Zoltan Fehervari

CTLA-4 Control over Foxp3+ Regulatory T Cell Function

Naturally occurring Foxp3+CD4+ regulatory T cells (Tregs) are essential for maintaining immunological self-tolerance and immune homeostasis. Here, we show that a specific deficiency of cytotoxic T lymphocyte antigen 4 (CTLA-4) in Tregs results in spontaneous development of systemic lymphoproliferation, fatal T cell–mediated autoimmune disease, and hyperproduction of immunoglobulin E in mice, and it also produces potent tumor immunity. Treg-specific CTLA-4 deficiency impairs in vivo and in vitro suppressive function of Tregs—in particular, Treg-mediated down-regulation of CD80 and CD86 expression on dendritic cells. Thus, natural Tregs may critically require CTLA-4 to suppress immune responses by affecting the potency of antigen-presenting cells to activate other T cells.

Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease.

Summary:u2002 Naturally arising CD25+CD4+ regulatory T (Treg) cells, most of which are produced by the normal thymus as a functionally mature T‐cell subpopulation, play key roles in the maintenance of immunologic self‐tolerance and negative control of a variety of physiological and pathological immune responses. Natural Tregs specifically express Foxp3, a transcription factor that plays a critical role in their development and function. Complete depletion of Foxp3‐expressing natural Tregs, whether they are CD25+ or CD25–, activates even weak or rare self‐reactive T‐cell clones, inducing severe and widespread autoimmune/inflammatory diseases. Natural Tregs are highly dependent on exogenously provided interleukin (IL)‐2 for their survival in the periphery. In addition to Foxp3 and IL‐2/IL‐2 receptor, deficiency or functional alteration of other molecules, expressed by T cells or non‐T cells, may affect the development/function of Tregs or self‐reactive T cells, or both, and consequently tip the peripheral balance between the two populations toward autoimmunity. Elucidation of the molecular and cellular basis of this Treg‐mediated active maintenance of self‐tolerance will facilitate both our understanding of the pathogenetic mechanism of autoimmune disease and the development of novel methods of autoimmune disease prevention and treatment via enhancing and re‐establishing Treg‐mediated dominant control over self‐reactive T cells.

Foxp3+ natural regulatory T cells preferentially form aggregates on dendritic cells in vitro and actively inhibit their maturation

Naturally occurring CD4+CD25+ regulatory T cells (Treg) suppress in vitro the proliferation of other T cells in a cell-contact-dependent manner. Dendritic cells (DCs) appear to be a target of Treg-mediated immune suppression. We show here that, in coculture of dye-labeled Treg cells and CD4+CD25− naïve T cells in the presence of T cell receptor stimulation, Treg cells, which are more mobile than naïve T cells in vitro, out-compete the latter in aggregating around DCs. Deficiency or blockade of leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) abrogates Treg aggregation, whereas that of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (CD152) does not. After forming aggregates, Treg cells specifically down-regulate the expression of CD80/86, but not CD40 or class II MHC, on DCs in both a CTLA-4- and LFA-1-dependent manner. Notably, Treg exerts this CD80/86-down-modulating effect even in the presence of strong DC-maturating stimuli, such as GM-CSF, TNF-α, IFN-γ, type I IFN, and lipopolysaccharide. Taken together, as a possible mechanism of in vitro Treg-mediated cell contact-dependent suppression, we propose that antigen-activated Treg cells exert suppression by two distinct steps: initial LFA-1-dependent formation of Treg aggregates on immature DCs and subsequent LFA-1- and CTLA-4-dependent active down-modulation of CD80/86 expression on DCs. Both steps prevent antigen-reactive naïve T cells from being activated by antigen-presenting DCs, resulting in specific immune suppression and tolerance.

CD4 + Tregs and immune control

Recent years have seen Tregs become a popular subject of immunological research. Abundant experimental data have now confirmed that naturally occurring CD25+CD4+ Tregs in particular play a key role in the maintenance of self tolerance, with their dysfunction leading to severe or even fatal immunopathology. The sphere of influence of Tregs is now known to extend well beyond just the maintenance of immunological tolerance and to impinge on a host of clinically important areas from cancer to infectious diseases. The identification of specific molecular markers in both human and murine immune systems has enabled the unprecedented investigation of these cells and should prove key to ultimately unlocking their clinical potential.

ICER/CREM‐mediated transcriptional attenuation of IL‐2 and its role in suppression by regulatory T cells

Here, we report that inducible cAMP early repressor/cAMP response element modulator (ICER/CREM) is induced early in CD25+CD4+ regulatory T cell (TR) assays mainly in activated Foxp3– effector T cells and this induction correlates with sharp decrease in number of IL‐2‐expressing T cells. Importantly, RNAi targeting of ICER/CREM in responder CD25–CD4+ T cells antagonizes TR‐mediated suppression. Moreover, forced expression of Foxp3 in naive CD25– T cells induces constitutive expression of ICER/CREM in T cells with a regulatory phenotype. Foxp3 facilitates expression of ICER/CREM both in Foxp3 transductants as well as CD25– responder T cells suggesting that induction of TR function in suppression assays may utilize contact‐dependent interaction. Indeed, CTLA‐4 blockade or use of B7‐deficient CD25– responder T cells prevents ICER/CREM accumulation and leads to the rescue of IL‐2 expression. Therefore, we propose that CTLA‐4 binding to B7 ligands expressed on activated ligand‐bearing Foxp3– effector T cells results in ICER/CREM‐mediated transcriptional attenuation of IL‐2. Collectively, these data suggest that Foxp3 expression in TR cells imposes suppression in contact‐dependent fashion by induction of constitutive ICER/CREM expression in activated CD25+ Foxp3– T cell effectors thus preventing them from producing IL‐2.

Regulatory T cell-mediated suppression: potential role of ICER

How regulatory T (TR) cells dampen T cell responses remains unclear. Multiple modes of action have been proposed, including cell contact‐dependent and/or cytokine‐dependent mechanisms. Suppression may involve direct contact between TR cells and responder T cells. Alternatively, TR cells may act on dendritic cells to reduce their abiity to prime T cells by modulating costimulation, inducing the secretion of suppressive cytokines or the increase of tryptophan metabolism. Here, we review emerging, novel mechanisms involved in contact‐dependent, TR‐mediated suppression of IL‐2 production in responder CD25− T lymphocytes and the potential involvement of inducible cAMP early repressor (ICER) in this suppression. Finally, cytokines such as TGF‐β and IL‐10, produced by TR cells or other cells, may exert local suppression, which can be conveyed by basic mechanism(s) acting in a similar manner as contact‐dependent, TR‐mediated suppression.

A paragon of self-tolerance: CD25+CD4+ regulatory T cells and the control of immune responses

The interest in naturally arising regulatory T (TR) cells as a paradigm for maintaining immunological self-tolerance has undergone an explosive re-emergence in recent years. This renaissance was triggered by several key experimental observations and the identification of specific molecular markers that have enabled the isolation and experimental manipulation of these cells. Although their existence was once controversial, a large body of evidence now highlights the critical roles of TR cells in maintaining immunological self-tolerance. Furthermore, abnormality of natural TR cells can be a primary cause of autoimmune and other inflammatory diseases in humans.

CD4+ regulatory cells as a potential immunotherapy

CD4+ regulatory T (TR) cells represent a unique lineage of thymically generated lymphocytes capable of powerfully suppressing immune responses. A large body of experimental data has now confirmed the key role played by these cells in the maintenance of self-tolerance. Increasingly, the importance of these cells is also being recognized in a host of other clinically relevant areas such as transplantation, tumour immunity, allergy and microbial immunity. Additionally, it is also possible to generate TR cells by using a variety of ex vivo experimental approaches. We will focus here on harnessing the suppressive abilities of both these families of regulatory cells and how this should give us access to a potent cell-based immunotherapy appropriate for clinical application.

Differential control of allo-antigen-specific regulatory T cells and effector T cells by anti-CD4 and other agents in establishing transplantation tolerance.

Donor-specific graft tolerance can be established by a combination of allo-antigen exposure and manipulation of T cell function, for example by donor-specific transfusion (DST) under the cover of a non-depleting anti-CD4 mAb. Yet, the cellular basis of this graft tolerance is still obscure. This report shows that T cell-deficient BALB/c nude mice reconstituted with naive unfractionated T cells are specifically tolerized to DBA/2 skin grafts by DST and anti-CD4 mAb treatment, whereas those transferred with T cell suspensions depleted of all Foxp3(+)CD25(+)CD4(+) natural regulatory T cells (Tregs) are not. The treatment inhibits Mls-1(a) allo-antigen-specific expansion of CD4(+) non-Tregs expressing Vbeta6 TCR subfamily but leaves the expansion of Vbeta6-expressing Tregs unaffected, allowing the latter to selectively expand and establish donor-specific tolerance. Furthermore, anti-CD4 mAb inhibits in vitro the selective expansion of allo-antigen-specific CD4(+) non-Tregs but not natural Tregs, as observed with in vitro anti-CD154 [CD40 ligand (CD40L)] mAb or rapamycin treatment. The results collectively indicate that the differential effect of biologicals and pharmacological substances on the expansion of allo-antigen-specific Tregs and effector T cells and resulting dominance of the former can be a key general mechanism underlying dominant transplantation tolerance.

The mucosa: at the frontlines of immunity

The immune system is presented with many challenges in its day-to-day duties, but no part of it is subjected to greater demands than those associated with the body’s mucosal surfaces. Not only are mucosal surfaces generally the first point of entry for pathogens and hence require a prompt and robust immune response, but they are also exposed to an enormous volume of irrelevant or innocuous antigens that need to be effectively ignored. Mounting an appropriate response therefore represents a key decision-making dilemma for the mucosal immune system. The mucosal surfaces of the gut, respiratory tract, reproductive tract, and eye represent a complex immunological network structured to resolve this dilemma. With this single topic issue of Trends in Immunology we have gathered together nine articles covering the breadth of mucosal immunology including the development of mucosal lymphoid tissues, lymphocyte recruitment, probiotics, vaccination, immunopathology, and tolerance mechanisms. It is heartening to see how investigators from diverse backgrounds have been drawn to this field and this should surely lead to fertile research and novel insights.The gut is arguably the body’s largest lymphoid compartment both in terms of cell numbers and size. This is likely a reflection not only of the gut’s large surface area but also the heavy antigen traffic passing through it. A particularly striking property of the gut is its ability to induce so-called oral tolerance, whereby harmless antigens presented to the gut immune system result in systemic unresponsiveness specific for that antigen. This ability enables gut immunity to navigate a path between inappropriate activation and dangerous inaction. Noriko Tsuji and colleagues (Tsukuba, Japan) detail the unique populations of antigen presenting cells, lymphocytes and effector molecules present within the gut associated lymphoid tissue (GALT) that initiate and control the process of oral tolerance. How the gut recruits and becomes populated by lymphocytes is the core theme of the article by William Agace (Lund, Sweden).The gut also plays host to a huge and fantastically diverse population of microbes. Some of these species are simple commensals neither helping nor harming the host, whereas others seem to play a more active and positive role by aiding digestion, hindering colonisation by pathogenic microbes, or fine tuning the immune system. Probiotics are cultured strains of bacteria that attempt to replicate the mutualistic functions of particular gut microbes and much has been reported about their potential benefits to human health. There is already a large and growing body of literature related to the beneficial effects of probiotics in infectious disease, atopy, and various inflammatory ailments, however some aspects of the data remain murky. Much of the confusion seems to arise from variability in the probiotic strains, dosing regimen, and differences in patient microflora. Another problem is surely an incomplete understanding of the immune response triggered by probiotics. Hironobu Nanno and colleagues (Yakult, Tokyo, Japan) take an encompassing look at this topic and attempt to offer immunological insights into how the immune system recognises and responds to probiotics. What seems to be clear though is that gut microfloral diversity and number needs to be kept at an optimum level to maintain human health. Sidonia Fagarasan and colleagues (RIKEN, Yokohama, Japan) describe here how the synthesis of IgA by B cells of the GALT regulates and is in turn regulated by the host microflora. Indeed the development of some key immunological architecture responsible for IgA production in the gut is dependent on microbial signals. This IgA feedback mechanism not only fine-tunes the gut microflora but also acts as a critical barrier to colonisation by pathogenic bacteria. Another important but more evolutionarily ancient mechanism of defence at the gut mucosa is via the production of antimicrobial proteins. Geraldine Canny (Lausanne, Switzerland) and colleagues describe one such protein, BPI (bactericidal/permeability-increasing protein), which is produced by gut epithelial cells and has both potent antimicrobial and endotoxin neutralising effects. These properties make BPI and related proteins attractive targets for biopharmaceutical development.The respiratory tract represents another major mucosal frontline and Hiroshi Kiyono and colleagues (Tokyo, Japan) illustrate how it is subjected to many of the same pathogenic and antigenic challenges as the gut. Indeed, they reveal that many of the induction and regulation systems for immunological responses appear to be shared between the gut and respiratory mucosa even though the developmental processes differ. The eye provides a further interesting example, which is often overlooked despite it being another critical mucosal surface. Joan Stein-Streilein describes how the eye’s delicate nature and its key impact on evolutionary fitness have imposed exacting constraints on the kinds of immune responses that can occur here; namely it needs to resist infection but collateral damage needs to be kept to an absolute minimum. The eye achieves this in part via a process called ACAID (anterior chamber associated immune deviation); a phenomenon uncannily similar to that of oral tolerance. Therefore both the eye and respiratory mucosa present striking examples of the parsimonious nature of the immune system.A failure of oral tolerance coupled with the heavy microbial burden of the gut, can lead to a number of debilitating conditions including inflammatory bowel disease (IBD). Although much is known about the lymphocyte culprits and pathology of IBD the role of other cell types has been less studied. Silvio Danese (Milan, Italy) attempts to address this in a wide-ranging review that draws together the current state of knowledge on the role of nonlymphoid components in the induction and pathogenesis of IBD. Clearly we need to look in often surprising and hitherto unexplored areas before we can obtain a comprehensive picture of this disease.Due to their heavy exposure to the external environment, mucosal surfaces tend to be primary targets for pathogens and often act as a nidus for infection and microbial persistence. For instance the reproductive mucosa is generally the initial site of HIV infection and the gut subsequently becomes the most important reservoir of viral replication. The regional specialisation of the immune system implies that targeted boosting of specific immune compartments should therefore be more effective than systemic immunisation, and indeed this fact might underscore the failure of the current generation of HIV vaccines. Igor Belyakov and colleagues (NIAID, USA) demonstrate how a number of promising mucosal immunisation strategies might more efficiently target and control HIV infection. Another related and appealing vaccine approach described by Hiroshi Kiyono and colleagues is the incorporation of vaccines into genetically engineered cereal plants such as rice. This approach could obviate the need for a vaccine cold-chain as well as ensure the timely and regular boosting of immunity without using needle and syringe. In the years to come, targeting the various mucosal surfaces of the body should lead to a more efficacious second generation of vaccines. Such a future however depends on a detailed understanding of the interrelated cells and mechanisms that comprise the mucosal immune system. We hope this single topic issue on mucosal immunology can convey some of the excitement and potential of this fascinating topic.