[Applications of artificial intelligence to new drug development]. / Applications de l'intelligence artificielle au développement de nouveaux médicaments.

Artificial intelligence (AI) encompasses technologies recapitulating four dimensions of human intelligence, i.e. sensing, thinking, acting and learning. The convergence of technological advances in those fields allows to integrate massive data and build probabilistic models of a problem. The latter can be continuously updated by incorporating new data to inform decision-making and predict the future. In support of drug discovery and development, AI allows to generate disease models using data obtained following extensive molecular profiling of patients. Given its superior computational power, AI can integrate those big multimodal data to generate models allowing: (i) to represent patient heterogeneity; and (ii) identify therapeutic targets with inferences of causality in the pathophysiology. Additional computational analyses can help identifying and optimizing drugs interacting with these targets, or even repurposing existing molecules for a new indication. AI-based modeling further supports the identification of biomarkers of efficacy, the selection of appropriate combination therapies and the design of innovative clinical studies with virtual placebo groups. The convergence of biotechnologies, drug sciences and AI is fostering the emergence of a computational precision medicine predicted to yield therapies or preventive measures precisely tailored to patient characteristics in terms of their physiology, disease features and environmental risk exposure.

Characterization of epitope specificities of reference antibodies used for the quantification of the birch pollen allergen Bet v 1.

BACKGROUND: Accurate allergen quantification is needed to document the consistency of allergen extracts used for immunotherapy. Herein, we characterize the epitope specificities of two monoclonal antibodies used in an ELISA for the quantification of the major birch pollen allergen Bet v 1, established as a reference by the BSP090 European project. METHODS: The ability of mAbs 5B4 and 6H4 to recognize Bet v 1 isoforms was addressed by immunochromatography. The capacity of each mAb to compete with patients' IgE for binding to Bet v 1 was measured by ELISA inhibition. Epitope mapping was performed by pepscan analysis, site-directed mutagenesis, and hydrogen/deuterium exchange-mass spectrometry. RESULTS: The 5B4 epitope corresponds to a peptide sequence (I56-K68) overlapping with the binding sites of patients' serum IgEs. Mutation of residues P59, E60, and K65 abolishes 5B4 binding to Bet v 1 and reduces the level of IgE recognition. In contrast, 6H4 recognizes a conformational epitope lying opposite to the 5B4 binding site, involving residues located in segments I44-K55 and R70-F79. Substitution of E45 reduces the binding capacity of 6H4, confirming that it is critical for the interaction. Both mAbs interact with >90% of Bet v 1 content present in the birch pollen extract, while displaying a weak cross-reactivity with other allergens of the PR-10 family. CONCLUSIONS: MAbs 5B4 and 6H4 recognize structurally distinct epitopes present in the vast majority of Bet v 1 isoforms. These results support the relevance as a reference method of the Bet v 1-specific quantitative ELISA adopted by the European Pharmacopoeia.

Novel approaches and perspectives in allergen immunotherapy.

In this review, we report on relevant current topics in allergen immunotherapy (AIT) which were broadly discussed during the first Aarhus Immunotherapy Symposium (Aarhus, Denmark) in December 2015 by leading clinicians, scientists and industry representatives in the field. The aim of this symposium was to highlight AIT-related aspects of public health, clinical efficacy evaluation, mechanisms, development of new biomarkers and an overview of novel therapeutic approaches. Allergy is a public health issue of high socioeconomic relevance, and development of evidence-based action plans to address allergy as a public health issue ought to be on national and regional agendas. The underlying mechanisms are in the focus of current research that lays the ground for innovative therapies. Standardization and harmonization of clinical endpoints in AIT trials as well as current knowledge about potential biomarkers have substantiated proof of effectiveness of this disease-modifying therapeutic option. Novel treatments such as peptide immunotherapy, intralymphatic immunotherapy and use of recombinant allergens herald a new age in which AIT may address treatment of allergy as a public health issue by reaching a large fraction of patients.

Patterns of IgE sensitization in house dust mite-allergic patients: implications for allergen immunotherapy.

BACKGROUND: Understanding patterns of IgE sensitization in Dermatophagoides-allergic patients living in various geographical areas is necessary to design a product suitable for worldwide allergen immunotherapy (AIT). METHODS: Using a HIFI Allergy customized microarray assay, IgEs specific for 12 purified allergens from Dermatophagoides pteronyssinus or D. farinae were assessed in sera from 1302 house dust mite (HDM)-allergic patients living in various areas. Comprehensive mass spectrometric (MS) analyses were conducted to characterize HDM extracts, as well as purified bodies and feces. RESULTS: Patterns of IgE reactivity to HDM allergens are comparable in all cohorts of patients analyzed, encompassing adults and 5- to 17-year-old children, as well as American, Canadian, European, and Japanese patients. Overall, >70% and >80% of HDM-allergic patients are sensitized to group 1 and group 2 allergens, respectively, from D. pteronyssinus and/or D. farinae species. Furthermore, 20-47% of patients also have IgEs to allergens from groups 4, 5, 7, 13, 15, 21, and 23. All patients have IgEs to allergens present in mite bodies and feces. MS-based analyses confirmed the presence of mite allergens recorded by IUIS in D. pteronyssinus and D. farinae extracts, with groups 2, 8, 10, 11, 14, and 20 prominent in bodies and groups 1, 6, 18, and 23 well represented in feces. CONCLUSIONS: Mite-specific AIT should rely upon a mixture of D. pteronyssinus and D. farinae extracts, manufactured from both feces and bodies. Such a combination is appropriate to treat children and adult Dermatophagoides-allergic patients from Asia, Europe, and North America.

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

BACKGROUND: The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. METHODS: Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. RESULTS: In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. CONCLUSIONS: In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

Development and evaluation of a sublingual tablet based on recombinant Bet v 1 in birch pollen-allergic patients.

BACKGROUND: Sublingual immunotherapy (SLIT) applied to type I respiratory allergies is commonly performed with natural allergen extracts. Herein, we developed a sublingual tablet made of pharmaceutical-grade recombinant Bet v 1.0101 (rBet v 1) and investigated its clinical safety and efficacy in birch pollen (BP)-allergic patients. METHODS: Following expression in Escherichia coli and purification, rBet v 1 was characterized using chromatography, capillary electrophoresis, circular dichroism, mass spectrometry and crystallography. Safety and efficacy of rBet v 1 formulated as a sublingual tablet were assessed in a multicentre, double-blind, placebo-controlled study conducted in 483 patients with BP-induced rhinoconjunctivitis. RESULTS: In-depth characterization confirmed the intact product structure and high purity of GMP-grade rBet v 1. The crystal structure resolved at 1.2 Å documented the natural conformation of the molecule. Native or oxidized forms of rBet v 1 did not induce the production of any proinflammatory cytokine by blood dendritic cells or mononuclear cells. Bet v 1 tablets were well tolerated by patients, consistent with the known safety profile of SLIT. The average adjusted symptom scores were significantly decreased relative to placebo in patients receiving once daily for 5 months rBet v 1 tablets, with a mean difference of 17.0-17.7% relative to the group treated with placebo (P < 0.025), without any influence of the dose in the range (12.5-50 µg) tested. CONCLUSION: Recombinant Bet v 1 has been produced as a well-characterized pharmaceutical-grade biological drug. Sublingual administration of rBet v 1 tablets is safe and efficacious in patients with BP allergic rhinoconjunctivitis.

Characterization of oral immune cells in birch pollen-allergic patients: impact of the oral allergy syndrome and sublingual allergen immunotherapy on antigen-presenting cells.

BACKGROUND: A detailed characterization of human oral immune cells is needed to better understand local mechanisms associated with allergen capture following oral exposure. METHODS: Oral immune cells were characterized by immunohistology and immunofluorescence in biopsies obtained from three healthy individuals and 23 birch pollen-allergic patients with/without oral allergy syndrome (OAS), at baseline and after 5 months of sublingual allergen immunotherapy (AIT). RESULTS: Similar cell subsets (i.e., dendritic cells, mast cells, and T lymphocytes) were detected in oral tissues from healthy and birch pollen-allergic individuals. CD207+ Langerhans cells (LCs) and CD11c+ myeloid dendritic cells (DCs) were found in both the epithelium and the papillary layer of the Lamina propria (LP), whereas CD68+ macrophages, CD117+ mast cells, and CD4+ /CD8+ T cells were rather located in both the papillary and reticular layers of the LP. Patterns of oral immune cells were identical in patients with/without OAS, except lower numbers of CD207+ LCs found in oral tissues from patients with OAS, when compared to OAS- patients (P < 0.05). A 5-month sublingual AIT had a limited impact on oral immune cells, with only a significant increase in IgE+ cells in patients from the active group. Colocalization experiments confirmed that such IgE-expressing cells mostly encompass CD68+ macrophages located in the LP, and to a lesser extent CD207+ LCs in the epithelium. CONCLUSION: Two cell subsets contribute to antigen/allergen uptake in human oral tissues, including (i) CD207+ LCs possibly involved in the physiopathology of OAS and (ii) CD68+ macrophages likely critical in allergen capture via IgE-facilitated mechanisms during sublingual AIT.

Respiratory allergen from house dust mite is present in human milk and primes for allergic sensitization in a mouse model of asthma.

There is an urgent need to identify environmental risk and protective factors in early life for the prevention of allergy. Our study demonstrates the presence of respiratory allergen from house dust mite, Der p 1, in human breast milk. Der p 1 in milk is immunoreactive, present in similar amounts as dietary egg antigen, and can be found in breast milk from diverse regions of the world. In a mouse model of asthma, oral exposure to Der p through breast milk strongly promotes sensitization rather than protect the progeny as we reported with egg antigen. These data highlight that antigen administration to the neonate through the oral route may contribute to child allergic sensitization and have important implications for the design of studies assessing early oral antigen exposure for allergic disease prevention. The up-to-now unknown worldwide presence of respiratory allergen in maternal milk allows new interpretation and design of environmental control epidemiological studies for allergic disease prevention.

Heterogeneity of antibody responses among clinical responders during grass pollen sublingual immunotherapy.

BACKGROUND: During allergen-specific sublingual immunotherapy (SLIT), the relevance of changes in specific IgE and IgG antibody titres to treatment efficacy remains to be evaluated at an individual patient level. OBJECTIVE: To investigate whether antibody responses can be used as biomarkers for SLIT efficacy. METHODS: Comprehensive quantitative, qualitative and functional analyses of allergen-specific IgA, IgE, IgG1-4 and IgM responses were performed using purified Phl p 1 to 12 allergens in sera, saliva and nasal secretions from 82 grass pollen allergic patients. These patients were enrolled in a randomized, double-blind placebo-controlled study and assessed in an allergen challenge chamber (ClinicalTrials.gov NCT00619827). Antibody responses were monitored in parallel to clinical responses before and after daily sublingual treatment for 4 months with either a grass pollen or a placebo tablet. RESULTS: A significant mean improvement (i.e. 33-40.6%) in rhinoconjunctivitis total symptom scores was observed in SLIT recipients, irrespective of their baseline patterns of IgE sensitization (i.e. narrow, intermediate, broad) to grass pollen allergens. SLIT did not induce any de novo IgE sensitization. Clinical responders encompassed both immunoreactive patients who exhibited strong increases in titres, affinity and/or blocking activity of grass-pollen-specific IgGs (representing 17% of treated patients), as well as patients with no detectable antibody responses distinguishing them from the placebo group. No significant changes were detected in antibody titres in saliva and nasal washes, even in clinical responders. CONCLUSIONS AND CLINICAL RELEVANCE: Sublingual immunotherapy with a grass pollen tablet is efficacious irrespective of the patients' baseline sensitization to either single or multiple grass pollen allergens. Seric IgG responses may contribute to SLIT-induced clinical tolerance in a fraction (i.e. 17%) of patients, but additional immune mechanisms are involved in most patients. Consequently, antibody responses cannot be used as a marker of SLIT efficacy at an individual patient level.

Advances in the quantification of relevant allergens in allergenic extracts.

Relevant allergens are major contributors to the safety and efficacy of allergenic extracts used in allergen immunotherapy (AIT). As such, they should be accurately quantified, as recommended by the 2008 European guidelines on allergen products. Until now, the quantification of relevant allergens was mainly performed by using immunoassays (e.g. ELISA) that relying upon specific antibodies. Although antibody-based quantification is commonly used to assess the concentration of relevant allergens in allergenic extracts, results must be taken with caution in the light of the inherent limitations of such techniques. In the present study, we discuss how those limitations can be overcome by using comprehensive mass spectrometry-based techniques.

Absence of IgE neosensitization in house dust mite allergic patients following sublingual immunotherapy.

BACKGROUND: The impact of sublingual immunotherapy (SLIT) on IgE neosensitization remains to be evaluated in large cohorts of patients. OBJECTIVES: The aim of this study was to assess the dynamics of antibody responses induced in patients with allergic rhinitis during a 12-month treatment with sublingual tablets of house dust mites (HDM) allergen extracts. METHODS: Antibody responses were assessed in relationship with neosensitization and clinical benefit in sera from 509 European house dust mite-allergic patients before and after 1 year of daily sublingual immunotherapy, using tablets containing Dermatophagoides pteronyssinus plus D farinae extracts or placebo (ClinicalTrials.gov NCT00674700). Patients were followed for one additional year after treatment cessation. IgE and IgG4 antibodies specific for mite extracts or purified group 1, 2 and 10 allergens were assessed using Immulite, Immunocap and ISAC assays. RESULTS: After 1 year of SLIT, mite-specific IgE and IgG4 titres increased by 1.5-fold and fourfold, respectively, in the active, but not in the placebo group. A strong IgG4 induction occurred in a subgroup (i.e. 10-15%) of "immunoreactive" patients, without any correlation with improvement in the average adjusted symptom score. Pre-existing IgE levels to purified mite allergens were not impacted during immunotherapy, and no de novo IgE responses to group 1, 2, 10 allergens were induced in patients who were unsensitized prior to immunotherapy. Similarly, no IgE neosensitization to wheat germ or yeast components used in the mite culture medium was observed. CONCLUSIONS AND CLINICAL RELEVANCE: We document in a cohort of 509 patients followed over a 2-year period that SLIT does not induce any IgE neosensitization to allergens contained in the vaccine, such as groups 1, 2 as well as the food-related group 10 allergen. This observation further corroborates the safer safety profile of SLIT over SCIT.

Allergen-specific CD4+ T cell responses in peripheral blood do not predict the early onset of clinical efficacy during grass pollen sublingual immunotherapy.

BACKGROUND: Surrogate biomarkers of efficacy are needed in support of allergen-specific immunotherapy. OBJECTIVE: The aim of this study was to relate changes in peripheral CD4(+) T cell responses to clinical efficacy during sublingual immunotherapy (SLIT). METHODS: Allergen-specific CD4(+) T cell responses were assessed in peripheral blood mononuclear cells (PBMCs) from 89 grass pollen-allergic individuals enrolled in a double-blind placebo-controlled SLIT study conducted in an allergen exposure chamber (ClinicalTrials.gov NCT00619827). Surface phenotype, proliferative responses, cytokine production and gene expression were analysed in coded samples at baseline, and after 2 and 4 months of SLIT, in PBMCs after in vitro allergen stimulation or among MHC class II/peptide (pMHCII)-tetramer-positive CD4(+) T cells. RESULTS: SLIT induced a 29.3% improvement of the average rhinoconjunctivitis total symptom score in the active group, when compared to the placebo group. In parallel, only minor changes in proportions of CD4(+) T cells expressing Th1 (CCR5(+), CXCR3(+)), Th2 (CRTh2(+), CCR4(+)) and Treg (CD25(+), CD127(-), Foxp3(+)) markers were detected. A down-regulation of IL-4 and IL-10 gene expression and IL-10 secretion (P < 0.001) were observed, as well as a decrease in the frequency of potential "pro-allergic" CD27(-) Th2 cells from patients receiving active tablets (P < 0.001), but without any correlation with clinical benefit. pMHCII-tetramer analyses failed to document any major impact in both numbers and polarization of circulating Phl p 1- and Phl p 5-specific CD4(+) T cells, confirming that early clinical improvement during SLIT is not associated with dramatic alterations in T lymphocyte responses. CONCLUSION & CLINICAL RELEVANCE: Changes in patterns of peripheral CD4(+) T cells are not markers for the early onset of efficacy during SLIT.

Sublingual allergen immunotherapy: mode of action and its relationship with the safety profile.

Allergen immunotherapy reorients inappropriate immune responses in allergic patients. Sublingual allergen immunotherapy (SLIT) has been approved, notably in the European Union, as an effective alternative to subcutaneous allergen immunotherapy (SCIT) for allergic rhinitis patients. Compared with SCIT, SLIT has a better safety profile. This is possibly because oral antigen-presenting cells (mostly Langerhans and myeloid dendritic cells) exhibit a tolerogenic phenotype, despite constant exposure to danger signals from food and microbes. This reduces the induction of pro-inflammatory immune responses leading to systemic allergic reactions. Oral tissues contain relatively few mast cells and eosinophils (mostly located in submucosal areas) and, in comparison with subcutaneous tissue, are less likely to give rise to anaphylactic reactions. SLIT-associated immune responses include the induction of circulating, allergen-specific Th1 and regulatory CD4+ T cells, leading to clinical tolerance. Although 40-75% of patients receiving SLIT experience mild, transient local reactions in the oral mucosa, these primary reactions rarely necessitate dose reduction or treatment interruption. We discuss 11 published case reports of anaphylaxis (all nonfatal) diagnosed according to the World Allergy Organization criteria and relate this figure to the approximately 1 billion SLIT doses administered worldwide since 2000. Anaphylaxis risk factors associated with SCIT and/or SLIT should be characterized further.

Evaluation of therapeutic sublingual vaccines in a murine model of chronic house dust mite allergic airway inflammation.

BACKGROUND: Second generation therapeutic vaccines based upon recombinant allergens or natural extracts, potentially formulated in vector systems or adjuvants, are being developed. To this aim, preclinical studies in relevant animal models are needed to select proper allergens, formulations and administration schemes. OBJECTIVE: To develop a chronic house dust mite (HDM) allergy model to evaluate sublingual therapeutic vaccine candidates. METHODS: The BABL/c mice that were used were sensitized with Dermatophagoides pteronyssinus (Dpte) and Dermatophagoides farinae (Dfar) mite extracts by intraperitoneal injections followed by aerosol exposures. Animals subsequently underwent sublingual immunotherapy (SLIT) with either Dpte, Dfar or Dpte/Dfar extracts, twice a week for 8 weeks. SLIT efficacy was assessed by whole body plethysmography, lung histology and broncho-alveolar lavages cell counts. Specific T cell and antibody responses to major and minor HDM allergens were monitored in tissues and serum/saliva, respectively. RESULTS: Mice sensitized to Dpte and Dfar allergens exhibited strong airway hyperresponsiveness (AHR) and lung inflammatory infiltrates including eosinophils. Sensitized animals mounted Th2-biased cellular and humoral responses specific for group 1 and 2 major allergens, as well as group 5, 7 and 10 minor allergens. This phenotype was sustained for at least 2 months, allowing the evaluation of immunotherapeutic protocols with HDM extracts-based vaccines. In this model, SLIT decreased AHR and Th2 responses and induced HDM-specific IgAs in saliva. The Dpte/Dfar mix proved the most efficacious when compared to Dpte or Dfar extracts alone. CONCLUSIONS AND CLINICAL RELEVANCE: The efficacy of a sublingual vaccine based on a Dpte/Dfar allergen extract mix was demonstrated in a well standardized murine model of chronic allergic airway inflammation based on clinically relevant mite allergens. The latter will be used as a benchmark for evaluation of future vaccines, including recombinant allergens. This HDM allergic airway inflammation animal model is a useful tool to design and select candidate vaccines to be tested in humans.

Comparison between major histocompatibility complex class II tetramer staining and surface expression of activation markers for the detection of allergen-specific CD4⁺ T cells.

BACKGROUND: Major histocompatibility complex (MHC) class II tetramers (tetramers) allow to detect allergen-specific CD4(+) T cells at a single-cell level. Limits to this technology include HLA restriction and the need to identify immunodominant T cell epitopes. OBJECTIVE: Assessing the expression of various activation markers following allergen stimulation to replace tetramer staining. METHODS: Peripheral blood mononuclear cells (PBMCs) from 25 birch pollen, grass pollen or house dust mite allergic individuals were stimulated with peptide mixes encompassing immunodominant epitopes from corresponding major allergens. After 2 weeks of in vitro amplification, cells were stained with both the appropriate tetramer and antibodies directed to CD25, CD30, CD39, CD69, CD137, CD154, GITR, HLA-DR and ICOS, before FACS analysis. RESULTS: Following allergen stimulation, percentages of tetramer(+) cells among CD4(+) CD154(+) cells range from 5% to 87%, depending upon donors. As for CD154, a large inter-individual variability is observed in terms of surface expression for all activation markers tested in allergen-stimulated PBMCs. T cells reactive with either tetramers (0.4-10.4% CD4(+) T cells) or anti-marker antibodies (2.2-32.7% CD4(+) T cells), but not both, are observed, reflecting the presence of anergic as well as non-specifically activated cells. Tetramer(+) /marker(+) , tetramer(+) /marker(-) and tetramer(-) /marker(+) cells were compared for their capacity to express cytokines, demonstrating that only the former represent bona fide allergen-specific activated CD4(+) T cells, based upon a higher expression of cytokines or corresponding genes in presence of the allergen. CONCLUSION AND CLINICAL RELEVANCE: No strict correlation exists between tetramer staining and the expression of multiple activation markers in stimulated CD4(+) T cells. Dual staining allows to discriminate functional tetramer(+) /marker(+) vs. anergic (tetramer(+) /marker(-) ) allergen-specific T cells or non-specifically activated (tetramer(-) /marker(+) ) T cells. Combining tetramer staining with the detection of activation markers helps understanding patient heterogeneity regarding specific CD4(+) T cell responses. This approach has immediate relevance for monitoring immune changes induced during specific immunotherapy.

Distinct characteristics of seasonal (Bet v 1) vs. perennial (Der p 1/Der p 2) allergen-specific CD4(+) T cell responses.

BACKGROUND: A better understanding of allergen-specific CD4(+) T cell responses is needed to help improving immunological therapies. Objective To compare CD4(+) T cell responses against seasonal (Bet v 1) and perennial (Der p 1, Der p 2) allergens. METHODS: Major histocompatibility complex class II peptide tetramers were engineered to monitor allergen-specific T cell responses. After in vitro expansion, tetramer(+) cells were tested for surface markers using cytofluorometry. Cytokine gene expression and production were assessed using quantitative PCR and cytokine surface capture assays, respectively. RESULTS: Tetramer(+) cells were detected in 19 patients allergic to house dust mites (HDM), seven allergic to birch pollen, 13 allergic to both and nine non-allergics with either an HLA-DRB1(*) 0101, (*) 0301, (*) 1501 or an HLA-DPB1(*) 0401 background. High-avidity T cells are elicited against the immunodominant Bet v 1(141-155) epitope, whereas broader low-avidity T cell responses are induced against Der p 1(16-30) ,(110-124) ,(171-185) and Der p 2(26-40,107-121) epitopes. Responses against Bet v 1 involve effector (CDL62 low, CCR7 low) or central (CD62L(+) , CCR7(+) ) memory cells in allergic and non-allergic individuals, respectively, whereas central memory cells are mostly detected against mite allergens. In non-allergics, both mite and Bet v 1-specific T cells produce IFN-γ and IL-10. In contrast to Bet v 1-driven Th2 responses, mite allergens induce highly polymorphic responses in allergics, including Th1, Th2/Th17 or mixed Th1/Th2 profiles. Mite-specific T cell frequencies in the blood remain in the range of 1-6 × 10(-4) CD4(+) T cells throughout the year. CONCLUSION: Different memory CD4(+) T cell responses are elicited in the context of chronic vs. seasonal stimulation with the allergen(s). The heterogeneity in the patterns of CD4(+) T cell responses observed in patients allergic to HDMs should be taken into account for specific immunotherapy.

Changes in basophil activation during grass-pollen sublingual immunotherapy do not correlate with clinical efficacy.

BACKGROUND: Biomarkers predicting the safety and efficacy of sublingual immunotherapy (SLIT) remain to be established. METHODS: Eighty-nine patients with allergic rhinoconjunctivitis to grass pollen received either a placebo or five-grass-pollen daily tablet sublingually for 4 months. Following exposure in an allergen challenge chamber, clinical responders and nonresponders were identified individually by evaluating their rhinoconjunctivitis total symptom score (RTSS). Activation of peripheral blood basophils was measured by cytofluorometry before and after 2 or 4 months of immunotherapy, based on CD203c surface expression following allergen stimulation. RESULTS: Patients receiving the grass-pollen tablet had a relative mean improvement of 29.3% vs placebo in the average RTSS after 4 months of SLIT (P < 0.0003). No significant changes in basophil activation were noticed after 2 or 4 months of SLIT despite induction of specific IgGs. Among individual clinical responders, basophil activation was either decreased, increased, or unmodified during SLIT. Levels of basophil activation prior to immunotherapy were not predictive of local adverse reactions associated with immunotherapy. A moderate association was found between basophil activation and allergen-specific IgE levels, skin reactivity, or RTSS, suggesting that the former is, to some extent, indicative of disease severity. As such, patients with the highest level of basophil activation before treatment were more likely to benefit clinically from SLIT. CONCLUSIONS: Allergen reactivity of peripheral blood basophils is not a biomarker for adverse events or early onset of clinical responses to SLIT.

Expression of a functional CD3-Ti antigen/MHC receptor in the absence of surface CD2. Analysis with clonal Jurkat cell mutants.

To investigate the requirement for CD2 expression in activation of T lymphocytes via the CD3-Ti antigen/MHC receptor complex, we produced and characterized a series of CD2- Jurkat variants. These mutants lack detectable surface CD2 as determined by indirect immunofluorescence, immunoprecipitation analysis, and specific radiolabeled antibody binding assay, but nevertheless, expressed normal numbers of CD3-Ti receptors. As expected, the combination of anti-CD2 antibodies, termed anti-T112 and anti-T113, which are mitogenic for resting T lymphocytes, failed to stimulate activation of these variants. In contrast, triggering of their CD3-Ti components resulted in the normal set of T lymphocyte-associated activation events, including phosphoinositide turnover, elevation in intracellular free calcium, early gene-induction events, and IL-2 production. Assuming that the Jurkat cell line is representative of normal cycling human T lymphocytes, we conclude that the presence of the CD2 molecule on the plasma membrane is not in itself a requirement for an operational CD3-Ti-alpha/beta receptor.

Dissection of the human CD2 intracellular domain. Identification of a segment required for signal transduction and interleukin 2 production.

To evaluate those residues in the 117 amino acids of the CD2 cytoplasmic domain required for transduction of T lymphocyte activation signals, a full-length human CD2 cDNA and a series of deletion and substitution mutants were inserted into the ovalbumin-specific, I-Ad-restricted murine T cell hybridoma 3DO54.8 using a retroviral system. The resulting cells express surface CD2 protein and unlike the parental murine line, are reactive with murine anti-human CD2 antibodies. Anti-T11(2) plus anti-T11(3) antibody stimulation of cells expressing a full-length CD2 cDNA results in a characteristic rise in cytosolic-free calcium [( Ca2+]i), and subsequent IL-2 secretion that accompany CD2 stimulation in human T lymphocytes. Transfectants expressing CD2 delta C98 and CD2 delta C77, partially deleted CD2 molecules containing the entire extracellular and transmembrane CD2 segments but only 98 and 77 amino acids of the cytoplasmic domain, respectively, are also activated by anti-CD2 mAbs. In contrast, clones expressing more severely truncated CD2 structures, CD2 delta C43 and CD2 delta C18, are not stimulated. These data show that the cytoplasmic domain plays an essential role in transduction of activation signals via CD2, and that the segment between amino acid residues 253 and 278 is necessary for activation. This region contains two tandem repeats of the sequence PPPGHR, thought to form part of a putative cationic site. Disruption of the latter by site-directed mutagenesis does not affect IL-2 gene induction, suggesting that only one of the repeats is required for activating this function of the CD2 molecule.

Involvement of the PPPGHR motif in T cell activation via CD2.

Prior studies identified a segment of the CD2 cytoplasmic domain between amino acid (aa) residues 253 and 287 as important in T lymphocyte signal transduction. This region contains two repeats of the sequence motif PPPGHR, thought to form a "cage" structure involved in CD2-mediated signaling. To evaluate this segment, a series of mutant human CD2 molecules were produced by oligonucleotide-directed mutagenesis and inserted into the ovalbumin-specific, I-Ad-restricted murine T-T hybridoma 3DO54.8 using the DOL retroviral system. CD2 M1 (271-272), CD2 M2 (278-279), and CD2 M4 (264-265) mutants replaced the positively charged adjacent aa histidine and arginine (HR) in the wild-type CD2 sequence with aspartic and glutamic acid (DE) at positions 271-272, 278-279, and 264-265, respectively. In addition, a truncation mutant, CD2 M3 (268), containing only 57 of the 117 cytoplasmic aa and terminating before the second PPPGHR sequence, was generated. Stimulation of transfectants CD2 FL, CD2 M1 (271-272), and CD2 M2 (278-279) with anti-T11(2) + anti-T11(3) antibodies resulted in a rise in cytosolic-free calcium [( Ca2+]i) and subsequent interleukin 2 (IL-2) secretion. In contrast, CD2 M4 (264-265) transfectants could not be activated in either assay. Thus, alteration of histidine 264 and/or arginine 265 within the first PPPGHR motif affects the process of signal transduction via CD2, whereas identical mutations in residues at 271-272 or 278-279 were individually without effect. Consistent with these data, CD2 M3 (268) transfectants were able to generate a detectable amount of IL-2 via CD2 triggering. These data support the notion that the PPPGHR motif at aa 260-265 is important for activation of T lymphocytes via the CD2 molecule.