CD4⁺ [20], CD8⁺ T cells [21] and B cells [22] in vitro. Most studies show that CD4⁺CD25⁺ cells could affect the activation of target lymphocytes, their expansion and differentiation into effector cells, probably within secondary lymphoid organs. One study suggests that these cells could equally regulate the effector responses at the level of the peripheral tissues. Indeed, the transfer of CD4⁺CD25⁺ cells prevents the development of effector cells responsible for auto-immune manifestations in mice thymectomized three days after birth, but also prevents the pathology induced by the transfer of cloned auto-reactive cells to athymic mice [23].

Although data concerning the site of CD4⁺CD25⁺ mediated regulation are very limited, studies of their responsiveness to chemokines suggest that these regulatory cells might be recruited both within secondary lymphoid organs and in inflamed tissues. Indeed, CD4⁺CD25⁺ T cells express CCR8 and CCR4 and preferentially respond in vitro to CCL4/MIP-1beta, CCL17/TARC and CCL22/MDC [24, 25]. The appearance of comparable auto-immune manifestations in mice lacking CCL4 or CD25 confirms the critical role of this chemokine in the in vivo recruitment of regulatory cells [25]. Importantly, these chemokines are produced by mature DC suggesting a critical role of these APCs in the attraction of regulatory CD4⁺CD25⁺ cells [24]. In addition, the expression of the integrin alphabeta7 on a sub-population of CD4⁺CD25⁺ [26-28], which recognize epithelial cadherin, might indicate that these regulatory cells navigate between epithelial tissues and secondary lymphoid organs to convey suppressive signals.

In most in vitro coculture systems, the suppressive effect of CD4⁺CD25⁺ cells appeared essentially due to a direct effect on lymphocyte targets, occurring even in the absence of antigen presenting cells [7]. However, the regulatory process could also involve an indirect effect of CD4⁺CD25⁺ cells on antigen presenting cells (such as dendritic cells), through down-regulation of the costimulatory molecules CD80 and CD86 [29].

The suppressive effect requires the activation of CD4⁺CD25⁺ cells via their T cell receptor, but once activated these cells can exert bystander suppression against lymphocytes with a different antigen specificity or MHC haplotype [7]. CD4⁺CD25⁺ T cell activation does not involve the CD28 pathway [30]. One of the key characteristics of CD4⁺CD25⁺ cells is their constitutive expression of CTLA-4 [30-32], which may play a critical role in the suppressive function of these cells. Indeed, two groups have shown that anti-CTLA-4 antibodies reversed the suppressive activity of CD4⁺CD25⁺ cells in vitro [10, 30]. Moreover, administration of anti-CTLA-4 antibodies leads to the development of auto-immune manifestations in normal mice [30] and inhibits the protector effect of CD4⁺CD25⁺ cells in experimental colitis models induced by the transfer of CD4⁺CD45RB^high cells in SCID mice [31]. Nevertheless the role of CTLA-4 in CD4⁺CD25⁺-dependent suppression remains controversial and several in vitro studies failed to identify any role for this molecule in immune suppression [8, 20, 33]. Moreover, CD4⁺CD25⁺ cells isolated from CTLA-4 deficient mice displayed a suppressive activity, which was, however, slightly reduced as compared to that of normal cells [30].

After activation, CD4⁺CD25⁺ cells are capable of producing immuno-suppressive cytokines like IL-10 and TGFbeta and to a lesser extent, IL-4 [10, 20, 22]. Most in vitro studies failed to show a role of these two cytokines in suppression, which appears to be dependent on cellular contact between CD4⁺CD25⁺ cells and the target cells [20, 34-37]. However, several groups have shown that high doses of anti-TGFbeta or soluble receptors for IL-10 and TGFbeta could reverse suppression [10, 22]. The nature of the membrane molecules implicated in the suppression remains to be defined and a role for membrane-bound TGFbeta1 has been suggested [22]. Alternatively, a recent study clearly indicated that CD4⁺CD25⁺ suppressor function can occur independently of TGF-beta 1 [38]. These discrepancies suggest multiple regulatory mechanisms by CD4⁺CD25⁺ cells. Indeed, a critical role for TGF-beta and IL-10 has been demonstrated in the regulation of experimental colitis induced by the transfer of naive CD4⁺CD45RB^high cells to T cell deficient mice [31, 39], whereas these two cytokines do not seem to be implicated in the auto-immune gastritis model [3, 40]. It is possible that distinct sub-populations of CD4⁺CD25⁺ cells regulate IBD and auto-immune gastritis and/or that the mechanism of CD4⁺CD25⁺ mediated regulation might depend on the target organ, the site of regulation and the initial inflammatory inducing agent (IBD, but not gastritis, critically depends on bacterial products).

Recent studies using DNA array technology allowed the identification of genes preferentially expressed in CD4⁺CD25⁺ cells, among which GITR ("Glucocorticoid-induced TNF receptor") seems to be implicated in their suppressive function [27].

Two separate groups added another piece to the complex puzzle of regulation by showing that human CD4⁺CD25⁺ cells can convert conventional CD4⁺ T cells into Tr1-like regulatory cells [41] or TGFbeta secreting cells [42]. Thus, CD4⁺CD25⁺ may fulfil their in vivo suppressive function both locally by a contact-dependent induction of T cell anergy and systemically by the induction of regulatory T cells (infectious tolerance), endowed with cytokine-mediated suppressive activity.

CD4⁺CD25⁺ cells and the regulation of contact sensitivity

Very few patho-physiological models using non-genetically modified animals have been studied, which makes it difficult to evaluate the regulatory role of CD4⁺CD25⁺ cells in patho-physiological situations. Among the various models described to date, contact sensitivity (CS) to haptens might prove useful to better appreciate the role of CD4⁺CD25⁺ cells in the regulation of a CD8⁺ T cell mediated skin inflammatory response.

CS to DNFB: a model of cutaneous inflammation initiated by cytotoxic CD8⁺ T cells

Contact sensitivity (CS) to haptens is one of the best models to study the mechanisms of induction and regulation of antigen-specific cutaneous inflammation. CS can be induced in the mouse by skin sensitization and subsequent challenge with haptens such as dinitrofluorobenzene (DNFB) and oxazolone. The inflammatory response starts 12 hours after challenge, reaches its maximum intensity at 24 to 48 hours, then resolves progressively within a few days.

In CS to DNFB, a clear functional dichotomy has been established between CD4⁺ and CD8⁺ T cells. CD8⁺ cells are effector cells and can develop in the absence of CD4⁺ T cell help [43], whereas CD4⁺ T cells have a regulatory role and control the intensity and resolution of the inflammation [44-47]. The inflammation is initiated by migration of cytotoxic CD8⁺ cells to the site of challenge, followed by recruitment of inflammatory cells. This cytotoxic activity requires a functional FAS/FAS-L or perforin pathway [48] and is directed against hapten-bearing keratinocytes [49].

CS is regulated by CD4⁺ T cells

CD4⁺ T lymphocytes most likely regulate the two phases of CS (Fig. 1). Within secondary lymphoid organs, following sensitization, CD4⁺ cells limit the size of the CD8⁺ effector cells [50] or modify their functional properties. After migrating to the challenge site, these cells probably contribute to the control of inflammation and its resolution [49]. Indeed, in the absence of CD4⁺ T cells, mice develop a more pronounced and persistent inflammation [44-47]. Limited information is currently available regarding whether a particular subset of regulatory cells is involved in the regulation of CS. Nickel specific Tr1 cells have been cloned from skin lesions of allergic contact dermatitis patients suggesting that this subset of regulatory cells might contribute to the regulation of the efferent phase of contact sensitivity [51]. Indirect evidence for the implication of CD4⁺CD25⁺ cells comes from the observation that IL-2-IgG2b fusion protein inhibited contact sensitivity associated with an increase of the size of the CD4⁺CD25⁺ T cell compartment [52]. Our own data, in the model of contact sensitivity to DNFB support a role for CD4⁺CD25⁺ regulatory T cells in the control of the response and in the establishment of oral tolerance (B. Dubois and D. Kaiserlian, submitted).

CONCLUSION

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