Oral immunotherapy with omalizumab reverses the Th2 cell-like programme of regulatory T cells and restores their function.

BACKGROUND: Oral immunotherapy (OIT) successfully desensitizes patients with food allergies, but the immune mechanisms mediating its efficacy remain obscure. OBJECTIVES: We tested the hypothesis that allergen-specific regulatory T (Treg) cell function is impaired in food allergy and is restored by anti-IgE antibody (omalizumab)-supplemented OIT. METHODS: Peanut-specific T effector (Teff) and Treg cell proliferative responses, activation markers and cytokine expression were analysed by flow cytometry in 13 peanut-allergic subjects before the start of omalizumab-supplemented OIT and periodically in some subjects thereafter for up to 2 years. Peripheral blood regulatory T cells (Treg cells) were analysed for their peanut-specific suppressor function before and at 1 year following OIT. This study was registered on ClinicalTrials.gov (NCT01290913). RESULTS: Proliferation of allergen-specific Teff and Treg cells precipitously declined following the initiation of omalizumab therapy prior to OIT, followed by partial recovery after the initiation of OIT. At baseline, peanut-specific Treg cells exhibited a Th2 cell-like phenotype, characterized by increased IL-4 expression, which progressively reversed upon OIT. Peanut-specific Treg cell suppressor activity was absent at the start of omalizumab/OIT therapy but became robust following OIT. Absent peanut-specific Treg cell function could also be recovered by the acute blockade of IL-4/IL-4R receptor signalling in Treg cells, which inhibited their IL-4 production. CONCLUSIONS AND CLINICAL RELEVANCE: OIT supplemented by omalizumab promotes allergen desensitization through an initial omalizumab-dependent step that acutely depletes allergen-reactive T cells, followed by an increase in allergen-specific Treg cell activity due to the reversal of their Th2 cell-like programme. Improved Treg cell function may be a key mechanism by which OIT ameliorates food allergy.

Interleukin 4 production by CD4+ T cells from allergic individuals is modulated by antigen concentration and antigen-presenting cell type.

We have previously shown that CD4+ T cells from allergic individuals are predisposed to produce interleukin (IL)-4 in response to allergens, and that allergen immunotherapy greatly reduced IL-4 production in an allergen-specific fashion. The mechanism that results in the reduction of IL-4 synthesis in treated individuals is unknown, but because clinical improvement during immunotherapy is associated with the administration of the highest doses of allergen, we hypothesized that high concentration of allergen results in the downregulation of IL-4 synthesis in CD4+ T cells. In this report, we demonstrated that CD4+ T cells from allergic donors produced high levels of IL-4 when stimulated with low concentrations of allergen (0.003-0.01 micrograms/ml), particularly when B cell-enriched populations presented the antigen. In contrast, the same responding CD4+ T cell population produced little IL-4 when stimulated with high concentrations of allergen (10-30 micrograms/ml), especially when monocytes were used as antigen-presenting cells (APC). The quantity of IL-4 produced was also found to be inversely related to the extent of proliferation of the CD4+ T cells in response to allergen/antigen; maximal proliferation of CD4+ T cells occurred in response to high concentrations of antigen when IL-4 production was minimal. Antigen presentation by B cell-enriched populations, instead of monocytes, induced less CD4+ T cell proliferation, but induced much greater IL-4 synthesis. Moreover, the addition of increasing numbers of APC (either B cells or monocytes) to cultures containing a constant number of responder T cells resulted in increased T cell proliferation and decreased IL-4 production. These results indicate that the circumstances under which memory T cells are activated, as well as the strength of the proliferative signal to T cells, greatly affect the quantity of IL-4 produced. Thus, our observations that the cytokine profile of allergen-specific memory CD4+ T cells can indeed be modulated by the antigen dose and APC type suggest that methods that preferentially enhance allergen uptake by monocytes and that enhance T cell proliferation will improve the clinical efficacy of immunotherapy in the treatment of allergic disease.

Allergen immunotherapy decreases interleukin 4 production in CD4+ T cells from allergic individuals.

Allergen specific CD4+ T cell clones generated from allergic individuals have been shown to produce increased levels of the cytokine interleukin 4 (IL-4), compared to allergen specific clones generated from nonallergic individuals. This difference between CD4+ T cells from allergic and nonallergic individuals with regard to cytokine production in response to allergen is thought to be responsible for the development of allergic disease with increased IgE synthesis in atopic individuals. We examined the production of IL-4 in subjects with allergic rhinitis and in allergic individuals treated with allergen immunotherapy, a treatment which involves the subcutaneous administration of increasing doses of allergen and which is highly effective and beneficial for individuals with severe allergic rhinitis. We demonstrated that the quantity of IL-4 produced by allergen specific memory CD4+ T cells from allergic individuals could be considerably reduced by in vivo treatment with allergen (allergen immunotherapy). Immunotherapy reduced IL-4 production by allergen specific CD4+ T cells to levels observed with T cells from nonallergic subjects, or to levels induced with nonallergic antigens such as tetanus toxoid. In most cases the levels of IL-4 produced were inversely related to the length of time on immunotherapy. These observations indicate that immunotherapy accomplishes its clinical effects by reducing IL-4 synthesis in allergen specific CD4+ T cells. In addition, these observations indicate that the cytokine profiles of memory CD4+ T cells can indeed be altered by in vivo therapies. Thus, the cytokine profiles of memory CD4+ T cells are mutable, and are not fixed as had been suggested by studies of murine CD4+ memory T cells. Finally, treatment of allergic diseases with allergen immunotherapy may be a model for other diseases which may require therapies that alter inappropriate cytokine profiles of memory CD4+ T cells.

Induction of human IgE synthesis by CD4+ T cell clones. Requirement for interleukin 4 and low molecular weight B cell growth factor.

We have analyzed in detail the precise requirements for the induction of human IgE synthesis using several experimental approaches with purified B cells and well-characterized alloantigen-specific CD4+ T cell clones expressing different profiles of lymphokine secretion. Using these clones under cognate conditions in which the B cells expressed alloantigens recognized by the cloned T cells, we have confirmed that IL-4 is required for the induction of IgE synthesis, but we have clearly demonstrated that IL-4 by itself is not sufficient. With several cloned CD4+ T cell lines, including an IL-4-producing clone that could not induce IgE synthesis, and cloned T cells pretreated with cyclosporin A to inhibit lymphokine synthesis, we showed that Th cell-B cell interactions are necessary for IgE synthesis, and that low molecular weight B cell growth factor (LMW-BCGF) and IL-4, in combination, are lymphokines of major importance in the induction of IgE synthesis. Together our results indicate that optimal induction of an IgE-specific response requires the exposure of B cells to a particular complex of signals that include (a) a signal(s) involving Th-B cell interaction that primes B cells to receive additional signals from soluble lymphokines, (b) a specific B cell proliferative signal provided by LMW-BCGF, and (c) a specific B cell differentiation signal provided by IL-4.

Differential requirements of B cells from normal and allergic subjects for the induction of IgE synthesis by an alloreactive T cell clone.

Human T cell helper/inducer clones were used to induce IgE synthesis in B cells from both allergic and nonallergic donors. An alloreactive T cell clone, activated by recognition of specific HLA-DR antigens, stimulated peripheral blood B cells from both allergic and nonallergic donors to synthesize IgE antibody. B cells of allergic donors differed from those of nonallergic donors in their requirements for induction of IgE synthesis. Induction of IgE synthesis in B cells from nonallergic individuals occurred only under conditions of cognate interaction, in which the B cells expressed the alloantigen recognized by the T cells. In contrast, IgE synthesis in B cells from allergic donors occurred under conditions of cognate interaction with T cells as well as bystander conditions where the B cells did not express the alloantigen recognized by the T cell clones and where the T cell clones were stimulated by third-party monocytes bearing the relevant alloantigens. Furthermore, bystander stimulation of IgE synthesis in allergic donors occurred in the presence of tetanus toxoid (TT) antigen-specific T cell clones activated by the appropriate TT-pulsed monocytes. In contrast to the differing requirements of B cells from normal vs. allergic subjects for the induction of IgE synthesis, these B cells did not differ in their requirements for the induction of IgG synthesis. IgG synthesis was induced in all B cells under conditions of cognate interaction with the T cells as well as under conditions of bystander stimulation. These results suggest that cognate T-B cell interactions may be important in the development of IgE immune responses in the normal host.

Novel genetic regulation of T helper 1 (Th1)/Th2 cytokine production and encephalitogenicity in inbred mouse strains.

Development of T helper cell (Th)1 or Th2 cytokine responses is essential for effector and regulatory functions of T helper cells. We have compared cytokine profiles of myelin basic protein (MBP) Ac1-16 peptide-specific T helper cells from inbred mouse strains expressing identical k haplotype-derived MHC class II molecules B10.A and B10.BR, B10.BR T cell lines (TCL) produced Th1 cytokines (including high levels of TNF-alpha) and induced experimental autoimmune encephalomyelitis after adoptive transfer. In contrast, B10.A TCL produced Th2 cytokines (including low levels of TNF-alpha) and were poorly encephalitogenic. The contributions of the genetic origin of the T cells and the APC were explored. Serial restimulations of the B10.BR TCL with B10.A or (B10.A x B10.BR) F1 splenic antigen presenting cells (APC) during the establishment of TCL markedly reduced both Th1 cytokine production and encephalitogenicity. In addition, a single restimulation with B10. A splenic APC reduced IFN-gamma and TNF-alpha production by established Th1 MBP-specific Ak-restricted B10.BR TCL and by a Th1 KLH-specific, Ek-restricted B10.BR T cell clone. These studies suggest that B10.A and B10.BR APC differ in their ability to stimulate IFN-gamma and TNF-alpha production by mature Th1 cells and also influence their Th1/Th2 commitment in vivo. The nature of the downregulatory activity of B10.A APC on IFN-gamma and TNF-alpha production was explored. 2-hour supernatants from antigen-activated B10.A APC/TCL cultures or from B10.A APC activated by LPS had the same inhibitory effects on IFN-gamma and TNF-alpha production by B10.BR TCL. The downregulatory effects of B10.A APC are independent of TNF-alpha, IL-4, IL-10, IL-12p40, IFN-gamma, IL-13, TGF-beta, and PGE2. Thus, genetic difference(s) between B10.A and B10.BR APC appear(s) to control the production or activity of a novel soluble cytokine regulatory factor that influences Th1/Th2 commitment and controls production of IFN-gamma and TNF-alpha by mature Th1 cells.

99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: microbes, apoptosis and TIM-1 in the development of asthma.

Asthma is a complex disorder which has increased dramatically in prevalence over the past three decades. Current therapies, based on the T helper type 2 (Th2) paradigm, have not been able to control this disease. Epidemiological studies have demonstrated an association between infection with the hepatitis A virus (HAV) and protection against the development of asthma, and genetic studies have shown that the HAV receptor, TIM-1 (T cell, immunoglobulin domain and mucin domain), is an important atopy susceptibility gene. Furthermore, recent studies indicate that TIM-1 is a receptor for phosphatidylserine, an important marker of apoptotic cells. These studies together suggest that HAV and TIM-1 may potently regulate asthma through novel non-Th2-mediated mechanisms. Further study of the immunobiology of TIM-1 and its involvement in the clearance of apoptotic cells is likely to provide important insight into the mechanisms that lead to, and those that protect against, asthma, and how infection affects immunity and the development of asthma.

TIM gene family and their role in atopic diseases.

The TIM gene family was discovered seven years ago by positional cloning in a mouse model of asthma and allergy. Three of the family members (TIM-1, TIM-3, and TIM-4) are conserved between mouse and man, and have been shown to critically regulate adaptive immunity. In addition, TIM-1 has been shown to play a major role as a human susceptibility gene for asthma, allergy and autoimmunity. Recently, TIM-4 has been identified as a ligand of phosphatidylserine and to control the uptake of apoptotic cells. These studies together suggest that the TIM gene family evolved to regulate immune responses by managing survival and cell death of hematopoetic cells.

iNKT cells in allergic disease.

Recent studies indicate that invariant TCR+ CD1d-restricted natural killer T (iNKT) cells play an important role in regulating the development of asthma and allergy. iNKT cells can function to skew adaptive immunity toward Th2 responses, or can act directly as effector cells at mucosal surfaces in diseases such as ulcerative colitis and bronchial asthma. In mouse models of asthma, NKT cell-deficient strains fail to develop allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma, and NKT cells are found in the lungs of patients with chronic asthma, suggesting a critical role for NKT cells in the development of AHR. However, much work remains in characterizing iNKT cells and their function in asthma, and in understanding the relationship between the iNKT cells and conventional CD4+ T cells.

Allergen-specific Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation.

Allergic asthma, which is present in as many as 10% of individuals in industrialized nations, is characterized by chronic airway inflammation and hyperreactivity induced by allergen-specific Th2 cells secreting interleukin-4 (IL-4) and IL-5. Because Th1 cells antagonize Th2 cell functions, it has been proposed that immune deviation toward Th1 can protect against asthma and allergies. Using an adoptive transfer system, we assessed the roles of Th1, Th2, and Th0 cells in a mouse model of asthma and examined the capacity of Th1 cells to counterbalance the proasthmatic effects of Th2 cells. Th1, Th2, and Th0 lines were generated from ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice and transferred into lymphocyte-deficient, OVA-treated severe combined immunodeficiency (SCID) mice. OVA-specific Th2 and Th0 cells induced significant airway hyperreactivity and inflammation. Surprisingly, Th1 cells did not attenuate Th2 cell-induced airway hyperreactivity and inflammation in either SCID mice or in OVA-immunized immunocompetent BALB/c mice, but rather caused severe airway inflammation. These results indicate that antigen-specific Th1 cells may not protect or prevent Th2-mediated allergic disease, but rather may cause acute lung pathology. These findings have significant implications with regard to current therapeutic goals in asthma and allergy and suggest that conversion of Th2-dominated allergic inflammatory responses into Th1-dominated responses may lead to further problems.

Human alveolar macrophages present antigen ineffectively due to defective expression of B7 costimulatory cell surface molecules.

Alveolar macrophages, resident phagocytic cells in the lung that derive from peripheral blood monocytes, are paradoxically ineffective in presenting antigen to T cells. We found that antigen presentation by alveolar macrophages could be restored by the addition of anti-CD28 mAb to cultures of T cells and macrophages, indicating that costimulation by alveolar macrophages via the CD28 pathway was defective. In addition, we found that alveolar macrophages activated with IFN-gamma failed to express B7-1 or B7-2 antigens, which normally ligate CD28 on T cells and provide a costimulatory signal required for the activation of T cells. These observations are the first to demonstrate the inability of a "professional" antigen-presenting cell type to effectively express the costimulatory molecules B7-1 and B7-2. Inasmuch as immune reactions within the lung are inevitably associated with inflammatory injury to pulmonary tissue, these observations suggest that reduced expression of B7-1 and B7-2 by alveolar macrophages may be advantageous, as a critical mechanism involved in the induction of peripheral tolerance to the abundance of antigens to which mucosal tissues are continuously exposed.

CD4(+) T helper cells engineered to produce latent TGF-beta1 reverse allergen-induced airway hyperreactivity and inflammation.

T helper 2 (Th2) cells play a critical role in the pathogenesis of asthma, but the precise immunological mechanisms that inhibit Th2 cell function in vivo are not well understood. Using gene therapy, we demonstrated that ovalbumin-specific (OVA-specific) Th cells engineered to express latent TGF-beta abolished airway hyperreactivity and airway inflammation induced by OVA-specific Th2 effector cells in SCID and BALB/c mice. These effects correlated with increased concentrations of active TGF-beta in the bronchoalveolar lavage (BAL) fluid, demonstrating that latent TGF-beta was activated in the inflammatory environment. In contrast, OVA-specific Th1 cells failed to inhibit airway hyperreactivity and inflammation in this system. The inhibitory effect of TGF-beta-secreting Th cells was antigen-specific and was reversed by neutralization of TGF-beta. Our results demonstrate that T cells secreting TGF-beta in the respiratory mucosa can indeed regulate Th2-induced airway hyperreactivity and inflammation and suggest that TGF-beta-producing T cells play an important regulatory role in asthma.

Human dermal fibroblasts present tetanus toxoid antigen to antigen-specific T cell clones.

Cultured human dermal fibroblasts treated with immune interferon express HLA-DR antigens. We report here that DR-positive fibroblasts present tetanus toxoid (TT) to autologous TT-specific monoclonal helper T cells vigorously depleted of monocytes by passage over Sephadex G10 columns followed by treatment with the monoclonal antibodies (mAb) OKM1 and Leu M1 plus complement. The extent of T cell proliferation in response to TT presented by DR-positive fibroblasts was similar to that elicited using monocytes as antigen-presenting cells. The proliferative response was TT dependent, antigen specific, depended upon DR expression by fibroblasts, appeared MHC restricted, and was completely blocked by mouse mAb to HLA-DR but not by mAb to HLA-A,B, or DQ. DR-positive fibroblasts pulsed with TT were similarly effective in antigen presentation. In summary, immune interferon-stimulated human dermal fibroblasts can substitute for classical antigen-presenting cells in antigen-specific proliferative responses. Since fibroblasts are a ubiquitous cell type in the body, they may play a significant role in the immunobiology of the host.

When engineered to produce latent TGF-beta1, antigen specific T cells down regulate Th1 cell-mediated autoimmune and Th2 cell-mediated allergic inflammatory processes.

To determine whether T cells which produce large amounts of latent TGF-beta1 are capable of down-regulating autoimmune and allergic disease, myelin basic protein (MBP)-specific and ovalbumin (OVA)-specific BALB/c cloned Th1 cells were transduced with cDNA for murine TGF-beta1 by coculture with fibroblasts producing a genetically engineered retrovirus. The transduced MBP-specific Th1 cells were found to lose the capacity to provoke EAE in BALB/c mice, and to gain instead the ability to protect against EAE in (SJLxBALB/c) F1 mice immunized with proteolipid protein (PLP). This protective effect was not obtained with OVA-specific TGF-beta1 transduced Th1 cells. The transduced OVA-specific Th1 cells did protect against airway hyperreactivity induced by Th2-cell mediated responses to inhaled OVA. This effect was again antigen specific and it also could not be obtained with untransduced OVA-specific Th1 cells. In both cases these effects of antigen specific TGF-beta1 transduced T cells were nullified by administration of neutralizing anti-TGF-beta mAb. Thus, the antigen specificity of the cloned T cells allows the site-specific local delivery of therapeutic active TGF-beta1 to both Th1 and Th2 cell-mediated inflammatory infiltrates.

TIM-4, expressed by medullary macrophages, regulates respiratory tolerance by mediating phagocytosis of antigen-specific T cells.

Respiratory exposure to antigen induces T cell tolerance via several overlapping mechanisms that limit the immune response. While the mechanisms involved in the development of Treg cells have received much attention, those that result in T cell deletion are largely unknown. Herein, we show that F4/80(+) lymph node medullary macrophages expressing TIM-4, a phosphatidylserine receptor, remove antigen-specific T cells during respiratory tolerance, thereby reducing secondary T cell responses. Blockade of TIM-4 inhibited the phagocytosis of antigen-specific T cells by TIM-4 expressing lymph node medullary macrophages, resulting in an increase in the number of antigen-specific T cells and the abrogation of respiratory tolerance. Moreover, specific depletion of medullary macrophages inhibited the induction of respiratory tolerance, highlighting the key role of TIM-4 and medullary macrophages in tolerance. Therefore, TIM-4-mediated clearance of antigen specific T cells represents an important previously unrecognized mechanism regulating respiratory tolerance.

Changes in antigen-specific T-cell number and function during oral desensitization in cow's milk allergy enabled with omalizumab.

Food allergy is a major public health problem, for which there is no effective treatment. We examined the immunological changes that occurred in a group of children with significant cow's milk allergy undergoing a novel and rapid high-dose oral desensitization protocol enabled by treatment with omalizumab (anti-immunoglobulin (Ig)E monoclonal antibodies). Within a week of treatment, the CD4(+) T-cell response to milk was nearly eliminated, suggesting anergy in, or deletion of, milk-specific CD4(+) T cells. Over the following 3 months while the subjects remained on high doses of daily oral milk, the CD4(+) T-cell response returned, characterized by a shift from interleukin-4 to interferon-γ production. Desensitization was also associated with reduction in milk-specific IgE and a 15-fold increase in milk-specific IgG4. These studies suggest that high-dose oral allergen desensitization may be associated with deletion of allergen-specific T cells, without the apparent development of allergen-specific Foxp3(+) regulatory T cells.

PD-L1 and PD-L2 modulate airway inflammation and iNKT-cell-dependent airway hyperreactivity in opposing directions.

Interactions of the inhibitory receptor programmed death-1 (PD-1) with its ligands, programmed death ligand (PD-L)1 and PD-L2, regulate T-cell activation and tolerance. In this study, we investigated the role of PD-L1 and PD-L2 in regulating invariant natural killer T (iNKT)-cell-mediated airway hyperreactivity (AHR) in a murine model of asthma. We found that the severity of AHR and airway inflammation is significantly greater in PD-L2(-/-) mice compared with wild-type mice after either ovalbumin (OVA) sensitization and challenge or administration of alpha-galactosylceramide (alpha-GalCer). iNKT cells from PD-L2(-/-) mice produced significantly more interleukin (IL)-4 than iNKT cells from control mice. Moreover, blockade of PD-L2 interactions of wild-type iNKT cells in vitro with monoclonal antibodies (mAbs) resulted in significantly enhanced levels of IL-4 production. In contrast, PD-L1(-/-) mice showed significantly reduced AHR and enhanced production of interferon-gamma (IFN-gamma) by iNKT cells. iNKT-deficient Jalpha18(-/-) mice reconstituted with iNKT cells from PD-L2(-/-) mice developed high levels of AHR, whereas mice reconstituted with iNKT cells from PD-L1(-/-) mice developed lower levels of AHR compared with control. As PD-L2 is not expressed on iNKT cells but rather is expressed on lung dendritic cells (DCs), in which its expression is upregulated by allergen challenge or IL-4, these findings suggest an important role of PD-L2 on lung DCs in modulating asthma pathogenesis. These studies also indicate that PD-L1 and PD-L2 have important but opposing roles in the regulation of AHR and iNKT-cell-mediated activation.

Natural killer T cells and the regulation of asthma.

A crucial role has been suggested for invariant natural killer T cells (iNKT) in regulating the development of asthma, a complex and heterogeneous disease characterized by airway inflammation and airway hyperreactivity (AHR). iNKT cells constitute a unique subset of T cells responding to endogenous and exogenous lipid antigens, rapidly secreting a large amount of cytokines, which amplify both innate and adaptive immunity. Herein, we review recent studies showing a requirement for iNKT cells in various models of asthma in mice and monkeys as well as studies in human patients. Surprisingly, in several different murine models of asthma, distinct subsets of iNKT cells were required, suggesting that iNKT cells serve as a common critical pathogenic element for many different forms of asthma. The importance of iNKT cells in both allergic and non-allergic forms of asthma, which are independent of adaptive immunity and associated with airway neutrophils, may explain situations previously found to be incompatible with the Th2 paradigm of asthma.