Safety and efficacy of amlitelimab, a fully human nondepleting, noncytotoxic anti-OX40 ligand monoclonal antibody, in atopic dermatitis: results of a phase IIa randomized placebo-controlled trial.

BACKGROUND: Atopic dermatitis (AD) is an inflammatory skin disease with significant unmet need. Blockade of the OX40-OX40 ligand (OX40L) costimulation pathway by targeting OX40L on antigen-presenting cells (APCs) with a fully human noncytotoxic, nondepleting anti-OX40L monoclonal antibody (amlitelimab; SAR445229; KY1005) is a novel way to modulate persistent inflammation. OBJECTIVES: To assess the safety and efficacy of amlitelimab over 16 weeks in adults with AD in a phase IIa double-blind placebo-controlled study. METHODS: The study was conducted at 19 hospitals in Germany, Poland, Spain and the UK. Eligible patients with moderate-to-severe AD were randomized (1 : 1 : 1) to low-dose intravenous (IV) amlitelimab (200 mg), high-dose IV amlitelimab (500 mg) or placebo, followed by three maintenance doses (50% of loading dose) at 4, 8 and 12 weeks, with safety follow-up to week 36. The co-primary endpoints were the incidence of treatment-emergent adverse events (all patients who received ≥ 1 dose of the study drug) and mean percentage change in Eczema Area and Severity Index (EASI) to week 16 (full analysis set). RESULTS: Between 13 December 2018 and 12 May 2020, 89 patients were randomly assigned to low- (n = 29) or high-dose amlitelimab (n = 30) or placebo (n = 29), of whom 88 proceeded to treatment [37 women (42%), 51 (58%) men; mean (SD) age 33.6 (11.9) years]. Amlitelimab was generally well tolerated with an unremarkable safety profile; no hypersensitivity events were reported. For the primary endpoint, the least square mean percentage change in EASI from baseline to week 16 was -80.12% [95% confidence interval (CI) -95.55 to -64.68; P = 0.009 vs. placebo] and -69.97% (95% CI -85.04 to -54.60; P = 0.07 vs. placebo) for the low- (n = 27) and high-dose (n = 27) amlitelimab groups, respectively, vs. -49.37% (95% CI -66.02 to -32.72) for placebo (n = 24). Numerically greater reductions in EASI were observed for amlitelimab vs. placebo from weeks 2 to 16. CONCLUSIONS: Novel targeting of OX40L-expressing APCs with amlitelimab was well tolerated and resulted in clinically meaningful improvements in AD.

ICOS-Positive Regulatory T Cells in Hepatocellular Carcinoma: The Perspective from Digital Pathology Analysis.

INTRODUCTION: Inducible co-stimulator (ICOS), an important co-stimulatory receptor on effector T cells (Teffs), may also contribute to tumor growth due to its high expression on regulatory T cells (Tregs). This study explored the clinical significance of ICOS-expressing Tregs in hepatocellular carcinoma (HCC). METHODS: Tumor tissues from HCC patients who received curative hepatectomy were obtained at a referral center. Dual immunohistochemistry was performed to evaluate the expression of ICOS and Foxp3. The cell densities and proximities between stained cells in regions of interest were measured by digital pathology and the associations with clinical outcome were analyzed. RESULTS: A total of 142 patients (male:female = 112: 30, median age of 61.0 years) were enrolled. Among them, 87 (61.3%) had chronic hepatitis B virus infection and 33 (23.2%) had chronic hepatitis C infection. Low α-fetoprotein level (<20 ng/mL) and early-stage were significantly associated with improved overall survival (OS). The density of ICOS+Foxp3+ cells and the ratio of ICOS+Foxp3+/total Foxp3+ cells were significantly higher (p < 0.001) in the tumor center than in the peritumor area. Patients with a high density of ICOS+Foxp3+ cells or a high ratio of ICOS+Foxp3+/total Foxp3+ cells in the tumor center trended to have a shorter OS. A shorter distance between ICOS+Foxp3+ cells and ICOS+Foxp3- cells (likely Teffs) in the tumor center was significantly associated with a shorter OS (p = 0.030), suggesting active immunosuppression of ICOS+ Tregs on ICOS+ Teffs. CONCLUSION: An increased abundance of ICOS+ Tregs in the tumor center in comparison to the peritumor area indicates a strong immunosuppressive tumor microenvironment of HCC. A high proportion of ICOS+Foxp3+ cells and a shorter distance between ICOS+ Tregs and other ICOS+ cells were associated with a poor OS, suggesting that depleting ICOS+ Tregs might provide clinical benefit for patients with HCC.

Activation-induced cell death of self-reactive regulatory T cells drives autoimmunity.

Activation of self-reactive T cells is a major driver to autoimmunity and is suppressed by mechanisms of regulation. In a humanized model of autoimmune thyroiditis, we investigated the mechanism underlying break of tolerance. Here, we found that a human TCR specific for the self-antigen thyroid peroxidase (TPO) is positively selected in the thymus of RAG KO mice on both T effector (Teff) and T regulatory (Treg) CD4+Foxp3+ cells. In vivo Teff are present in all immune organs, whereas the TPO-specific Treg are present in all lymphoid organs with the exception of the thyroid-draining lymph nodes. We suggest that the presence of TPO in the thyroid draining lymph nodes induces the activation of Teff and the depletion of Treg via activation-induced cell death (AICD). Our findings provide insights on the failure of the mechanisms of immune tolerance, with potential implications in designing immunotherapeutic strategies.

New Approaches in Immunotherapy for the Treatment of Lung Cancer.

Despite the several advances in the last few years into treatment of advanced lung cancer, the 5-year survival remains extremely low. New therapeutic strategies are currently under investigation, and immunotherapy seems to offer a promising treatment alternative. In the last decade, therapeutic cancer vaccines in lung cancer have been rather disappointing, mainly due to the lack of efficient predictive biomarkers. A better refinement of the patient population that might respond to treatment might finally lead to a success story. For the first time, the immune checkpoint inhibitors are demonstrating sustained antitumor response and improved survival and they may be the first immunotherapeutics available for patients with lung cancer.

NHS-IL2 combined with radiotherapy: preclinical rationale and phase Ib trial results in metastatic non-small cell lung cancer following first-line chemotherapy.

BACKGROUND: NHS-IL2 (selectikine, EMD 521873, MSB0010445) consists of human NHS76 (antibody specific for necrotic DNA) fused to genetically modified human interleukin 2 (IL-2) and selectively activates the high-affinity IL-2 receptor. Based on an evolving investigational concept to prime the tumor microenvironment with ionizing radiation prior to initiating immunotherapy, 2 related studies were conducted and are reported here. The first, a preclinical study, tests the systemic effect of the immunocytokine NHS-IL2 and radiotherapy in a lung carcinoma animal model; the second, a phase Ib trial in patients with metastatic non-small cell lung carcinoma (NSCLC), was designed to determine the safety and tolerability of NHS-IL2 in combination with radiotherapy directly following first-line palliative chemotherapy. METHODS: Tumor-bearing C57Bl/6 mice were treated with NHS-IL2 alone (5 mg/kg; days 7-9), fractionated radiotherapy (3.6 Gy; days 0-4) plus cisplatin (4 mg/kg; day 0), or the triple combination. Metastatic NSCLC patients who achieved disease control with first-line palliative chemotherapy were enrolled in the phase Ib trial. Patients received local irradiation (5x 4 Gy) of a single pulmonary nodule. Dose-escalated NHS-IL2 was administered as 1-h intravenous infusion on 3 consecutive days every 3 weeks. RESULTS: NHS-IL2 plus radiotherapy induced immune response activation and complete tumor growth regressions in 80%-100% of mice. In patients with metastatic NSCLC treated with NHS-IL2 (3, 3, and 7 patients in the 0.15-mg/kg, 0.30-mg/kg, and 0.45-mg/kg cohorts, respectively), maximum tolerated dose was not reached. Most frequently reported adverse events were fatigue, anorexia, and rash. Transient increases in leukocyte subsets were observed. In 3 patients, thyroid gland dysfunction occurred. No objective responses were reported; long-term survival was observed in 2 patients, including 1 patient with long-term tumor control. CONCLUSIONS: Combining NHS-IL2 with radiotherapy achieved synergistic antitumor activity in preclinical studies, supporting the use in lung cancer patients. This combination was well tolerated and 2 of 13 patients achieved long-term survival. TRIAL REGISTRATION: ClinicalTrials.gov NCT00879866.

T-cell activation by treatment of cancer patients with EMD 521873 (Selectikine), an IL-2/anti-DNA fusion protein.

BACKGROUND: EMD 521873 (Selectikine or NHS-IL2LT) is a fusion protein consisting of modified human IL-2 which binds specifically to the high-affinity IL-2 receptor, and an antibody specific for both single- and double-stranded DNA, designed to facilitate the enrichment of IL-2 in tumor tissue. METHODS: An extensive analysis of pharmacodynamic (PD) markers associated with target modulation was assessed during a first-in-human phase I dose-escalation trial of Selectikine. RESULTS: Thirty-nine patients with metastatic or locally advanced tumors refractory to standard treatments were treated with increasing doses of Selectikine, and nine further patients received additional cyclophosphamide. PD analysis, assessed during the first two treatment cycles, revealed strong activation of both CD4+ and CD8+ T-cells and only weak NK cell activation. No dose response was observed. As expected, Treg cells responded actively to Selectikine but remained at lower frequency than effector CD4+ T-cells. Interestingly, patient survival correlated positively with both high lymphocyte counts and low levels of activated CD8+ T-cells at baseline, the latter of which was associated with enhanced T-cell responses to the treatment. CONCLUSIONS: The results confirm the selectivity of Selectikine with predominant T-cell and low NK cell activation, supporting follow-up studies assessing the clinical efficacy of Selectikine for cancer patients.

OX40L Inhibition Suppresses KLH-driven Immune Responses in Healthy Volunteers: A Randomized Controlled Trial Demonstrating Proof-of-Pharmacology for KY1005.

The safety, tolerability, immunogenicity, and pharmacokinetic (PK) profile of an anti-OX40L monoclonal antibody (KY1005, currently amlitelimab) were evaluated. Pharmacodynamic (PD) effects were explored using keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT) immunizations. Sixty-four healthy male subjects (26.5 ± 6.0 years) were randomized to single doses of 0.006, 0.018, or 0.05 mg/kg, or multiple doses of 0.15, 0.45, 1.35, 4, or 12 mg/kg KY1005, or placebo (6:2). Serum KY1005 concentrations were measured. Antibody responses upon KLH and TT immunizations and skin response upon intradermal KLH administration were performed. PD data were analyzed using repeated measures analysis of covariances (ANCOVAs) and post hoc exposure-response modeling. No serious adverse events occurred and all adverse events were temporary and of mild or moderate severity. A nonlinear increase in mean serum KY1005 concentrations was observed (median time to maximum concentration (Tmax ) ~ 4 hours, geometric mean terminal half-life (t½) ~ 24 days). Cutaneous blood perfusion (estimated difference (ED) -13.4 arbitrary unit (AU), 95% confidence interval (CI) -23.0 AU to -3.8 AU) and erythema quantified as average redness (ED -0.23 AU, 95% CI -0.35 AU to -0.11 AU) decreased after KY1005 treatment at doses of 0.45 mg/kg and above. Exposure-response analysis displayed a statistically significant treatment effect on anti-KLH antibody titers (IgG maximum effect (Emax ) -0.58 AU, 95% CI -1.10 AU to -0.06 AU) and skin response (erythema Emax -0.20 AU, 95% CI -0.29 AU to -0.11 AU). Administration of KY1005 demonstrated an acceptable safety and tolerability profile and PK analyses displayed a nonlinear profile of KY1005. Despite the observed variability, skin challenge response after KY1005 treatment indicated pharmacological activity of KY1005. Therefore, KY1005 shows potential as a novel pharmacological treatment in immune-mediated disorders.

SUMOylation of tissue transglutaminase as link between oxidative stress and inflammation.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CF is characterized by chronic bacterial lung infections and inflammation, and we have previously reported that tissue transglutaminase (TG2), a multifunctional enzyme critical to several diseases, is constitutively up-regulated in CF airways and drives chronic inflammation. Here, we demonstrate that the generation of an oxidative stress induced by CFTR-defective function leads to protein inhibitor of activated STAT (PIAS)y-mediated TG2 SUMOylation and inhibits TG2 ubiquitination and proteasome degradation, leading to sustained TG2 activation. This prevents peroxisome proliferator-activated receptor (PPAR)gamma and IkBalpha SUMOylation, leading to NF-kappaB activation and to an uncontrolled inflammatory response. Cellular homeostasis can be restored by small ubiquitin-like modifier (SUMO)-1 or PIASy gene silencing, which induce TG2 ubiquitination and proteasome degradation, restore PPARgamma SUMOylation, and prevent IkBalpha cross-linking and degradation, thus switching off inflammation. Manganese superoxide dismutase overexpression as well as the treatment with the synthetic superoxide dismutase mimetic EUK-134 control PIASy-TG2 interaction and TG2 SUMOylation. TG2 inhibition switches off inflammation in vitro as well as in vivo in a homozygous F508del-CFTR mouse model. Thus, TG2 may function as a link between oxidative stress and inflammation by driving the decision as to whether a protein should undergo SUMO-mediated regulation or degradation. Targeting TG2-SUMO interactions might represent a new option to control disease evolution in CF patients as well as in other chronic inflammatory diseases, neurodegenerative pathologies, and cancer.

Degenerate self-reactive human T-cell receptor causes spontaneous autoimmune disease in mice.

Thyroid autoimmune disorders comprise more than 30% of all organ-specific autoimmune diseases and are characterized by autoantibodies and infiltrating T cells. The pathologic role of infiltrating T cells is not well defined. To address this issue, we generated transgenic mice expressing a human T-cell receptor derived from the thyroid-infiltrating T cell of a patient with thyroiditis and specific for a cryptic thyroid-peroxidase epitope. Here we show that mouse major histocompatibility complex molecules sustain selection and activation of the transgenic T cells, as coexpression of histocompatibility leukocyte antigen molecules was not needed. Furthermore, the transgenic T cells had an activated phenotype in vivo, and mice spontaneously developed destructive thyroiditis with histological, clinical and hormonal signs comparable with human autoimmune hypothyroidism. These results highlight the pathogenic role of human T cells specific for cryptic self epitopes. This new 'humanized' model will provide a unique tool to investigate how human pathogenic self-reactive T cells initiate autoimmune diseases and to determine how autoimmunity can be modulated in vivo.

Lysosomal accumulation of gliadin p31-43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARgamma downregulation in intestinal epithelial cells and coeliac mucosa.

BACKGROUND: An unresolved question in coeliac disease is to understand how some toxic gliadin peptides, in particular p31-43, can initiate an innate response and lead to tissue transglutaminase (TG2) upregulation in coeliac intestine and gliadin sensitive epithelial cell lines. Aim We addressed whether the epithelial uptake of p31-43 induces an intracellular pro-oxidative envoronment favouring TG2 activation and leading to the innate immune response. METHODS: The time course of intracellular delivery to lysosomes of p31-43, palpha-2 or palpha-9 gliadin peptides was analysed in T84 and Caco-2 epithelial cells. The effects of peptide challenge on oxidative stress, TG2 and peroxisome proliferator-activated receptor (PPAR)gamma ubiquitination and p42/44-mitogen activated protein (MAP) kinase or tyrosine phosphorylation were investigated in cell lines and cultured coeliac disease biopsies with/without anti-oxidant treatment or TG2 gene silencing by immunoprecipitation, western blot, confocal microscopy and Fluorenscence Transfer Resonance Energy (FRET) analysis. RESULTS: After 24 h of challenge p31-43, but not palpha-2 or palpha-9, is still retained within LAMP1-positive perinuclear vesicles and leads to increased levels of reactive oxygen species (ROS) that inhibit TG2 ubiquitination and lead to increases of TG2 protein levels and activation. TG2 induces cross-linking, ubiquitination and proteasome degradation of PPARgamma. Treatment with the antioxidant EUK-134 as well as TG2 gene silencing restored PPARgamma levels and reversed all monitored signs of innate activation, as indicated by the dramatic reduction of tyrosine and p42/p44 phosphorylation. CONCLUSION: p31-43 accumulation in lysosomes leads to epithelial activation via the ROS-TG2 axis. TG2 works as a rheostat of ubiquitination and proteasome degradation and drives inflammation via PPARgamma downregulation.

An Antibody Targeting ICOS Increases Intratumoral Cytotoxic to Regulatory T-cell Ratio and Induces Tumor Regression.

The immunosuppressive tumor microenvironment constitutes a significant hurdle to immune checkpoint inhibitor responses. Both soluble factors and specialized immune cells, such as regulatory T cells (Treg), are key components of active intratumoral immunosuppression. Inducible costimulatory receptor (ICOS) can be highly expressed in the tumor microenvironment, especially on immunosuppressive Treg, suggesting that it represents a relevant target for preferential depletion of these cells. Here, we performed immune profiling of samples from tumor-bearing mice and patients with cancer to demonstrate differential expression of ICOS in immune T-cell subsets in different tissues. ICOS expression was higher on intratumoral Treg than on effector CD8 T cells. In addition, by immunizing an Icos knockout transgenic mouse line expressing antibodies with human variable domains, we selected a fully human IgG1 antibody called KY1044 that bound ICOS from different species. We showed that KY1044 induced sustained depletion of ICOShigh T cells but was also associated with increased secretion of proinflammatory cytokines from ICOSlow effector T cells (Teff). In syngeneic mouse tumor models, KY1044 depleted ICOShigh Treg and increased the intratumoral TEff:Treg ratio, resulting in increased secretion of IFNγ and TNFα by TEff cells. KY1044 demonstrated monotherapy antitumor efficacy and improved anti-PD-L1 efficacy. In summary, we demonstrated that using KY1044, one can exploit the differential expression of ICOS on T-cell subtypes to improve the intratumoral immune contexture and restore an antitumor immune response.

Anti tumour necrosis-alpha therapy increases the number of FOXP3 regulatory T cells in children affected by Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Its pathogenesis is not completely understood, though the prevailing model is that the intestinal flora drives a strong intestinal T helper 1 (Th1)/Th17 type immune response and inflammation in the genetically susceptible host. This leads to overly aggressive T-cell responses to normal bacteria causing tissue damage. Intestinal homeostasis and maintenance of tolerance to harmless antigens in the intestine has been shown to be maintained by CD4+ CD25+ T regulatory cells (Treg) in animal models of inflammatory bowel diseases. Here we investigated whether Infliximab, a chimeric monoclonal antibody directed against tumour necrosis factor (TNF)-alpha shown to be highly effective in the treatment of CD, has any effect on mucosal CD4+ CD25+ (FOXP3+) Tregs. Colonic mucosal biopsies from children with active Crohn's disease treated in vivo with Infliximab and controls were analysed to determine FOXP3 expression by immunofluorescence and reverse transcription-polymerase chain reaction. We observed that FOXP3+ T cells were significantly reduced in mucosa of CD patients with active disease compared with controls and restored to normal following Infliximab treatment. The frequency of FOXP3+ cells and mRNA expression was significantly increased in CD mucosa from patients treated in vivo with Infliximab compared with CD patients treated with conventional therapies. In conclusion, we show that Infliximab treatment does not solely neutralize soluble TNF-alpha, but also affects activation and possibly expansion of mucosal regulatory T cells. We suggest that anti TNF-alpha immunotherapy can also restore mucosal homeostasis in Crohn's disease.

Prospects for combining targeted and conventional cancer therapy with immunotherapy.

Over the past 25 years, research in cancer therapeutics has largely focused on two distinct lines of enquiry. In one approach, efforts to understand the underlying cell-autonomous, genetic drivers of tumorigenesis have led to the development of clinically important targeted agents that result in profound, but often not durable, tumour responses in genetically defined patient populations. In the second parallel approach, exploration of the mechanisms of protective tumour immunity has provided several therapeutic strategies - most notably the 'immune checkpoint' antibodies that reverse the negative regulators of T cell function - that accomplish durable clinical responses in subsets of patients with various tumour types. The integration of these potentially complementary research fields provides new opportunities to improve cancer treatments. Targeted and immune-based therapies have already transformed the standard-of-care for several malignancies. However, additional insights into the effects of targeted therapies, along with conventional chemotherapy and radiation therapy, on the induction of antitumour immunity will help to advance the design of combination strategies that increase the rate of complete and durable clinical response in patients.

Gliadin as a stimulator of innate responses in celiac disease.

In celiac disease (CD) we have the prototype of an immune mediated response dominated by the activation of the adaptive immune system and in particular of CD4+ HLA class II restricted T cells. Various seminal studies have established the precise mechanism of how antigen (prolamine) specific activation of CD4+ mucosal T cells occurs. Thus, CD is a condition in which T cells and their activation is the essential hinge in the pathogenic process. These functional studies have provided the explanation for the genetic association between CD and certain HLA alleles (HLA DQ2 and DQ8). These genetic, molecular and functional studies have permitted the clarification of a powerful Th1 dominated pro-inflammatory response that characterises the small intestine of active CD patients. Despite this unassailable set of information and reports there are some intriguing points that have been raised by a series of studies which have indicated that CD is not only defined by an aberrant prolamine-induced activation of the adaptive immune system. New evidence and re-assessments of old studies, point to a more complex pathogenic cascade, which may help to unravel some of the residual obscure points of CD pathogenesis. Here, we outline the current concepts that indicate a direct involvement of the adaptive immune system and we discuss all the evidence supporting a direct activation of the innate immune system by fragments of prolamines, which are not recognized T cell epitopes and how they could influence CD. The gliadin-induced activation of the 'innate' immune system might also have a significant role in the induction and persistence of many CD complications and most definitively for the most aggressive one, namely mucosal T cell lymphomas. We further suggest a novel way to harness the unwanted immune response to toxic prolamine, and thus indicate new potential therapeutic strategies to treat or at least control CD.

Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease.

BACKGROUND: The adaptive immune system is central to the development of coeliac disease. Adaptive immune responses are, however, controlled by a preceding activation of the innate immune system. We investigated whether gliadin, a protein present in wheat flour, could activate an innate as well as an adaptive immune response in patients with coeliac disease. METHODS: Duodenal biopsy samples from 42 patients with untreated coeliac disease, 37 treated patients, and 18 controls, were cultured in vitro for 3 h or 24 h, in the presence of either immunodominant gliadin epitopes (p(alpha)-2 and p(alpha)-9) or a non-immunodominant peptide (p31-43) known to induce small intestine damage in coeliac disease. We also incubated biopsy samples from nine untreated and six treated patients with a non-immunodominant peptide for 3 h, before incubation with immunodominant gliadin epitopes. Different combinations of interleukin-15 or signal transduction inhibitors were added to selected incubations. FINDINGS: Only the non-immunodominant peptide induced rapid expression of interleukin-15, CD83, cyclo-oxygenase (COX)-2, and CD25 by CD3- cells (p=0.005 vs medium alone) and enterocyte apoptosis (p<0.0001). Only the non-immunodominant peptide induced p38 MAP kinase activation in CD3- cells. Pre-incubation with the non-immunodominant peptide enabled immunodominant epitopes to induce T-cell activation (p=0.001) and enterocyte apoptosis. Inhibition of interleukin-15 or of p38 MAP kinase controlled such activity. INTERPRETATION: A gliadin fragment can activate the innate immune system, affecting the in situ T-cell recognition of dominant gliadin epitopes. Although our findings emphasise the key role of gliadin-specific T cells, they suggest a complex pathogenic situation, and show that inhibition of interleukin-15 or p38 MAP kinase might have the potential to control coeliac disease.

Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition.

Accumulation of unwanted/misfolded proteins in aggregates has been observed in airways of patients with cystic fibrosis (CF), a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show how the defective CFTR results in defective autophagy and decreases the clearance of aggresomes. Defective CFTR-induced upregulation of reactive oxygen species (ROS) and tissue transglutaminase (TG2) drive the crosslinking of beclin 1, leading to sequestration of phosphatidylinositol-3-kinase (PI(3)K) complex III and accumulation of p62, which regulates aggresome formation. Both CFTR knockdown and the overexpression of green fluorescent protein (GFP)-tagged-CFTR(F508del) induce beclin 1 downregulation and defective autophagy in non-CF airway epithelia through the ROS-TG2 pathway. Restoration of beclin 1 and autophagy by either beclin 1 overexpression, cystamine or antioxidants rescues the localization of the beclin 1 interactome to the endoplasmic reticulum and reverts the CF airway phenotype in vitro, in vivo in Scnn1b-transgenic and Cftr(F508del) homozygous mice, and in human CF nasal biopsies. Restoring beclin 1 or knocking down p62 rescued the trafficking of CFTR(F508del) to the cell surface. These data link the CFTR defect to autophagy deficiency, leading to the accumulation of protein aggregates and to lung inflammation.

Tissue transglutaminase activation modulates inflammation in cystic fibrosis via PPARgamma down-regulation.

Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor gamma and increase of the classic parameters of inflammation, such as TNF-alpha, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-gamma and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.

High incidence of spontaneous autoimmune thyroiditis in immunocompetent self-reactive human T cell receptor transgenic mice.

Autoantigen-specific TCR transgenic mice allow us to assess the role of T cells in autoimmunity. We have recently generated humanized TAZ10 transgenic mice expressing the human TCR specific for the immunodominant epitope of thyroid peroxidase (TPO). We have shown that these transgenic mice do not undergo tolerance in vivo and that on Rag deficient background they are susceptible to spontaneous autoimmune thyroiditis. Here we show that, in contrast to other transgenic models of autoimmunity, almost all TCR(+)Rag1+ (T+R+) T cells are activated in vivo leading to the development of spontaneous autoimmune thyroiditis. In these mice, disease is also accompanied by a significant reduction of CD4+CD25hi regulatory T cells. These data indicate that the pathogenic activity of the self-reactive TCR can circumvent the regulatory function operated by the non-transgenic T cells that are normally present in T+R+ mice, leading to autoimmunity.

Human autoantibodies modulate the T cell epitope repertoire but fail to unmask a pathogenic cryptic epitope.

Abs can tune the responses of Ag-specific T cells by influencing the nature of the epitope repertoire displayed by APCs. We explored the interaction between human self-reactive T cells and human monoclonal autoantibodies from combinatorial Ig-gene libraries derived from autoimmune thyroiditis patients and specific for the main autoantigen thyroid peroxidase (TPO). All human mAbs extensively influenced the T cell epitope repertoire recognized by different TPO-specific T cell clones. The action of the human mAbs was complex, because sometimes the same Ab suppressed or enhanced the epitopes recognized by the 10 different TPO-specific T cell clones. The human mAbs could modulate the epitope repertoire when TPO was added exogenously and when expressed constitutively on the surface of APCs. However, they could not unmask an immunodominant cryptic TPO epitope. In this study, we show that human autoantibodies influence the activity of self-reactive T cells and prove their relevance in concealing or exposing epitopes recognized by self-reactive T cells. However, our results further stress the biological significance of the immunodominant cryptic epitope we have defined and its potential importance in the evolution of autoimmunity.

Protein conformation significantly influences immune responses to prion protein.

In prion diseases, such as variant Creutzfeldt-Jakob disease normal cellular prion protein (PrPC), a largely alpha-helical structure is converted to an abnormal conformational isoform (PrPSc) that shows an increase in beta-sheet content. Similarly, the recombinant form of PrPC (ralpha-PrP) can be converted to a conformation dominated by beta-sheet (rbeta-PrP) by reduction and mild acidification in vitro, a process that may mimic in vivo conversion following PrPC internalization during recycling. Despite PrPSc accumulation and prion propagation in the lymphoreticular system before detectable neuroinvasion, no Ab response to PrP has been detected, probably due to immune tolerance. To investigate how the immune system may respond to alpha- and beta-PrP, we immunized Prnp(0/0) mice that are not tolerant of PrP with ralpha-PrP and rbeta-PrP. In this study, we show that although T cells stimulated by these differently folded conformers PrP recognize similar immunodominant epitopes (residues 111-130 and 191-210) the cytokine profile in response to ralpha- and rbeta-PrP was different. Challenge with ralpha-PrP elicited a strong response of IL-5 and IL-10, whereas rbeta-PrP led to an early increased production of IFN-gamma. In addition, immunization with ralpha-PrP led to production of predominantly IgG1 isotype Ab in the sera, whereas after immunization with rbeta-PrP, IgG2b was significantly produced. Thus, both humoral and cellular responses to these differently folded isoforms of the same protein are different, indicating a possible involvement of Th1 and Th2 pathway activation. These differences may be exploitable diagnostically and therapeutically for prion diseases, such as variant Creutzfeldt-Jakob disease.