Simultaneous Genetic Ablation of PD-1, LAG-3, and TIM-3 in CD8 T Cells Delays Tumor Growth and Improves Survival Outcome.

Immune checkpoint inhibitors (ICI) represented a step forward in improving the outcome of patients with various refractory solid tumors and several therapeutic regimens incorporating ICI have already been approved for a variety of tumor entities. However, besides remarkable long-term responses, checkpoint inhibition can trigger severe immune-related adverse events in some patients. In order to improve safety of ICI as well as T cell therapy, we tested the feasibility of combining T cell-based immunotherapy with genetic disruption of checkpoint molecule expression. Therefore, we generated H-Y and ovalbumin antigen-specific CD8+ T cells with abolished PD-1, LAG-3, and TIM-3 expression through CRISPR/Cas9 technology. CD8+ T cells, subjected to PD-1, LAG-3, and TIM-3 genetic editing, showed a strong reduction in immune checkpoint molecule expression after in vitro activation, while no relevant reduction in responsiveness to in vitro stimulation was observed. At the same time, in B16-OVA tumor model, transferred genetically edited OT-1 CD8+ T cells promoted longer survival compared to control T cells and showed enhanced expansion without associated toxicity. Our study supports the notion that antigen-specific adoptive T cell therapy with concomitant genetic disruption of multiple checkpoint inhibitory receptors could represent an effective antitumor immunotherapy approach with improved tolerability profile.

Characterization of the effects on pruritus by novel treatments for atopic dermatitis.

Chronic pruritus is a common and debilitating symptom in patients with atopic dermatitis and contributes to impairment of quality of life. Effective treatment of pruritus should therefore be one of the main treatment goals in patients with atopic dermatitis. Pathophysiologically, the histamine-independent pruritogens interleukin-31, interleukin-13, and interleukin-4, have been shown to play a major role in atopic dermatitis. All three cytokines can mediate chronic pruritus via Janus kinase 1/2 signaling pathways. Novel drugs target these pathways and have shown rapid and sustained reduction of pruritus in patients with atopic dermatitis in clinical use and in phase II and III clinical trials. Here we summarize the published data on the effects of these drugs on itch parameters such as overall reduction in pruritus intensity and percent of patients with atopic dermatitis achieving a relevant reduction in itch. Each of the novel drugs shows very good effects on pruritus. These data offer hope for an even better and possibly more specific treatment of pruritus in patients with atopic dermatitis in the future. In addition, the different pharmacological approaches give us the chance to learn more about the pathophysiology of pruritus in atopic dermatitis.

Spectrum of Genetic Autoinflammatory Diseases Presenting with Cutaneous Symptoms.

Autoinflammatory diseases comprise a group of chronic disabling entities characterized by inflammation without the presence of infectious agents, auto-antibodies or antigen-specific T-cells. Many autoinflammatory diseases are caused by monogenic defects, which lead to disturbed immune signalling with release of proinflammatory mediators. In addition to interleukin-1ß and interleukin-18, interferons play a key role in the pathophysiology of these disorders. Patients with autoinflammatory diseases show a broad variety of clinical symptoms, including skin involvement. Wheals, pustules and ulcerative lesions are the most common cutaneous findings observed. Knowledge of the clinical presentation of autoinflammatory diseases is crucial for establishing the diagnosis and guiding appropriate treatment. This review focuses on the dermatological findings in selected autoinflammatory disorders based on their distinct pathomechanisms.

Inhibition of interleukin-1 with rilonacept is not effective in cold urticaria-Results of a randomized, placebo-controlled study.

BACKGROUND: Cold urticaria (ColdU) is characterized by pruritic wheals following exposure of the skin to cold. Many patients show insufficient response to antihistamines, the first line treatment. Based on the high efficacy of interleukin-1(IL-1)-inhibition in cold-induced urticarial autoinflammatory diseases, we assessed the effects of rilonacept, an IL-1 inhibitor, in ColdU patients unresponsive to standard treatment. METHODS: In this randomized, double-blind, placebo-controlled two-center study, we included 20 patients with ColdU. In the first part, patients received 320 mg rilonacept or placebo (1:1) followed by weekly doses of 160 mg rilonacept or placebo for 6 weeks. In the second part, all patients received weekly 160 mg or 320 mg rilonacept for 6 weeks, open-label. The primary endpoint was change in critical temperature threshold (CTT). Secondary endpoints included changes in quality of life impairment (Dermatology Life Quality Index, DLQI), differences of inflammatory mediators upon cold provocation and safety assessment over the study period. RESULTS: Baseline mean CTTs were 20.2°C (placebo) and 17.3°C (rilonacept). Mean CTTs did not change significantly during the 6-week double-blind treatment (placebo - 0.45°C; rilonacept +0.89°C). IL-6, IL-18 and HSP-70 blood levels showed interindividual variability without significant changes during hand cold water bath provocation in placebo- or rilonacept-treated patients. In contrast, DLQI significantly improved in the rilonacept (mean DLQI reduction of 3.8; p = 0.002) but not in the placebo group (mean DLQI reduction of 0). Comparing baseline with the rilonacept open-label treatment, there were no changes in CTTs or DLQI scores. CONCLUSION: IL-1 inhibition with rilonacept did not improve ColdU, but demonstrated a good safety profile. CLINICAL TRIAL REGISTRATION: EudraCT number: 2012-005726-30. CLINICALTRIALS: gov identifier: NCT02171416.

Soluble IgE-binding factors in the serum of food-allergic patients: Possible pathophysiological role of soluble FcεRI as protective factor.

BACKGROUND: IgE-mediated food allergy is the result of an aberrant immune response involving the interaction of a food allergen with its specific IgE bound to FcɛRI, the high affinity IgE receptor, on mast cells. Allergen-specific IgE also binds to soluble binding factors, but, their expression and role in food allergy is not well characterized. Here, we assess the prevalence and relevance of soluble IgE binding factors in food allergy and tolerance. METHODS: We measured serum levels of four IgE binding factors, that is, galectin-3, galectin-9, soluble FcɛRI (sFcεRI) and soluble CD23 (sCD23) in 67 adults sensitized to peanut or hazelnut and sFcɛRI in 29 children sensitized to hen's egg. Adults without food allergen sensitization (n = 17) served as healthy controls. We compared serum levels of patients and controls and assessed them, in the former, for links to clinical features including allergy and tolerance. RESULTS: Serum levels of sFcɛRI and sCD23, but not galectin-3 and galectin-9, significantly differ in food-sensitized patients as compared to healthy controls. A subgroup (28%) of peanut and hazelnut allergic patients had elevated sFcεRI levels, that were associated with higher total and specific IgE levels. Furthermore, sFcεRI levels were significantly higher in tolerant subjects compared to allergics. Among hazelnut allergic patients, those with high sFcεRI levels tolerated the highest protein amounts in the oral food challenge. CONCLUSION: sFcɛRI but not sCD23, galectin-3 and galectin-9 might play a role in the pathophysiology of food allergy. Its functional role or use as biomarker should be assessed in further studies.

In Urticarial Vasculitis, Long Disease Duration, High Symptom Burden, and High Need for Therapy Are Linked to Low Patient-Reported Quality of Life.

BACKGROUND: Urticarial vasculitis (UV) is a rare and difficult-to-treat chronic skin disease defined by long-lasting urticarial lesions and the histopathologic finding of leukocytoclastic vasculitis. As of yet, little is known about UV patients' perspective on the disease. OBJECTIVE: To assess UV patients' perspective on the clinical course, treatment response, greatest challenges, and quality-of-life (QOL) impairment. METHODS: A web-based questionnaire was disseminated in a Facebook group of patients with UV. Patients with UV confirmed by skin biopsy were included. RESULTS: Patients with UV had a mean age of 47.3 ± 12.3 years and were mostly female (94.3%; n = 82 of 87). The median delay in diagnosis was 8.1 months (interquartile range, 2.0-46.3). Normocomplementemia and hypocomplementemia were present in 54.0% (n = 27) and 46.0% (n = 23) of 50 patients, respectively. Most patients with UV (51.8%; n = 43 of 83) reported severely decreased QOL due to their disease. Low QOL was also the most frequently reported greatest challenge for patients with UV (40.7%), followed by the long-standing course of UV with frequent relapses (14.8%). Low QOL correlated with long disease duration (r = 0.298; P = .02) and high numbers of clinical symptoms (r = 0.294; P = .007). Patients with UV with allergies, lung diseases, and chronic infections reported lower QOL. Patients with UV with low QOL were treated with analgesics, dapsone, montelukast, omalizumab, and colchicine more often than patients with UV with higher QOL (P < .05 for all). CONCLUSIONS: Our results show a considerable impairment in QOL in patients with UV associated with long disease duration, high symptom burden, and a high need for therapy. Improvement of the management of UV by further research is necessary.

Generation of murine monoclonal antibodies with specificity against conventional camelid IgG1 and heavy-chain only IgG2/3.

Camelids possess antibodies with a conventional four-chain structure consisting of two heavy and two light chains (of subclass IgG1) but further they also generate heavy-chain only antibodies (of subclass IgG2 and 3) which are fully functional in antigen binding. In this study subclass-specific murine monoclonal antibodies specific to conventional camelid IgG1 and heavy-chain only IgG2/3 were generated and validated for the use as potent secondary detection reagents. The monoclonal antibodies are able to differentiate between all camelid IgGs, conventional four-chain camelid antibodies (of subclass IgG1) and exclusively heavy chain-only antibodies (of subclasses IgG2 and IgG3). Further these antibodies were used to detect specific immune responses after vaccination of Camelids against bovine corona- and rotavirus strains and different E.coli and Clostridia - antigens and to identify Erysipelothrix rhusiopathiae infected animals within a herd. The described antibodies are suitable as new secondary agents for the detection of different camelid subclasses and the validation of camelid immune reactions.

Generation and validation of murine monoclonal and camelid recombinant single domain antibodies specific for human pancreatic glycoprotein 2.

Pancreatic secretory zymogen-granule membrane glycoprotein 2 (GP2) has been identified as a major autoantigenic target in Crohn's disease patients. It was reported recently that a long (GP2a) and a short (GP2b) isoform of GP2 exist and that in the outcome of inflammatory bowel diseases (IBD) GP2-specific autoantibodies probably appear as new serological markers for diagnosis and therapeutic monitoring. To investigate this further and in order to establish diagnostic tools for the discrimination of both GP2 isoforms, a set of different murine monoclonal and camelid recombinant single domain antibodies (camelid VHH) was generated and validated in various enzyme-linked immunosorbent assay (ELISA) formats, immunofluorescence on transgenic cell lines and immunohistochemistry on monkey pancreas tissue sections. Out of six binders identified, one was validated as highly specific for GP2a. This murine monoclonal antibody (mAb) was used as capture antibody in construction of a sandwich ELISA for the detection of GP2a. Camelid VHHs or a second murine mAb served as detection antibodies in this system. All antibodies were also able to stain GP2a or GP2b on transgenic cell lines as well as on pancreatic tissue in immunohistochemistry. The KD values measured for the camelid VHHs were between 7 nM and 23pM. This set of specific binders will enable the development of suitable diagnostic tools for GP2-related studies in IBD.