The universal effects of low-dose interleukin-2 across 13 autoimmune diseases in a basket clinical trial.

BACKGROUND: A Tregs insufficiency is central to autoimmune and inflammatory diseases pathophysiology and low dose interleukin-2 (IL-2LD) can specifically activate Tregs. OBJECTIVE: To assess IL-2LD therapeutic potential and select diseases for further clinical development, we performed an open-label, phase 2a, disease-finding, "basket trial" involving patients with one of 13 different autoimmune diseases. METHODS: 81 patients treated with IL-2LD (1 million IU/day) for 5 days, followed by fortnightly injections. The first 48 patients received diluted Proleukin®, while the subsequent 33 received ready-to-use ILT-101®. The primary endpoint was the change in Tregs at day-8 compared to baseline. Key secondary endpoints included clinical efficacy assessments using the Clinical Global Impression (CGI) scale, disease-specific scores, and EuroQL-5D-5L. RESULTS: Our study unveiled a universal and significant expansion and activation of Tregs, without concomitant Teffs activation, across all 13 autoimmune diseases. Both Proleukin® and ready-to-use ILT-101® demonstrated identical effects on Tregs. CGI scores reflecting activity, severity, and efficacy were significantly reduced in the overall patient population. Disease-specific clinical scores improved in five of the six disease cohorts with at least six patients, namely ankylosing spondylitis, systemic lupus erythematosus, Behçet's disease, Sjögren's syndrome, and systemic sclerosis. Urticaria was the only severe adverse event related to treatment. CONCLUSION: IL-2LD was well-tolerated, exhibiting specific Treg activation and clinical improvements across the 13 autoimmune diseases. CLINICAL IMPLICATION: Tregs stimulation by IL-2LD is a promising therapeutic strategy and IL-2LD holds considerable promise for integration into combinatorial therapeutic approaches.

Deep immunophenotyping reveals that autoimmune and autoinflammatory disorders are spread along two immunological axes capturing disease inflammation levels and types.

OBJECTIVES: Based on genetic associations, McGonagle and McDermott suggested a classification of autoimmune and autoinflammatory diseases as a continuum ranging from purely autoimmune to purely autoinflammatory diseases and comprising diseases with both components. We used deep immunophenotyping to identify immune cell populations and molecular targets characterising this continuum. METHODS: We collected blood from 443 patients with one of 15 autoimmune or autoinflammatory diseases and 71 healthy volunteers. Deep phenotyping was performed using 13 flow cytometry panels characterising over 600 innate and adaptive cell populations. Unsupervised and supervised analyses were conducted to identify disease clusters with their common and specific cell parameters. RESULTS: Unsupervised clustering categorised these diseases into five clusters. Principal component analysis deconvoluted this clustering into two immunological axes. The first axis was driven by the ratio of LAG3+ to ICOS+ in regulatory T lymphocytes (Tregs), and segregated diseases based on their inflammation levels. The second axis was driven by activated Tregs and type 3 innate lymphoid cells (ILC3s), and segregated diseases based on their types of affected tissues. We identified a signature of 23 cell populations that accurately characterised the five disease clusters. CONCLUSIONS: We have refined the monodimensional continuum of autoimmune and autoinflammatory diseases as a continuum characterised by both disease inflammation levels and targeted tissues. Such classification should be helpful for defining therapies. Our results call for further investigations into the role of the LAG3+/ICOS+ balance in Tregs and the contribution of ILC3s in autoimmune and autoinflammatory diseases. TRIAL REGISTRATION NUMBER: NCT02466217.

Particulate antigens administrated by intranasal and intravaginal routes in a prime-boost strategy improve HIV-specific TFH generation, high-quality antibodies and long-lasting mucosal immunity.

Mucosal surfaces serve as the primary entry points for pathogens such as SARS- CoV-2 coronavirus or HIV in the human body. Mucosal vaccination plays a crucial role to successfully induce long-lasting systemic and local immune responses to confer sterilizing immunity. However, antigen formulations and delivery methods must be properly selected since they are decisive for the quality and the magnitude of the elicited immune responses in mucosa. We investigated the significance of using particulate antigen forms for mucosal vaccination by comparing VLP- or protein- based vaccines in a mouse model. Based on a mucosal prime-boost immunization protocol combining (i) HIV- pseudotyped recombinant VLPs (HIV-VLPs) and (ii) plasmid DNA encoding HIV- VLPs (pVLPs), we demonstrated that combination of intranasal primes and intravaginal boosts is optimal to elicit both humoral and cellular memory responses in mucosa. Interestingly, our results show that in contrast to proteins, particulate antigens induce high-quality humoral responses characterized by a high breadth, long-term neutralizing activity and cross-clade reactivity, accompanying with high T follicular helper cell (TFH) response. These results underscore the potential of a VLP-based vaccine in effectively instigating long-lasting, HIV-specific immunity and point out the specific role of particulate antigen form in driving high-quality mucosal immune responses.

Low-dose interleukin-2 therapy in active systemic lupus erythematosus (LUPIL-2): a multicentre, double-blind, randomised and placebo-controlled phase II trial.

OBJECTIVES: A regulatory T cell (Treg) insufficiency due to shortage of interleukin-2 (IL-2) is central to the pathophysiology of systemic lupus erythematosus (SLE). We performed a multicentre, double-blinded, randomised, placebo-controlled phase II proof-of-concept trial to evaluate the efficacy of low-dose IL-2 therapy in patients with SLE having moderate-to-severe disease activity while receiving standard treatment. METHODS: We randomly assigned 100 patients in a 1:1 ratio to receive either 1.5 million IU/day of subcutaneous IL-2 (ILT-101) or placebo for 5 days followed by weekly injections for 12 weeks. Clinical efficacy was assessed at week 12 in a predefined hierarchical analysis of (1) the SLE responder index-4 (SRI-4) response as a primary end point, and of (2) relative and (3) absolute changes in the Safety of Estrogens in Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index scores as key secondary end points. RESULTS: The primary end point was not met in the intention-to-treat population (ILT-101: 68%, placebo: 58%; p=0.3439), due to a 100% SRI-4 response rate in the placebo group from the two sites from Bulgaria. A post hoc per-protocol analysis on a prespecified population that excluded patients from these two sites (n=53) showed a statistically significant difference for the SRI-4 response rate (ILT-101: 83.3%; placebo: 51.7%; p=0.0168), and for the two key secondary end points, accompanied by differences in several secondary exploratory end points. ILT-101 was well tolerated and there was no generation of antidrug antibodies. CONCLUSIONS: The post hoc hierarchical analysis of the primary and key secondary end points in a per-protocol population, complemented by the exploratory analyses of multiple other secondary end points, support that low-dose IL-2 is beneficial in active SLE. TRIAL REGISTRATION NUMBER: NCT02955615.

NCKAP1L defects lead to a novel syndrome combining immunodeficiency, lymphoproliferation, and hyperinflammation.

The Nck-associated protein 1-like (NCKAP1L) gene, alternatively called hematopoietic protein 1 (HEM-1), encodes a hematopoietic lineage-specific regulator of the actin cytoskeleton. Nckap1l-deficient mice have anomalies in lymphocyte development, phagocytosis, and neutrophil migration. Here we report, for the first time, NCKAP1L deficiency cases in humans. In two unrelated patients of Middle Eastern origin, recessive mutations in NCKAP1L abolishing protein expression led to immunodeficiency, lymphoproliferation, and hyperinflammation with features of hemophagocytic lymphohistiocytosis. Immunophenotyping showed an inverted CD4/CD8 ratio with a major shift of both CD4+ and CD8+ cells toward memory compartments, in line with combined RNA-seq/proteomics analyses revealing a T cell exhaustion signature. Consistent with the core function of NCKAP1L in the reorganization of the actin cytoskeleton, patients' T cells displayed impaired early activation, immune synapse morphology, and leading edge formation. Moreover, knockdown of nckap1l in zebrafish led to defects in neutrophil migration. Hence, NCKAP1L mutations lead to broad immune dysregulation in humans, which could be classified within actinopathies.

Immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial.

OBJECTIVE: Regulatory T cells (Tregs) prevent autoimmunity and control inflammation. Consequently, any autoimmune or inflammatory disease reveals a Treg insufficiency. As low-dose interleukin-2 (ld-IL2) expands and activates Tregs, it has a broad therapeutic potential. AIM: We aimed to assess this potential and select diseases for further clinical development by cross-investigating the effects of ld-IL2 in a single clinical trial treating patients with 1 of 11 autoimmune diseases. METHODS: We performed a prospective, open-label, phase I-IIa study in 46 patients with a mild to moderate form of either rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, psoriasis, Behcet's disease, granulomatosis with polyangiitis, Takayasu's disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis and sclerosing cholangitis. They all received ld-IL2 (1 million IU/day) for 5 days, followed by fortnightly injections for 6 months. Patients were evaluated by deep immunomonitoring and clinical evaluation. RESULTS: ld-IL2 was well tolerated whatever the disease and the concomitant treatments. Thorough supervised and unsupervised immunomonitoring demonstrated specific Treg expansion and activation in all patients, without effector T cell activation. Indication of potential clinical efficacy was observed. CONCLUSION: The dose of IL-2 and treatment scheme used selectively activate and expand Tregs and are safe across different diseases and concomitant treatments. This and preliminary indications of clinical efficacy should licence the launch of phase II efficacy trial of ld-IL2 in various autoimmune and inflammatory diseases. TRIAL REGISTRATION NUMBER: NCT01988506.

Deep phenotyping of immune cell populations by optimized and standardized flow cytometry analyses.

Multicolor flow cytometry is a technology of choice for phenotyping of immune cells, and it can be used routinely for the follow up of patients in clinical trials. But it is challenging to define combinations of conjugated antibodies that efficiently allow the detailed analysis of major immune cell subsets and the identification of rare cell populations. In a collaborative work among the Immunology, Immunopathology, Immunotherapy (I3 ) laboratory, and the laboratory of immunomonitoring in oncology (L.I.O), we developed and validated 12 different 10-color flow cytometry panels that allow the deep immunophenotyping of cells from whole blood for the follow up of autoimmune and cancer patients. Here, we describe these optimized flow cytometry panels, showing that they provide the advanced analysis of T cells (including regulatory T cells), B cells, NK cells, MAIT cells, myeloid cells, monocytes, and dendritic cells. Most of the panels have been dried to improve standardization of the labeling and the entire procedure can be performed on less than 2 ml of whole blood. These deep immunophenotyping flow cytometry panels constitute a powerful tool for the monitoring of immune blood cells and will hopefully lead to the discovery of new biomarkers and potential therapeutic targets in autoimmune and cancer clinical trials. © 2018 International Society for Advancement of Cytometry.

Clinical and multi-omics cross-phenotyping of patients with autoimmune and autoinflammatory diseases: the observational TRANSIMMUNOM protocol.

INTRODUCTION: Autoimmune and autoinflammatory diseases (AIDs) represent a socioeconomic burden as the second cause of chronic illness in Western countries. In this context, the TRANSIMMUNOM clinical protocol is designed to revisit the nosology of AIDs by combining basic, clinical and information sciences. Based on classical and systems biology analyses, it aims to uncover important phenotypes that cut across diagnostic groups so as to discover biomarkers and identify novel therapeutic targets. METHODS AND ANALYSIS: TRANSIMMUNOM is an observational clinical protocol that aims to cross-phenotype a set of 19 AIDs, six related control diseases and healthy volunteers . We assembled a multidisciplinary cohort management team tasked with (1) selecting informative biological (routine and omics type) and clinical parameters to be captured, (2) standardising the sample collection and shipment circuit, (3) selecting omics technologies and benchmarking omics data providers, (4) designing and implementing a multidisease electronic case report form and an omics database and (5) implementing supervised and unsupervised data analyses. ETHICS AND DISSEMINATION: The study was approved by the institutional review board of Pitié-Salpêtrière Hospital (ethics committee Ile-De-France 48-15) and done in accordance with the Declaration of Helsinki and good clinical practice. Written informed consent is obtained from all participants before enrolment in the study. TRANSIMMUNOM's project website provides information about the protocol (https://www.transimmunom.fr/en/) including experimental set-up and tool developments. Results will be disseminated during annual scientific committees appraising the project progresses and at national and international scientific conferences. DISCUSSION: Systems biology approaches are increasingly implemented in human pathophysiology research. The TRANSIMMUNOM study applies such approach to the pathophysiology of AIDs. We believe that this translational systems immunology approach has the potential to provide breakthrough discoveries for better understanding and treatment of AIDs. TRIAL REGISTRATION NUMBER: NCT02466217; Pre-results.

A standardized flow cytometry procedure for the monitoring of regulatory T cells in clinical trials.

BACKGROUND: Quantification of regulatory T cells (Tregs) is crucial in immunomonitoring in clinical trials as this cell population has been shown to be involved in a wide range of diseases, including cancers, autoimmune diseases, infections, and allergies. Human Tregs are defined as CD4+ CD25+ CD127low FoxP3+ cells, and the standardization of their staining by flow cytometry is a challenge, especially in multicenter clinical trials, notably because of the intracellular location of FoxP3. METHOD: A flow cytometry staining procedure was settled and standardized to measure human Tregs in peripheral whole blood using precoated dried antibodies in ready-to-use tubes. It was compared with reference methods and implemented and validated to be suitable with different cytometer platforms. RESULTS: The standardized protocol developed with dried antibodies and reduced volumes of whole blood allows an optimal identification of Tregs. Compared with classical staining procedure, it reduces the number of steps required, in a very fast and simple technique. The accuracy of the method was confirmed by a multicenter comparison with different cytometer brands. CONCLUSIONS: Our results highlight the reliability of this high-standard protocol that could become a reference method for the monitoring of Tregs in clinical trials. © 2018 International Clinical Cytometry Society.

Retrovirus-Based Virus-Like Particle Immunogenicity and Its Modulation by Toll-Like Receptor Activation.

Retrovirus-derived virus-like particles (VLPs) are particularly interesting vaccine platforms, as they trigger efficient humoral and cellular immune responses and can be used to display heterologous antigens. In this study, we characterized the intrinsic immunogenicity of VLPs and investigated their possible adjuvantization by incorporation of Toll-like receptor (TLR) ligands. We designed a noncoding single-stranded RNA (ncRNA) that could be encapsidated by VLPs and induce TLR7/8 signaling. We found that VLPs efficiently induce in vitro dendritic cell activation, which can be improved by ncRNA encapsidation (ncRNAVLPs). Transcriptome studies of dendritic cells harvested from the spleens of immunized mice identified antigen presentation and immune activation as the main gene expression signatures induced by VLPs, while TLR signaling and Th1 signatures characterize ncRNAVLPs. In vivo and compared with standard VLPs, ncRNAVLPs promoted Th1 responses and improved CD8+ T cell proliferation in a MyD88-dependent manner. In an HIV vaccine mouse model, HIV-pseudotyped ncRNAVLPs elicited stronger antigen-specific cellular and humoral responses than VLPs. Altogether, our findings provide molecular evidence for a strong vaccine potential of retrovirus-derived VLPs that can be further improved by harnessing TLR-mediated immune activation.IMPORTANCE We previously reported that DNA vaccines encoding antigens displayed in/on retroviral VLPs are more efficient than standard DNA vaccines at inducing cellular and humoral immune responses. We aimed to decipher the mechanisms and investigated the VLPs' immunogenicity independently of DNA vaccination. We show that VLPs have the ability to activate antigen-presenting cells directly, thus confirming their intrinsic immunostimulatory properties and their potential to be used as an antigenic platform. Notably, this immunogenicity can be further improved and/or oriented by the incorporation into VLPs of ncRNA, which provides further TLR-mediated activation and Th1-type CD4+ and CD8+ T cell response orientation. Our results highlight the versatility of retrovirus-derived VLP design and the value of using ncRNA as an intrinsic vaccine adjuvant.

Narcolepsy Type 1 Is Associated with a Systemic Increase and Activation of Regulatory T Cells and with a Systemic Activation of Global T Cells.

Narcolepsy is a rare neurologic disorder characterized by excessive daytime sleepiness, cataplexy and disturbed nocturnal sleep patterns. Narcolepsy type 1 (NT1) has been shown to result from a selective loss of hypothalamic hypocretin-secreting neurons with patients typically showing low CSF-hypocretin levels (<110 pg/ml). This specific loss of hypocretin and the strong association with the HLA-DQB1*06:02 allele led to the hypothesis that NT1 could be an immune-mediated pathology. Moreover, susceptibility to NT1 has recently been associated with several pathogens, particularly with influenza A H1N1 virus either through infection or vaccination. The goal of this study was to compare peripheral blood immune cell populations in recent onset pediatric NT1 subjects (post or non-post 2009-influenza A H1N1 vaccination) to healthy donors. We demonstrated an increased number of central memory CD4+ T cells (CD62L+ CD45RA-) associated to an activated phenotype (increase in CD69 and CD25 expression) in NT1 patients. Percentage and absolute count of regulatory T cells (Tregs) in NT1 patients were increased associated with an activated phenotype (increase in GITR and LAP expression), and of activated memory phenotype. Cytokine production by CD4+ and CD8+ T cells after activation was not modified in NT1 patients. In H1N1 vaccinated NT1 patients, absolute counts of CD3+, CD8+ T cells, and B cells were increased compared to non-vaccinated NT1 patients. These results support a global T cell activation in NT1 patients and thus support a T cell-mediated autoimmune origin of NT1, but do not demonstrate the pathological role of H1N1 prophylactic vaccination. They should prompt further studies of T cells, particularly of Tregs (such as suppression and proliferation antigen specific assays, and also T-cell receptor sequencing), in NT1.

Low-Dose IL-2 Induces Regulatory T Cell-Mediated Control of Experimental Food Allergy.

Regulatory T cells (Tregs) are pivotal for maintenance of immune self-tolerance and also regulate immune responses to exogenous Ags, including allergens. Both decreased Treg number and function have been reported in allergic patients, offering new therapeutic perspectives. We previously demonstrated that Tregs can be selectively expanded and activated by low doses of IL-2 (ld-IL-2) inducing immunoregulation without immunosuppression and established its protective effect in autoimmune diseases. In this study, we evaluated the ability of ld-IL-2 to control allergy in an experimental model of food allergy. Ld-IL-2 induced Treg expansion and activation that elicited protection against clinical manifestations of food allergy in two mouse models with OVA and peanut. This clinical effect was lost in Treg-depleted mice, demonstrating the major contribution of Tregs in ld-IL-2 efficacy. Mechanistic studies further indicated that protection from allergy could be explained by a Treg-dependent local modification of the Th1/Th2 balance and an inhibition of mast cell recruitment and activation. Preventive and therapeutic effects of ld-IL-2 were observed over a 7-mo-period, highlighting its long-term efficacy. This study demonstrated that ld-IL-2 is efficient to prevent and to treat allergic immune responses, and thus represents a promising therapeutic strategy for managing allergic diseases.

Human and Mouse CD8(+)CD25(+)FOXP3(+) Regulatory T Cells at Steady State and during Interleukin-2 Therapy.

In addition to CD4(+) regulatory T cells (Tregs), CD8(+) suppressor T cells are emerging as an important subset of regulatory T cells. Diverse populations of CD8(+) T cells with suppressive activities have been described. Among them, a small population of CD8(+)CD25(+)FOXP3(+) T cells is found both in mice and humans. In contrast to thymic-derived CD4(+)CD25(+)FOXP3(+) Tregs, their origin and their role in the pathophysiology of autoimmune diseases (AIDs) are less understood. We report here the number, phenotype, and function of CD8(+) Tregs cells in mice and humans, at the steady state and in response to low-dose interleukin-2 (IL-2). CD8(+) Tregs represent approximately 0.4 and 0.1% of peripheral blood T cells in healthy humans and mice, respectively. In mice, their frequencies are quite similar in lymph nodes (LNs) and the spleen, but two to threefold higher in Peyer patches and mesenteric LNs. CD8(+) Tregs express low levels of CD127. CD8(+) Tregs express more activation or proliferation markers such as CTLA-4, ICOS, and Ki-67 than other CD8(+) T cells. In vitro, they suppress effector T cell proliferation as well as or even better than CD4(+) Tregs. Owing to constitutive expression of CD25, CD8(+) Tregs are 20- to 40-fold more sensitive to in vitro IL-2 stimulation than CD8(+) effector T cells, but 2-4 times less than CD4(+) Tregs. Nevertheless, low-dose IL-2 dramatically expands and activates CD8(+) Tregs even more than CD4(+) Tregs, in mice and humans. Further studies are warranted to fully appreciate the clinical relevance of CD8(+) Tregs in AIDs and the efficacy of IL-2 treatment.

Enveloped virus-like particle platforms: vaccines of the future?

The techniques to produce effective vaccines have evolved, and the early vaccines (live, inactivated, subunit...) are no longer considered as the most appropriate for new vaccine development. We question here what will be the future vaccines, and argue that virus-like particle (VLP)-based vaccines are promising candidates. In addition to being effective vaccines against analogous viruses from which they are derived, VLPs can also be used to present foreign epitopes to the immune system. The achievement of this strategy can be illustrated by the recent development of malaria candidate vaccine. We point out recent VLP-based vaccine developments and discuss future perspectives.

Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms.

The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs) to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccines encoding the non-EV-associated antigen. Here, we compared our two approaches to associate the ovalbumin (OVA) antigen to EVs: (a) by fusion to the lipid-binding domain C1C2 of MFGE8(=lactadherin), which is exposed on the surface of secreted membrane vesicles; and (b) by fusion to retroviral Gag capsid protein, which is incorporated inside membrane-enclosed virus-like particles. Plasmids encoding either form of modified OVA were used as DNA-based vaccines (i.e. injected into mice to allow in vivo expression of the antigen associated to EVs). We show that both DNA vaccines induced, with similar efficiency, OVA-specific CD8(+) T cells and total IgG antibodies. By contrast, each vaccine preferentially stimulated different isotypes of immunoglobulins, and the OVA-C1C2-encoding vaccine favoured antigen-specific CD4(+) T lymphocyte induction as compared to the Gag-OVA vaccine. Nevertheless, both OVA-C1C2 and Gag-OVA vaccines efficiently prevented in vivo outgrowth of OVA-expressing tumours and reduced tumour progression when administered to tumour-bearing mice, although with variable efficacies depending on the tumour models. DNA vaccines encoding EV-associated antigens are thus promising immunotherapy tools in cancer but also potentially other diseases.

Efficacy of DNA vaccines forming e7 recombinant retroviral virus-like particles for the treatment of human papillomavirus-induced cancers.

Human papillomavirus (HPV) is involved in the development of anogenital tumors and also in the development of oropharyngeal head and neck carcinomas, where HPV-16, expressing the E6 and E7 oncoproteins, is the most frequent serotype. Although vaccines encoding L1 and L2 capsid HPV proteins are efficient for the prevention of HPV infection, they are inadequate for treating established tumors. Hence, development of innovative vaccine therapies targeting E6/E7 is important for controlling HPV-induced cancers. We have engineered a nononcogenic mutated E7-specific plasmo-retroVLP vaccine (pVLP-E7), consisting of plasmid DNA, that is able to form recombinant retrovirus-based virus-like particles (VLPs) that display E7 antigen into murine leukemia virus Gag proteins pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G). pVLP-E7 vaccinations were studied for their ability to generate specific immune responses and for induction of protective immunity against tumor cell challenge in preventive and therapeutic models. The produced VLPs induce the maturation of human dendritic cells in vitro and mount specific E7 T cell responses. Intradermic vaccinations of mice with pVLP-E7 show their efficacy to generate antigen-specific T cell responses, to prevent and protect animals from early TC-1 tumor development compared with standard DNA or VLP immunizations. The vaccine efficacy was also evaluated for advanced tumors in mice vaccinated at various time after the injection of TC-1 cells. Data show that pVLP-E7 vaccination can cure mice with already established tumors only when combined with Toll-like receptor-7 (TLR7) and TLR9 agonists. Our findings provide evidence that pVLPs, combining the advantages of DNA and VLP vaccines, appear to be a promising strategy for the treatment of HPV-induced cancers.