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.

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.

Low-dose IL-2 in children with recently diagnosed type 1 diabetes: a Phase I/II randomised, double-blind, placebo-controlled, dose-finding study.

AIMS/HYPOTHESIS: Low-dose IL-2 (ld-IL2) selectively activates and expands regulatory T cells (Tregs) and thus has the potential to skew the regulatory/effector T (Treg/Teff) cell balance towards improved regulation. We investigated which low doses of IL-2 would more effectively and safely activate Tregs during a 1 year treatment in children with recently diagnosed type 1 diabetes. METHODS: Dose Finding Study of IL-2 at Ultra-low Dose in Children With Recently Diagnosed Type 1 Diabetes (DF-IL2-Child) was a multicentre, double-blinded, placebo-controlled, dose-finding Phase I/II clinical trial conducted in four centres at university hospitals in France: 24 children (7-14 years old) with type 1 diabetes diagnosed within the previous 3 months were randomly assigned 1:1:1:1 to treatment by a centralised randomisation system, leading to a 7/5/6/6 patient distribution of placebo or IL-2 at doses of 0.125, 0.250 or 0.500 million international units (MIU)/m2, given daily for a 5 day course and then fortnightly for 1 year. A study number was attributed to patients by an investigator unaware of the randomisation list and all participants as well as investigators and staff involved in the study conduct and analyses were blinded to treatments. The primary outcome was change in Tregs, expressed as a percentage of CD4+ T cells at day 5. It pre-specified that a ≥60% increase in Tregs from baseline would identify Treg high responders. RESULTS: There were no serious adverse events. Non-serious adverse events (NSAEs) were transient and mild to moderate. In treated patients vs placebo, the commonest NSAE was injection site reaction (37.9% vs 3.4%), whereas other NSAEs were at the same level (23.3% vs 19.2%). ld-IL2 induced a dose-dependent increase in the mean proportion of Tregs, from 23.9% (95% CI -11.8, 59.6) at the lowest to 77.2% (44.7, 109.8) at the highest dose, which was significantly different from placebo for all dose groups. However, the individual Treg responses to IL-2 were variable and fluctuated over time. Seven patients, all among those treated with the 0.250 and 0.500 MIU m-2 day-1 doses, were Treg high responders. At baseline, they had lower Treg proportions in CD4+ cells than Treg low responders, and serum soluble IL-2 receptor α (sIL-2RA) and vascular endothelial growth factor receptor 2 (VEGFR2) levels predicted the Treg response after the 5 day course. There was no significant change in glycaemic control in any of the dose groups compared with placebo. However, there was an improved maintenance of induced C-peptide production at 1 year in the seven Treg high responders as compared with low responders. CONCLUSIONS/INTERPRETATION: The safety profile at all doses, the dose-dependent effects on Tregs and the observed variability of the Treg response to ld-IL2 in children with newly diagnosed type 1 diabetes call for use of the highest dose in future developments. The better preservation of insulin production in Treg high responders supports the potential of Tregs in regulating autoimmunity in type 1 diabetes, and warrants pursuing the investigation of ld-IL2 for its treatment and prevention. TRIAL REGISTRATION: ClinicalTrials.gov NCT01862120. FUNDING: Assistance Publique-Hôpitaux de Paris, Investissements d'Avenir programme (ANR-11-IDEX-0004-02, LabEx Transimmunom and ANR-16-RHUS-0001, RHU iMAP) and European Research Council Advanced Grant (FP7-IDEAS-ERC-322856, TRiPoD).

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.

Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients.

Most autoimmune diseases (AID) are linked to an imbalance between autoreactive effector T cells (Teffs) and regulatory T cells (Tregs). While blocking Teffs with immunosuppression has long been the only therapeutic option, activating/expanding Tregs may achieve the same objective without the toxicity of immunosuppression. We showed that low-dose interleukin-2 (ld-IL-2) safely expands/activates Tregs in patients with AID, such HCV-induced vasculitis and Type 1 Diabetes (T1D). Here we analyzed the kinetics and dose-relationship of IL-2 effects on immune responses in T1D patients. Ld-IL-2 therapy induced a dose-dependent increase in CD4(+)Foxp3(+) and CD8(+)Foxp3(+) Treg numbers and proportions, the duration of which was markedly dose-dependent. Tregs expressed enhanced levels of activation markers, including CD25, GITR, CTLA-4 and basal pSTAT5, and retained a 20-fold higher sensitivity to IL-2 than Teff and NK cells. Plasma levels of regulatory cytokines were increased in a dose-dependent manner, while cytokines linked to Teff and Th17 inflammatory cells were mostly unchanged. Global transcriptome analyses showed a dose-dependent decrease in immune response signatures. At the highest dose, Teff responses against beta-cell antigens were suppressed in all 4 patients tested. These results inform of broader changes induced by ld-IL-2 beyond direct effects on Tregs, and relevant for further development of ld-IL-2 for therapy and prevention of T1D, and other autoimmune and inflammatory diseases.

Induction of regulatory T cells and efficacy of low-dose interleukin-2 in systemic sclerosis: interventional open-label phase 1-phase 2a study.

BACKGROUND: Systemic sclerosis (SSc) is a chronic autoimmune disease, with impaired immune response, increased fibrosis and endothelial dysfunction. Regulatory T cells (Tregs), which are essential to control inflammation, tissue repair and autoimmunity, have a decreased frequency and impaired function in SSc patients. Low-dose interleukin-2 (IL-2LD) can expand and activate Tregs and has, therefore, a therapeutic potential in SSc. OBJECTIVE: We aimed to assess the safety and biological efficacy of IL-2LD in patients with SSc. METHODS: As part of the TRANSREG open-label phase IIa basket trial in multiple autoimmune diseases, we studied nine patients with SSc without severe organ involvement. Patients received 1 million international units (MIU)/day of IL-2 for 5 days, followed by fortnightly injections for 6 months. Laboratory and clinical evaluations were performed between baseline and month 6. RESULTS: At day 8, the primary endpoint (Treg frequency) was reached with a 1.8±0.5-fold increase of Treg levels among CD4+ T lymphocytes (p=0.0015). There were no significant changes in effector T cells nor in B cells. IL-2LD was well tolerated, and no serious adverse events related to treatment occurred. There was a globally stable measurement in the modified Rodnan skin score and Valentini score at month 6. Disease activity and severity measures, the quality of life evaluated by EuroQL-5D-5L and pulmonary function test parameters remained stable during the study period. CONCLUSION: IL-2LD at a dosage of 1 MIU/day safely and selectively activates and expands Tregs. Clinical signs remain stable during the study period. This opens the door to properly powered phase II efficacy trials investigating IL-2LD therapeutic efficacy in SSc.

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.

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.