Cytokine production by allergen-specific T cells is crucial in establishing and maintaining the tolerant or inflammatory context of allergen recognition, Production of Th2 cytokines is associated with allergic diseases including asthma. The nonallergic phenotype has historically been associated either with a failure to recognize the allergen (immunologic ignorance) or the expression of a “protective” Th1 cytokine profile. Indeed, Th1 cytokine profiles have been reported in nonallergic individuals in response to allergen. However, Th1 responses to allergens would be expected to give rise to inflammatory responses such as delayed-type hypersensitivity reactions, which is not generally the case. Thus, allergic individuals respond to allergen with an inflammatory Th2 response, whereas nonallergic individuals appear to make an immune response that is associated with Th1 cytokines but is noninflammatory.
Studies suggest that active regulation is an essential element in maintaining noninflammatory peripheral tolerance to allergens in healthy individuals. Blood T cells were stimulated with aeroallergens and/or food allergens and subsequently selected on the basis of allergen-induced cytokine production. The profile of allergen-specific interferon (IFN)-^ (Th1 marker), IL-4 (Th2 marker), and IL-10 (antiinflammatory cytokine and marker [Tr1] of a population of regulatory T cells [Treg]) production differed between allergic and nonallergic subjects, with the ratio of cell numbers secreting these three cytokines determining the development of a healthy or allergic immune response. Thus, lowTr1 numbers and high Th2 cell numbers resulted in an allergic response, whereas in nonallergic individuals a mixed Th1/Th2 response was associated with a strong IL-10 response.14 Similarly, T-cell clones derived from children persistently allergic to cow milk produced Th2 cytokines (IL-4, IL-13), whereas allergic control subjects without cow milk allergy (but allergic to another food) produced a mixed Th1/Th2 response associated with markedly elevated IL-10 levels.
Tri and T-Helper Type 3
Further examples of a role for IL-10-producing Tr1s in the maintenance of tolerance to allergens can be found in three groups of individuals exposed to relatively high doses of allergen: bee keepers, individuals naturally exposed to high ambient concentrations of cat dander, and allergic patients undergoing specific allergen immunotherapy (SIT). Bee keepers stung repeatedly during the bee-keeping season demonstrate a marked increase in allergen-specific IL-10 secretion from peripheral blood T cells as the season progresses. Many individuals possess allergen-specific IgE, and the first few stings of the season may elicit local allergic reactions. However, reactions to stings disappear rapidly as IL-10 increases. The rise in IL-10 appears to be transient and to require the presence of the allergen because levels return to background shortly after the bee-keeping season.
Individuals exposed to high levels of cat allergen also appear to be protected from allergic sensitivity through a mechanism that involves the induction of IL-10 and allergen-specific IgG4 rather than IgE. The induction of high levels of allergen-specific IgG4 in the relative absence of IgE has been referred to as a modified Th2 response. Interestingly, the relationship between high-dose exposure and the modified Th2 response does not appear to hold true for several other allergens including HDM. Higher doses of HDM exposure are associated with more frequent allergic sensitization. However, it should be appreciated that the relative exposure levels in biological terms may not be equivalent. In other words, high-dose HDM exposure may equate to only moderate cat dander exposure. Were individuals exposed to doses of HDM well in excess of those found in domestic environments, a similar “high-dose tolerance” may be observed.
SIT is associated with the induction of elements of both Th1 and Tr1 responses. Mechanisms of SIT have recently been reviewed elsewhere.18 Early studies identified a relative increase in the IFN-7: IL-4 ratio in both tissues and peripheral T-cell response following SIT. More recently, IL-10 production has been reported in T cells, B cells, and monocytes/macrophages following SIT. Thus, SIT appears to modify both antigen-presenting cell and T-cell responses through the induction of IL-10 and, in some reports, transforming growth factor (TGF)-(p.18 Peripheral blood T cells isolated after SIT were able to suppress allergen-specific proliferation of pretreatment T cells in an IL-10-dependent and TGF-P-dependent fashion because neutralizing antibodies to these cytokines abrogated suppression. Treatment of allergic asthmatic subjects with allergen-derived peptides, representing the major T-cell epitopes, resulted in the induction of IL-10 and of a population of functional CD4+ regulatory T cells capable of downregulating pretreatment T-cell responses to allergen. Changes in allergen-specific antibody isotypes have also been observed following SIT, and these are closely associated with increases in regulatory cytokines, since IL-10 enhances the production of IgG4 (the most prominent isotype induced during SIT) and TGF-P is an isotype switch factor for IgA (less prominently and less frequently observed following SIT).
A distinct Tr1 population expressing high levels of TGF-P has also been identified in the gut and has been labeled T-helper type 3 (Th3).19 Th3 cells suppress proinflammatory T-cell responses in the gut and promote IgA isotype switching. TGF-^ inhibits proliferation and cytokine secretion in resting T cells but not activated T cells. TGF-^ has been shown to induce IL-10 expression in T cells, the latter ameliorating TGF-^-dependent fibrosis.20 IL-10 and TGF-P appear to collaborate in regulating proinflammatory immune responses. Thus, T cells producing IL-10 and/or TGF-P appear to have a regulatory role in the response to allergen in both normal individuals and those exposed to high levels of certain allergens.
IL-10-secreting Tr1-like cells can also be generated with vitamin D3 (1,25(OH)2 VitD3) and corticosteroids such as dexamethasone.21 Treatment of allergic and nonallergic individuals with inhaled or systemic glucocorticosteroids resulted in increased expression of IL-10 messenger RNA together with induction of the forkhead (winged helix) transcription factor Forkhead box p3 (Fox p3). Levels of IL-10 and Fox p3 messenger RNA correlated closely.22 Preincubation of CD4+CD25+ peripheral blood T cells (Treg) with fluticasone propionate enhanced the ability of these cells to inhibit proliferative responses of CD4+CD25-negative T cells and was associated with increased IL-10 expression, Steroid enhancement of suppression was blocked with an antibody to IL-10.23 The role of IL-10-secreting Tr1s in allergy and asthma has been extensively reviewed elsewhere.24 However, IL-10-se-creting Tr1s and TGF-^-secreting Th3 cells constitute only two subsets of “adaptive” Tr1s, and there is evidence that other subsets, particularly thymus-derived or “natural” Treg, may also play a role in allergy and asthma. In the context of allergic diseases, Th1 cells may antagonize Th2 responses, and “immune deviation” from a Th2 to a Th1 response may be a legitimate regulatory strategy for the treatment of allergic disease. In addition, natu-ral-killer T cells and 78 T cells have also been shown to have regulatory roles.
Natural (Thymus-Derived) Treg (CD4+CD25hi Fox рз+)
The T-cell population bearing the CD4 + CD25 + phenotype houses both activated T-helper cells and Treg. A number of molecules have been identified that make it possible to differentiate these two populations both phenotypically and functionally. Natural Treg (CD4 + CD25-high Foxp3+) constitute 5 to 10% of the peripheral T-cell pool in humans and mice. They do not proliferate in response to either polyclonal anti-CD3 stimulation or antigenic stimulation and, furthermore, they can inhibit the proliferative responses of CD4+CD25-negative T cells. In addition to high levels of expression of CD25, many phenotypic markers have been associated with natural Treg, including cytotoxic T-lymphocyte-as-sociated antigen 4, neuropilin-1, haem oxygenase-1, notch 3, glucocorticoid-induced tumor necrosis factor receptor, CD38+CD45RB-low, lymphocyte activation gene-3, G protein-coupled receptor 83, and most consistently Fox p3.28 Deletion of the Fox p3 gene abrogates suppression by CD4 + CD25-high T cells, whereas ectopic expression of the gene in CD25-negative T cells rendered these suppressive. Mutations in the Fox p3 gene have been described in patients with IPEX (immune dysregulation, polyen-docrinopathy, enteropathy, X-linked syndrome), a syndrome that includes development of elevated IgE responses and atopic dermatitis.30 However, IL-10-producing Tr1s do not express Fox p3, indicating that regulation can occur through different pathways.
The majority of peripheral natural Treg develop in the thymus, but CD4+CD25-high Fox p3+ cells have also been induced in the periphery, in an antigen-specific, TGF-^-dependent fashion, through low-dose antigen (peptide) exposure.32 Thymic differentiation of these cells occurs through positive selection events facilitated by medullary epithelial cells (particularly within Hassall corpuscles) and thymic stromal lymphopoietin (TSLP)-activated medullary DCs.33 Interestingly, with respect to allergic disease, TSLP-activated DCs produce large quantities of the Th2 chemokines TARC and MDC. Unlike many DC subsets, they produce little in the way of proinflammatory Th1 cytokines such as IL-12 and tumor necrosis factor-a. In the thymus, TSLP-activated DCs induced expression of Fox p3 and CD25 in thymocytes. Furthermore, TSLP-activated DCs induced strong proliferative responses (in the absence of Th1 or Th2 cytokines) and regulatory function in these cells. However, in the periphery, TSLP-activated DCs strongly activated T cells but induced differentiation of Fox p3-negative Th2 T cells rather than Treg (Fig 1). Interestingly, TSLP is highly expressed by keratinocytes from patients with atopic dermatitis and is associated with the induction of strong Th2 responses and production of IL-4, IL-5, IL-13, and tumor necrosis factor-a, while downregulating expression of IL-10 and IFN-7.35 Numbers of CD4 + CD25-high Fox p3+ Treg are reduced in the skin of patients with atopic dermatitis36 despite apparently normal numbers of these cells in the peripheral blood.
Similar mechanisms appear to be associated with the development of natural CD4 + CD25-high Fox p3+ Treg in the thymus and Th2 T cells in the periphery. Understanding the different signals that lead to these divergent cell fates may be key to understanding the failure of peripheral regulation in allergic disease. Naive T cells activated by TSLP-activated DCs, or functionally immature DCs, may lead to Th2 differentiation in a low-TGF-P, high-IL-2 microenvironment in the skin (for example through bacterial superantigen-dependent T-cell activation and IL-2 production in atopic dermatitis), whereas the relative absence of IL-2 (and/or other growth factors) and the presence of TGF-P may lead to preferential differentiation of Treg.
Figure 1. TSLP drives development of Treg and Th2 cells. TSLP is secreted by epithelial cells in the periphery or in Hassall corpuscles in the thymus. TSLP-activated DCs in the thymus induce Treg (CD4 + CD25+ Foxp3 + ). TGF-P may contribute to conversion of developing thymocytes into Treg by slowing down T-cell proliferation. TSLP-activated DCs in the periphery induce Th2 T cells. IL-2 may contribute to the development of Th2 rather than Treg by enhancing proliferation. TSLP-activated DCs produce TARC and MDC that recruit T cells expressing CC chemokine receptor 4, which may include both Treg and Th2 cells.