Much is currently made of the control of asthma in therapeutic guidelines. Both the British guidelines and the Global Initiative for Asthma (GINA) define measures of control of the disease, and recent studies have defined strategies for control using available anti-inflammatory and bronchodilator therapy such as inhaled steroids and long acting β₂ agonists.^1,2 However, currently available treatments suppress inflammation but do not modify the underlying immunological predisposition to the disease.

Asthma is widely recognised as an inflammatory airway disease driven by activation of Th2-type T lymphocytes in both atopic allergic and intrinsic or non-allergic forms.^3–5 Recent advances in our understanding of the control of the immunological process have identified regulatory suppressive T cells which can prevent activation of self-reactive or pathological T cells in autoimmune or infectious disease models.^6–8 Does this understanding of immune regulation hold the prospect of disease control or even prevention for asthma?

MECHANISM OF ACTION

The interest of immunologists in actively suppressive T cells was re-awoken by the finding by Sakaguchi and co-workers that depletion of CD4+CD25+ T lymphocytes from mice led to development of autoimmune pathology which could be prevented by re-introduction of these cells.⁹ Such CD4+CD25+ regulatory T cells were active in many disease models and could reverse established inflammatory diseases such as colitis.¹⁰ These cells were shown to arise by high affinity selection in the thymus and are thought to form a “naturally occurring” regulatory population that is an important part of maintenance of tolerance or non-reactivity of the immune system against itself.^11,12 Shevach and co-workers established an in vitro system and showed that CD4+CD25+ T cells failed to proliferate to polyclonal or antigenic stimulation in culture; furthermore, they could suppress proliferation and cytokine production by CD4+CD25− T cells.¹³ This system showed that human peripheral blood CD4+CD25+ T cells also represent a non-proliferating suppressive T cell population.^14–19 The mechanism of suppression by CD4+CD25+ regulatory T cells remains unclear: in some in vivo models suppression is dependent on the immunosuppressive cytokines interleukin 10 (IL-10) and transforming growth factor β (TGFβ), whereas suppressive activity in vitro is not affected by the absence or neutralisation of these factors.^7,8 In vitro suppression by both mouse and human CD4+CD25+ T cells requires contact between regulating and responding cells and is partly dependent on negative co-stimulatory signals including CTLA4 and PD-1.⁷ As CD25 is the alpha chain of the IL-2 receptor and these cells do not make their own IL-2, one possibility is that regulation occurs through competition for this and other T cell growth factors as well as by competition for “space” in repopulation experiments in lymphopenic mice.²⁰ Since CD4+CD25+ T cells could regulate non-T cell dependent colitis in a mouse model, such interactions with responder T cells cannot be the sole mode of suppression and regulatory T cells are likely to influence cells of the innate immune system including dendritic cells.²¹ Can CD4+CD25+ T cells inhibit activation of Th2 cells in animal models or in vitro?

Animal models

Animal models of allergic airway sensitisation have been useful in defining potential immunological mechanisms in asthma and allergic disease.²² However, there are few data on CD4+CD25+ T cell regulation of mouse Th2 airway inflammation and airway hyperresponsiveness. When CD4+CD25+ T cells were depleted in one model airway, inflammation actually decreased.²³ This may be because CD25 is also a marker of recently activated T cells or memory effector cells, so both regulators and effectors had been removed. However, co-transfer of ex vivo expanded CD4+CD25+ ovalbumin specific T cells together with Th2 cells had no effect on subsequent inhaled ovalbumin challenge in another mouse model,²⁴ and ovalbumin specific airway CD4+CD25+ T cells in another complex double transgenic model reduced airway inflammation but not airway hyperresponsiveness in response to inhaled challenge.²⁵

In vitro experiments

Human peripheral blood CD4+CD25+ T cells were shown to be suppressive in allergen stimulated cultures.^26–28 We recently compared such suppressive activity in allergen stimulated in vitro cultures of CD4+CD25− and CD4+CD25+ T cells from non-atopic and atopic volunteers. CD4+CD25+ T cells suppressed proliferation and cytokine production by CD4+CD25− from non-atopic subjects almost completely, but this suppressive activity was significantly reduced in cultures from atopic volunteers, particularly when blood was taken from hay fever sufferers during the height of the pollen season.²⁸ Interestingly, removal of CD4+CD25+ T cells from peripheral blood of non-atopic individuals revealed proliferation and Th2 cytokine production to allergen stimulation which was similar to that from atopic volunteers. These data led to the suggestion that Th2 responses to allergen in non-atopic subjects may be actively suppressed by CD4+CD25+ T cells, and that this regulation is either deficient in atopic subjects or overcome by allergen exposure. Clearly, further work is required to determine whether such regulatory cells occur in the airway or are reduced in asthma.

Although CD4+CD25+ regulatory T cells have been isolated from lung tissue around lung cancers,²⁹ phenotypic identification of regulatory T cells is hampered by the lack of a specific cell marker. One important advance in understanding the regulatory function of these cells came from studies of a rare human immunodefiency (IPEX) which results in autoimmune and allergic disease and its murine counterpart, the “scurfy” mouse strain: both were shown to result from mutation of a transcription factor termed FoxP3.^30,31 Furthermore, in elegant experiments FoxP3 knockout mice were shown to lack CD4+CD25+ regulatory T cells, whereas ectopic expression of the transcription factor by retroviral transfer into CD4+CD25− T cells rendered these regulatory.^32–34 How FoxP3 influences suppression is unclear, and although we and others have confirmed relative overexpression of mRNA for FoxP3 by human CD4+CD25+ T cells,²⁸ this may not represent a specific marker for this cell type³⁵ nor does it appear to be active in suppression by other regulatory T cell subtypes. Recently, neuropilin-1 was identified as a potential surface marker for mouse CD4+CD25+ T cells.³⁶

It is therefore possible that atopy (and asthma) result from a failure to suppress inappropriate Th2 responses to environmental allergens. What factors determine the balance between regulatory and potentially immunopathogical Th2 responses to allergen exposure in the developing (or mature) immune system? Increasing interest has focused on the mode of activation of the innate immune system (including airway dendritic cells) as a major determinant of the type of T cell response to antigen exposure. As well as the route, dose and frequency of antigen exposure, co-activation of pattern recognition receptors such as Toll-like receptors (TLR) or co-stimulatory molecules act as important determinants of T cell activation.³⁷ It is possible that relative levels of activation of these receptors may be relevant—for example, low dose bacterial lipopolysaccharide (LPS) acting through TLR4 favours Th2 development³⁸ whereas higher doses drive Th1 development and exposure of dendritic cells to LPS can overcome regulation by CD4+CD25+ T cells.^39,40 Similarly, the balance of co-stimulation may be important as ICOS co-stimulation is active in supporting Th2 responses but can also drive development of IL-10 producing regulatory T cells in the mouse lung.⁴¹ Such considerations may provide an immunological basis for the hygiene hypothesis for the increasing prevalence of asthma and allergic disease: this may represent a failure of development of appropriate regulatory responses due to lack of appropriate TLR or co-stimulator activation at the time of allergen exposure.⁴² It may also underlie some of the genetic associations of asthma as with TLR2 or CD14 (an LPS receptor) polymorphisms.^43,44 It is noteworthy that high exposure to cat allergen reduces the risk of IgE sensitisation and induces an IL-10 predominant “modified Th2” response which may represent regulation.⁴⁵ However, such a protective effect of high level allergen exposure is not reported for house dust mite. Much more work is required before exposure to allergen or other factors can be manipulated to prevent allergic sensitisation.

CLINICAL IMPLICATIONS

How might regulatory T cells be manipulated or induced for treatment or prevention of asthma? Although CD4+CD25+ T cells do not proliferate in vitro in many systems, it has recently been shown that human cells can be expanded in response to antigen⁴⁶ and CD4+CD25+ T cells do proliferate upon in vivo transfer to mice.⁴⁷ It is suggested that suppression by CD4+CD25+ T cells is not antigen specific once these cells are activated, and it might be feasible to transfer cells expanded ex vivo. However, such cell therapy would be complex and potentially hazardous. Another approach is to induce a regulatory population in vivo. Such “adaptive” regulatory T cells have been described in vivo in mice and in vitro in mice and humans and include a range of subtypes that are distinct from the “naturally occurring” CD4+CD25+ T cells.¹² For example, IL-10 producing regulatory T cells were derived in vitro from both human and mouse T cells by activation in the presence of dexamethasone and vitamin D3 (which inhibit development of Th2 and Th1 cells, respectively).⁴⁸ This raises the possibility of inducing similar cells by in vivo allergen exposure in the face of immunosuppressive agents. We recently showed that in vitro exposure of CD4+CD25+ T cells to corticosteroids increased their suppressive activity in subsequent allergen stimulated cultures through increased IL-10 production.⁴⁹ IL-10 producing regulatory T cell clones were produced by activation in the presence of IL-10: these Tr1 cells prevented IgE and Th2 expansion in a mouse model of allergic airways disease but also produced both IL-5 and interferon γ.^50,51 Animal models of tolerance involving nasal or oral delivery of protein or peptide also induce regulatory T cell populations—either IL-10 producing T cells or Th3 cells making TGFβ.^52,53 For many years allergen injection immunotherapy has been used to control allergic diseases including rhinitis and seasonal asthma,^54,55 and this treatment produces long lasting clinical effects and a reduction in Th2 responses to allergen exposure. Allergen immunotherapy also induces a predominant IL-10 response to allergen, and this may be associated with development of regulatory T cells which were CD4+CD25+.^56,57 Whether this phenotype relates to CD4+CD25+ regulatory T cells or represents activation by allergen remains to be determined, although no difference was found in the suppressive activity of peripheral blood CD4+CD25+ T cells from hay fever patients who had or had not been treated with immunotherapy.⁵⁸ Allergen immunotherapy is not used for asthma treatment in the UK because of the risk of anaphylaxis,⁵⁹ but a number of modifications may allow its development for asthma. One approach is to break the allergen into short peptides which retain T cell reactivity but which no longer crosslink IgE (so it will not trigger anaphylaxis).⁶⁰ This approach shows some efficacy in reducing airway hyperresponsiveness and reduced peripheral blood Th2 responses to allergen, again with an increase in IL-10 production.^61,62 We recently showed that reduced CD4+ T cell responses to cat allergen following peptide therapy were not associated with changes in suppression by blood CD4+CD25+ T cells,⁶³ so this type of immunotherapy may induce other regulatory T cell subtypes (or work in another fashion). Other approaches are a combination of immunotherapy with adjuvants such as CpG oligonucleotides (which activate TLR9) or mycobacterial products.^64,65 Clearly, it will be important to establish both safety and bystander suppression of other allergens or antigens before these approaches can be used clinically. However, it is interesting that allergen immunotherapy reduced the development of both new allergen sensitisation and of asthma in trials in children with allergic rhinitis, which suggests that immune modulation may hold the potential to prevent asthma.^66,67

CONCLUSIONS

Current data support the suggestion that regulatory T cells may be important in preventing allergic sensitisation in non-allergic individuals. If the balance between regulation and activation of Th2 T cells can be manipulated, this holds great promise for treatment and prevention of asthma. Regulatory T cells may also be important for a number of other lung diseases: CD4+CD25+ T cells isolated from lung tissue around lung cancers suppressed anti-tumour responses and temporary inactivation of such suppression may be useful for treatment.²⁹ It is also possible that airway inflammation in chronic obstructive pulmonary disease results from a failure to suppress T cell responses to host antigens revealed or altered by smoking or infection. Understanding and manipulation of immune regulation will form a key part in the development of new treatments in coming years.