Low-dose IL-2 Therapy Research Articles

Metabolic impact of low dose IL-2 therapy for primary Sjögren’s Syndrome in a double-blind, randomized clinical trial

ObjectivesLow-dose interleukin 2 (Ld-IL2) is increasingly being explored as an immune-modulating treatment for autoimmune diseases which mainly affect T cell subsets. This study investigates the metabolic effects of Ld-IL2 therapy in patients with primary Sjögren’s syndrome (pSS).MethodA total of 60 patients were recruited to conduct a double-blind, randomized clinical trial. Of these patients, 50% (30/60) received Ld-IL2 therapy along with standard treatment for 12 weeks, followed by 12 weeks of follow-up. The effectiveness was evaluated by Sjögren's Tool for Assessing Response (STAR). An untargeted analysis was performed to profile hydrophilic metabolites.ResultsMetabolic profiling revealed significant alterations post-treatment, notably in metabolites like acetyl-CoA, ascorbic acid, and glutathione, which are beneficial in managing autoimmune diseases. In addition, the levels of metabolite accumulation were correlated with variations in immune cell subsets (p < 0.05), particularly Tregs. Moreover, patients exhibiting a specific metabolic profile, including lower serum levels of isoleucine, ADP, Thymidine 5'-triphosphate, and other metabolites, had a high response rate (91.7%-98.6%), as indicated by the receiver operating characteristic (ROC) curve.ConclusionsThese findings suggest that Ld-IL2 therapy influences metabolic pathways in pSS, offering insights into the systemic effects of Ld-IL2 therapy beyond immune modulation.Trial registration numberClinicalTrials.gov number, NCT02464319.Key Points• Metabolic alteration in pSS is significantly associated with Ld-IL2 therapy.• Metabolic changes correlate with variations in immune cell subsets, particularly Tregs.• Metabolic profiling could be a valuable tool in guiding Ld-IL2 therapy choices for pSS patients.

Predictive biomarkers for low-dose IL-2 therapy efficacy in systemic lupus erythematosus: a clinical analysis

BackgroundLow-dose IL-2 (Ld-IL2) has shown favorable therapeutic effects in systemic lupus erythematosus (SLE) therapy. However, previous clinical trials reported an SLE Responder Index-4 (SRI-4) response rate of 65.52%-68%, with approximately half failing to achieve the primary endpoint by week 24. Our study aims to determine the real-world use of Ld-IL2 and to identify determinants of its effectiveness in SLE.MethodsWe pooled data from 342 SLE patients undergoing sequential Ld-IL2 treatment, with 314 persisting for over 3 months were included in effectiveness and prediction analyses. All patients were categorized into responder (n = 136) and non-responder group (n = 178) according to SRI-4. Lupus Low Disease Activity State (LLDAS) was also analyzed to validate our results.ResultsRash, lower complement 3 (C3), and renal involvement including urine protein, urine occult blood and urine casts emerged as prominent predictors of achieving SRI-4. Adjusting for baseline values using the ratio of change to baseline revealed significant differences in CD4 + T cell immune profiles between responders and non-responders. ROC analysis confirmed a satisfactory performance of rash, renal involvement, percentage change of CD4 + T cells, and C3 in predicting SRI-4, yielding an AUC of 0.933. LLDAS analysis showed that hematological involvements and lower CLA + Treg were potent predictive markers in LLDAS attainment. Conversely, renal involvement failed to have significant association in achieving LLDAS. The analysis of background therapy in SLE patients showed that MMF was more likely to reach the SRI-4 response with the combination of Ld-IL2.ConclusionsThese findings uncovered the predictors of Ld-IL2 treatment efficacy in SLE patients and provided guidance to physicians for rational utilization.

BLIMP-1 and CEACAM1 cooperate to regulate human regulatory T cell proliferation and function

Abstract BLIMP-1, encoded by the PRDM1 gene, is a major transcriptional hub distinct from Foxp3 in human Tregs, but its mechanisms of action have not been fully determined. Here, using CRISPR/Cas9-mediated disruption of PRDM1, we show that BLIMP-1 limits Treg proliferation while supports Treg functional activity. PRDM1KO human Tregs are less suppressive and exhibit increased expansion in a humanized mouse model of xenogeneic GVHD. BLIMP-1 restrains Treg proliferation by lowering IL-2R signaling through downregulation of CD25 and by inhibiting responsiveness to TCR/CD28 signaling and mTOR activation through upregulation of CEACAM1, an immune checkpoint. Expression of CEACAM1 is induced by IL-2 in human, but not mouse, Tregs. Prolonged IL-2R signaling leads to optimal BLIMP-1 expression and chromatin opening of human CEACAM1, leading to high cell surface CEACAM1 through a 3’ regulatory region containing motifs for binding PRDM1 and STATs. Correspondingly, CEACAM1 is highly induced on Tregs from autoimmune patients undergoing low-dose IL-2 therapy, and these Tregs showed reduced proliferation. In vivo studies using xenogeneic GVHD mouse model show that CEACAM1KO human Tregs have higher suppressive activities, indicating a potential role of CEACAM1 in Treg function. Collectively, our findings uncover a novel IL-2-dependent regulatory loop whereby BLIMP-1 and CEACAM1 act to limit human Treg proliferation and contribute to Treg suppressive function.

Assessing the effectiveness of Interleukin-2 therapy in experimental type 1 diabetes

AimMuch focus of immunotherapy for type 1 diabetes (T1D) has been devoted on selectively boosting regulatory T (Treg) cells using low dose IL-2 due to their constitutive expression of IL-2Rα, CD25. However, several clinical trials using a low dose of IL-2 only showed a limited improvement of metabolic control. It can therefore be hypothesized that further decreasing IL-2 dosage may increase the selective responsiveness of Treg cells.MethodsWe induced experimental T1D using multiple low dose streptozotocin (STZ) injections and treated the mice with an ultra-low dose IL-2 (uIL-2, approximately 7-fold lower than low dose). Immune response was studied using multicolor flow cytometry.ResultsWe found that uIL-2 did not protect STZ mice from developing hyperglycemia. It did neither increase Treg cell proportions, nor did it correct the phenotypic shift of Treg cells seen in T1D. It only partially decreased the proportion of IFN-γ+ T cells. Likewise, uIL-2 also did not protect the dysfunction of regulatory B (Breg) cells. Strikingly, when administered in combination with an anti-inflammatory cytokine IL-35, uIL-2 abrogated IL-35’s protective effect. Low dose IL-2, on the other hand, protected half of the STZ mice from developing hyperglycemia. No difference was found in the Treg and Breg response, and it only tended to decrease CD80 expression in macrophages and dendritic cells.ConclusionIn conclusion, further decreasing IL-2 dosage may not be a suitable approach for T1D therapy, and the limited success suggests that an alternative low dose IL-2 therapy strategy or other immunotherapies should be considered.

Systematic literature review on early clinical evidence for immune-resolution therapies and potential benefits to patients and healthcare providers.

Several current therapies for autoimmune diseases do not provide sustained remission. Therapies that focus on the restoration of homeostasis within the immune system (i.e., immune resolution) could overcome the limitations of current therapies and provide more durable remission. However, there is no established consensus on appropriate clinical trial designs and endpoints to evaluate such therapies. Therefore, we conducted a systematic literature review (SLR) focusing on five index diseases (asthma, atopic dermatitis, rheumatoid arthritis, systemic lupus erythematosus [SLE], and ulcerative colitis) to explore published literature on 1) expert opinion on immune-resolution outcomes that should be measured in clinical trials; and 2) quantification of immune resolution in previous clinical trials. The SLR was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Embase and MEDLINE databases were systematically searched (2013-2023) for published English language articles. Conference proceedings (2020-2022) from American Academy of Dermatology, American College of Rheumatology, Digestive Disease Week, European Alliance of Associations for Rheumatology, and European Academy of Dermatology and Venereology were searched to include relevant abstracts. The study protocol was registered in PROSPERO (CRD42023406489). The SLR included 26 publications on 20 trials and 12 expert opinions. Expert opinions generally lacked specific recommendations on the assessment of immune resolution in clinical trials and instead suggested targets or biomarkers for future therapies. The targets included thymic stromal lymphopoietin (TSLP) in asthma; T helper (Th)2 and Th22 cells and their respective cytokines (interleukin [IL]-4R and IL-22) in atopic dermatitis; inhibitory/regulatory molecules involved in T-cell modulation, and protein tyrosine phosphatase, non-receptor type 22 (PTPN22) in rheumatoid arthritis; low-dose IL-2 therapy in SLE; and pro-resolution mediators in ulcerative colitis and asthma. In the interventional studies, direct biomarker assessments of immune resolution were the number/proportion of regulatory T-cells (Treg) and the ratio Th17/Treg in SLE and rheumatoid arthritis; the number of T follicular helper cells (Tfh), Th1, Th2, Th17, and Th22 in atopic dermatitis, rheumatoid arthritis, and SLE; and mucosal proinflammatory gene signatures (tumor necrosis factor [TNF], interleukin 1 alpha [IL1A], regenerating family member 1 alpha [REG1A], IL8, interleukin 1 beta [IL1B], and leukocyte immunoglobulin-like receptors A [LILRA]) in ulcerative colitis. Several studies reported a statistically significant relationship between clinical remission and immune-resolution biomarkers, suggesting a link between T-cell homeostasis, cytokine production, and disease activity in autoimmune diseases. Existing literature does not offer clear guidance on the evaluation of immune resolution in interventional studies. Further research and consensus are needed to assess a treatment's ability to induce long-term remission or low disease activity. https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023406489, identifier CRD42023406489.

Low-dose interleukin-2 therapy in systemic lupus erythematosus.

In systemic lupus erythematosus (SLE), T regulatory cells (Tregs) contribute to the inhibition of autoimmune responses by suppressing self-reactive immune cells. Interleukin (IL)-2 plays an essential role in the generation, function and homeostasis of the Tregs and is reduced in SLE. Several clinical studies, including randomized trials, have shown that low-dose IL-2 therapy in SLE patients is safe and effective and can reduce disease manifestations. This review discusses the rationale for the use of low-dose IL-2 therapy in SLE, the clinical responses in patients, and the effects of this therapy on different types of T cells. Considerations are made on the current and future directions of use of low-dose IL-2 regimens in SLE.

Pathogenesis and novel therapeutics of regulatory T cell subsets and interleukin-2 therapy in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a heterogeneous multisystem inflammatory disease with wide variability in clinical manifestations. Natural arising CD4+ regulatory T cells (Tregs) play a critical role in maintaining peripheral tolerance by suppressing inflammation and preventing autoimmune responses in SLE. Additionally, CD8+ regulatory T cells, type 1 regulatory T cells (Tr1), and B regulatory cells also have a less well-defined role in the pathogenesis of SLE. Elucidation of the roles of various Treg subsets dedicated to immune homeostasis will provide a novel therapeutic approach that governs immune tolerance for the remission of active lupus. Diminished interleukin (IL)-2 production is associated with a depleted Treg cell population, and its reversibility by IL-2 therapy provides important reasons for the treatment of lupus. This review focuses on the pathogenesis and new therapeutics of human Treg subsets and low-dose IL-2 therapy in clinical benefits with SLE.

How GRAIL controls Treg function to maintain self-tolerance.

Regulatory T cells (Tregs) normally maintain self-tolerance. Tregs recognize "self" such that when they are not working properly, such as in autoimmunity, the immune system can attack and destroy one's own tissues. Current therapies for autoimmunity rely on relatively ineffective and too often toxic therapies to "treat" the destructive inflammation. Restoring defective endogenous immune regulation (self-tolerance) would represent a paradigm shift in the therapy of these diseases. One recent approach to restore self-tolerance is to use "low dose IL-2" as a therapy to increase the number of circulating Tregs. However, studies to-date have not demonstrated that low-dose IL-2 therapy can restore concomitant Treg function, and phase 2 studies in low dose IL-2 treated patients with autoimmune diseases have failed to demonstrate significant clinical benefit. We hypothesize that the defect in self-tolerance seen in autoimmunity is not due to an insufficient number of available Tregs, but rather, due to defects in second messengers downstream of the IL-2R that normally control Treg function and stability. Previous studies from our lab and others have demonstrated that GRAIL (a ubiquitin E3 ligase) is important in Treg function. GRAIL expression is markedly diminished in Tregs from patients with autoimmune diseases and allergic asthma and is also diminished in Tregs of mice that are considered autoimmune prone. In the relevant pathway in Tregs, GRAIL normally blocks cullin ring ligase activity, which inhibits IL-2R desensitization in Tregs and consequently promotes Treg function. As a result of this defect in GRAIL expression, the Tregs of patients with autoimmune diseases and allergic asthma degrade IL-2R-associated pJAK1 following activation with low dose IL-2, and thus cannot maintain pSTAT5 expression. pSTAT5 controls the transcription of genes required for Treg function. Additionally, the GRAIL-mediated defect may also allow the degradation of the mTOR inhibitor, DEP domain-containing mTOR interacting protein (Deptor). This can lead to IL-2R activation of mTOR and loss of Treg stability in autoimmune patients. Using a monoclonal antibody to the remnant di-glycine tag on ubiquitinated proteins after trypsin digestion, we identified a protein that was ubiquitinated by GRAIL that is important in Treg function, cullin5. Our data demonstrate that GRAIL acts a negative regulator of IL-2R desensitization by ubiquitinating a lysine on cullin5 that must be neddylated to allow cullin5 cullin ring ligase activity. We hypothesize that a neddylation inhibitor in combination with low dose IL-2 activation could be used to substitute for GRAIL and restore Treg function and stability in the Tregs of autoimmune and allergic asthma patients. However, the neddylation activating enzyme inhibitors (NAEi) are toxic when given systemically. By generating a protein drug conjugate (PDC) consisting of a NAEi bound, via cleavable linkers, to a fusion protein of murine IL-2 (to target the drug to Tregs), we were able to use 1000-fold less of the neddylation inhibitor drug than the amount required for therapeutically effective systemic delivery. The PDC was effective in blocking the onset or the progression of disease in several mouse models of autoimmunity (type 1 diabetes, systemic lupus erythematosus, and multiple sclerosis) and a mouse model of allergic asthma in the absence of detectable toxicity. This PDC strategy represents targeted drug delivery at its best where the defect causing the disease was identified, a drug was designed and developed to correct the defect, and the drug was targeted and delivered only to cells that needed it, maximizing safety and efficacy.

Absolute decrease in regulatory T cells and low-dose interleukin-2 therapy: restoring and expanding regulatory T cells to treat systemic sclerosis: a 24-week study.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular lesions, immunological alterations and tissue fibrosis. There is some evidence of an imbalance between T-cell subsets in this disease. Interleukin (IL)-2 is a cytokine that can regulate the activity of immune cells and there is evidence that low-dose IL-2 therapy can be used to treat immune diseases. To investigate the changes of peripheral lymphocyte subsets, especially T helper (Th)17 and regulatory T (Treg) cells and the effects of low-dose IL-2 therapy in patients with SSc. In total, 66 patients with SSc and 49 sex- and age-matched healthy controls (HCs), were enrolled. The absolute numbers of peripheral lymphocyte subsets in these individuals were determined by flow cytometry. The 66 patients, were divided into 2 groups: 23 (the IL-2 group) were treated with low-dose (5.0 × 105 IU) IL-2 by subcutaneous injection daily for 5 days combined with conventional therapy, while the remaining 23 patients received conventional therapy only. Compared with HCs, the absolute numbers of peripheral T, CD4+ T, CD8+ T, natural killer and Treg cells were significantly lower in patients with SSc, with the most dramatic difference seen in both the absolute number and percentage of Treg cells in these patients, including new (previously untreated) cases, resulting in an imbalance (elevated ratio) between Th17 and Treg cells. At Week 24 after commencement of IL-2 treatment, Treg cells were markedly increased and tended to restore the balance of Th17 to Treg cells compared with baseline. Erythrocyte sedimentation rate, C-reactive protein, modified Rodnan Skin Score and visual analogue scale score were significantly decreased in both the IL-2 and non-IL-2 groups, indicating disease improvement. Notably, compared with those in the non-IL-2 group, patients treated with IL-2 had greater improvement. Our study showed that the absolute numbers of peripheral Treg cells together with total T, CD4+ T, CD8+ T and NK is significantly decreased, leading to an imbalance of Th17 to Treg cells in patients with SSc, and that low-dose IL-2 treatment could restore the balance of the two immune cells and reduce disease activity without obvious adverse effects.

Opportunities for correction of immunosuppression in patients with COVID-19

Here, we review thematic publications in available literature sources of the databases PubMed, Scopus, Web of Science, eLibrary, 49 of which were dated of the years 19972022. Analysis of such reports is aimed at assessing features of cytokine storm-induced hyperinflammatory reaction with signs of immunosuppression accompanied by pronounced lymphopenia and lowered count of CD4+T helpers during severe COVID-19. The prognostic factor for unfavorable prognosis was based on the marker of systemic inflammatory reaction correlating with the disease severity the soluble IL-2 receptor as well as the neutrophil-to-lymphocyte ratio and the lymphocyte subset imbalance. An immunosuppressive therapy of severe forms of COVID-19, aimed at weakening the inflammatory response, exacerbates immune dysfunction by suppressing the T cell function, mainly due to Th1 lymphocytes involved in recognizing and eliminating intracellular pathogens particularly viruses. Upon that, cell-mediated immunity becomes compromised that relies on cytotoxic T-lymphocytes, natural killer cells and macrophages. Timely and targeted immunocorrection is required to prevent or reduce the immunosuppression that accompanies a severe disease course and leads to serious and prolonged complications, as well as to association of secondary infections. In fight against the cytokine storm, it is important not to miss a time point of developing immunosuppressive condition that transitions into immunoparalysis as follows from recent publications covering the tactics of treating immune-mediated complications of coronavirus infection. The review discusses opportunities for immunosuppressive therapy along with glucocorticosteroids and monoclonal antibodies blocking IL-6 or cognate receptors. Studies using mesenchymal stem cells (MSCs) to reduce systemic inflammatory response at COVID-19 are outlined in the review. The use of antigen-specific Treg and their combinations with antagonists of tumor necrosis factor- (TNF), interferon- (IFN) as well as low-dose IL-2 in patients with SARS-CoV-2 infection were analyzed. The prognostic perspectives for CAR-T cells and CAR-NK cells technology have been considered as novel therapeutic approaches aimed at training effector cells to recognize the surface SARS-CoV-2 virus spike-like (S) protein. The feasibility of a therapeutic approach is also emphasized by comparatively analyzed of efficacy of using IL-7 or IL-15 during lymphopenia in patients with COVID-19. Here, side effects complicating immunocorrection come to the fore. Critical evaluation of corrected immunosuppressive conditions in patients with COVID-19 in the post-COVID-19 period by using low-dose IL-2 therapy revealed its ability to repair cellular immune response. As a result, a low-dose IL-2 therapy is recommended as a cytokine replacement therapy in such patients with COVID-19 during hyper-to-hypo-inflammatory phase transition in immune response.

NEO-TRA1: A CD25-Targeted De Novo non-Alpha Agonist of the IL-2 Receptor Selectively Expands Regulatory T Cells

Introduction: Regulatory T cells (Tregs) are crucial for maintaining immune tolerance and their dysfunction contributes to inflammatory and autoimmune conditions, such as graft versus host disease (GVHD). IL-2 is a pleiotropic cytokine that activates both immunosuppressive Tregs and inflammatory cells, including NK cells, cytotoxic T cells, and helper T cells. Although low dose IL-2 therapy effectively expands Tregs, this intervention is limited by coincident stimulation of conventional T cells and natural killer cells. Investigational therapeutics derived from directed mutagenesis of native IL-2 sequences have resulted in some improvements in Treg selectivity. To optimize the magnitude and selectivity of Treg expansion, we utilized de novo protein design technology to fine-tune the individual receptor interfaces of IL-2 mimetic proteins targeted to Tregs with CD25 antibodies. We found decoupling receptor agonism and targeting is an effective means of improving Treg stimulation and corresponding therapeutic candidates elicit robust expansion of Treg populations in vivo without detectable stimulation of inflammatory immune cells. Methods: We designed a series of de novo IL-2Ra independent agonists of the IL-2 receptor that demonstrate reduced binding affinity to CD122 and CD132, the beta and gamma chains of the IL-2 receptor complex, by up to 5 logs when compared to IL-2. These non-alpha IL-2 mimetic variants were fused to a humanized anti-CD25 mAb that lacks effector function for cis-targeting to Tregs. Candidate molecules were screened on primary human T cells to assess pSTAT5 signaling. Treg-specific molecules were administered to humanized NSG mice weekly (four times) and peripheral blood was collected for immune cell profiling via flow cytometry. Results: Screening of TRA candidates in primary human T cells identified several highly selective Treg agonists, including NEO-TRA1 (Fig1). At all concentrations, CD8+ and CD4+ non-Tregs showed low or negligible levels of pSTAT5 signaling upon treatment with NEO-TRA1. Nevertheless, CD4+CD25highCD127low Tregs show increasing pSTAT5 signaling at a wide range of concentrations starting at 20pM, and demonstrate preferential signaling in Tregs by NEO-TRA1. To establish the ability of our TRA to expand Treg in vivo, NSG mice engrafted with human hematopoietic stem cells to create a humanized immune system received weekly injections of NEO-TRA1, vehicle control, or a previously described Treg selective IL-2 mutein (IL-2v-Fc). Characterization of Day 13 CD4+CD25+Foxp3+ Treg percentages in peripheral blood mononuclear cells show that the Treg population expanded to ~30% of total CD4+ cells in mice receiving NEO-TRA1, while Treg percentages reached ~22% in mice receiving IL-2v-Fc (Fig2). Despite a slight decrease at Day 20, the Treg percentages remained higher in NEO-TRA1-treated cohorts than in control groups throughout the study. Conclusions: NEO-TRA1 exhibits potent expansion of Treg populations within primary human samples and in humanized mouse models. The observed profile of STAT5 phosphorylation demonstrates selective activation of Tregs over other lymphocyte populations. NEO-TRA1 is the first Treg agonist based on de novo protein design. NEO-TRA1 is both specific and potent and has high therapeutic potential for the treatment of GVHD and other autoimmune or inflammation diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Responses of regulatory and effector T-cells to low-dose interleukin-2 differ depending on the immune environment after allogeneic stem cell transplantation.

CD4+Foxp3+ regulatory T cells (Tregs) play a central role in the maintenance of immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). Tregs promptly respond to low concentrations of IL-2 through the constitutive expression of high-affinity IL-2 receptors. It has been reported that low-dose IL-2 therapy increased circulating Tregs and improved clinical symptoms of chronic GVHD. Clinical studies of IL-2 therapy so far have mainly targeted patients in the chronic phase of transplantation when acute immune responses has subsided. However, the biological and clinical effects of exogenous IL-2 in an acute immune environment have not been well investigated. In the current study, we investigated the impact of exogenous IL-2 therapy on the post-transplant homeostasis of T cell subsets which influence the balance between GVHD and GVL in the acute phase, by setting the various immune environments early after HSCT in murine model. We initially found that 5,000 IU of IL-2 was enough to induce the active proliferation of Treg without influencing other conventional T cells (Tcons) when administered to normal mice. However, activated Tcons showed the response to the same dose of IL-2 in recipients after allogeneic HSCT. In a mild inflammatory environment within a threshold, exogenous IL-2 could effectively modulate Treg homeostasis with just limited influence to activated T cells, which resulted in an efficient GVHD suppression. In contrast, in a severely inflammatory environment, exogenous IL-2 enhanced activated T cells rather than Tregs, which resulted in the exacerbation of GVHD. Of interest, in an immune-tolerant state after transplant, exogenous IL-2 triggered effector T-cells to exert an anti-tumor effect with maintaining GVHD suppression. These data suggested that the responses of Tregs and effector T cells to exogenous IL-2 differ depending on the immune environment in the host, and the mutual balance of the response to IL-2 between T-cell subsets modulates GVHD and GVL after HSCT. Our findings may provide useful information in the optimization of IL-2 therapy, which may be personalized for each patient having different immune status.

CEACAM1 is an IL-2R-dependent biomarker in patients with multiple autoimmune-diseases undergoing low-dose IL-2 therapy

Abstract Low-dose (LD) IL-2 is a promising strategy to boost Tregs in patient with autoimmune diseases. This therapeutic strategy would benefit by identifying biomarkers that are highly and selectively responsive to IL-2R signaling in Treg and Teff cells. To initially address this point, RNA-seq was used to screen for genes that are induced in CD4+ Foxp3+ Treg and CD4+ CD45RO+ T effector/memory (TEM) cells from normal subjects in vitro by IL-2R, but not TCR/CD28, signaling. CEACAM1 was identified as a candidate gene in Tregs 4 and 24 hours post-stimulation. However, CEACAM1 was optimally upregulated in Treg and TEM cells after an extended 5 day culture with anti-CD3/CD28 and IL-2. Notably, anti-IL-2 essentially completely inhibited this upregulation, indicating that CEACAM1 expression was strictly IL-2 dependent. Tregs were shown to express the long isoform of CEACAM1 with inhibitory ITIM domains and the ITIMs may be functionally active as most in vitro activated CEACAM1+ Tregs did not express Ki67. To assess CEACAM1 as an IL-2-dependent biomarker during LD-IL-2 therapy, CD25 and CEACAM1 were evaluated at the protein and RNA levels for patient samples from eight distinct autoimmune diseases. In all samples, CEACAM1 was upregulated after a 5-day induction course of LD-IL-2 in Tregs but not TEM cells. This finding correlated with the effect of LD-IL-2 on CD25. CEACAM1 levels returned to baseline 14 days post-LD-IL-2 treatment. After the induction course of LD-IL-2, most CEACAM1+ Tregs were Ki67−, consisting with an inhibitory role for CEACAM1. Collectively, our findings indicate that CEACAM1 is a biomarker for LD-IL-2 therapy whose expression is highly dependent on IL-2R signaling and suggest that CEACAM1 may function to regulate Treg homeostasis. Supported by R01AI131643

Low-dose IL-2 therapy limits the reduction in absolute numbers of peripheral lymphocytes in systemic lupus erythematosus patients with infection

Background Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder characterized by disturbed cellular and humoral immune responses. Dysregulations of immune system and immunosuppressive medications predispose SLE patients to infection. This study aims to investigate the alterations and absolute concentrations of lymphocyte subpopulations in SLE patients with different infection and their responses of low-dose IL-2 therapy. Methods A total of 333 patients with SLE without recent infection, 162 patients suffering infection, and age and sex-matched 132 healthy controls (HCs) were recruited. Of them, 54 SLE patients (including 41 non-infected group and 13 infected group) received a 5-day course of low-dose IL-2 administration at a dose of 0.5 million IU per day. Lymphocyte subpopulations were analyzed by flow cytometry. Results Patients with SLE had lower levels of lymphocyte subpopulations in peripheral blood such as T, B, NK, CD4 + T, CD8+ T, Th1, Th2, Th17, and Treg cells, and the reduction in these cells was more obvious in patients with infection (p <.05 to p <.01). Low-dose IL-2 effectively expanded T (p <.001), B (p <.001), CD4 + T (p <.01), CD8 + T (p <.001), Th1 (p <.01), Th17 (p <.1), and Treg cells (p <.01) of SLE patients, these cells were comparable to that of HCs after the IL-2 treatment. Conclusions Patients with SLE had insufficiency of circulating lymphocyte subsets. This phenomenon was more obverse in those accompanying infection, suggesting the low concentration of lymphocytes may be used as indicators of high infection risk in SLE patients. Low-dose IL-2 induced expansion of Treg cells and NK cells, which may contribute to the restoration of immune homeostasis in SLE patients.

69 - Low Dose IL-2 Therapy in Patients with Chronic Gvhd Induces PD-1+treg That Are Highly Activated, Immune Suppressive, and Proapoptotic

69 - Low Dose IL-2 Therapy in Patients with Chronic Gvhd Induces PD-1+treg That Are Highly Activated, Immune Suppressive, and Proapoptotic

Ex Vivo IL-2 Treatment Increases the Immunosuppressive Subset of T Lymphocytes to Suppress Allo-Immune Response

T lymphocytes are critical players in graft-versus-host disease (GVHD); however, their specific roles and mechanisms in GVHD have not been clearly defined. Co-inhibitory receptors including PD1, TIM3, TIGIT, and LAG3 play a key role in regulating T cell responses and maintaining immune homeostasis. TIM3 expression on lymphocytes was identified to induce immune tolerance in a mouse GVHD model; in addition, PD1 +TIM3 + double-positive T cells displayed more potent immunosuppression compared to PD1 + T cells. However, despite the strong immunosuppression exerted by PD1 +TIM3 + double-positive T cells, their clinical potential is greatly limited by their low cell number in peripheral blood. In this study, we introduce a novel method to isolate CD3 + cells from G-CSF mobilized peripheral blood stem cells (G-PBSCs), and culture CD3 +PD1 +TIM3 + lymphocytes to treat GVHD.Methods. The donors were subcutaneously injected with G-CSF (10μg/kg) for five days. G-PBSCs were collected from the donors using a COBE spectra cell separator. Then, the highly purified CD3 + cells were isolated by positive selection with magnetic-activated cell sorting (MACS) from G-PBSCs using CD3 Dynabeads™. Isolated CD3 + cells were cultured with a low concentration of IL-2 (50U/mL), and PD1, TIM3, LAG3, and TIGIT expressions were assessed using flow cytometry (FACS). CD3 +PD1 +TIM3 +, CD3 +PD1 +TIM3 +LAG3 +TIGIT - and CD3 +PD1 +TIM3 +LAG3 +TIGIT +cells are sorted and cultured with irradiated allo-MNCs for 4 days (Mixed Lymphocyte Reaction; MLR). We used 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure of T cells proliferation.Results. In normal, untreated G-PBSCs, very low percentages of cells are CD3 +PD1 +TIM3 +, CD3 +PD1 +LAG3 + and CD3 +PD1 +TIGIT + (0.6±0.4, 0.3±0.2 and 1.3±0.5% in G-PBSC, respectively). However, after treating G-PBSC cells with a low concentration of IL-2 (50 U/mL), we discovered the percentages of CD3 +PD1 +TIM3 +, CD3 +PD1 +LAG3 +, and CD3 +PD1 +TIGT + lymphocytes in G-PBSCs markedly increased by 19.6±5.9, 18.5±4.3, 17.7±6.5%, respectively. Of note, there was a very small change (~1.2 fold increase) in the total number of CD3 + lymphocytes, which indicates that the low dose IL-2 therapy alters subpopulations of T lymphocytes, rather than increasing the proliferation rate. Next, we tested the immunosuppressive capacity of the cultured CD3 +PD1 +TIM3 +cells. To do this, we used CSFE-MLR to measure T cell proliferation. CD3 +PD1 +TIM3 + lymphocytes were depleted in CD3 + G-PBSCs by flow cytometry-based cell sorting. Then we cultured the CD3 + G-PBSCs with allo-MNCs and discovered that the CD3 + G-PBSCs that lacked CD3 +PD1 +TIM3 + lymphocytes demonstrated a significantly increased level of T-cell proliferation compared to CD3 + G-PBSCs, and thereby confirming the immunosuppressive function of CD3 +PD1 +TIM3 + lymphocytes in inhibiting T cell proliferation (% of unstimulated T cells in CD3 + G-PBSCs vs. in CD3 +PD1 +TIM3 + depleted G-PBSC CD3 + cells; 33.5±7.5% vs. 6.3±4.2%).After confirming the immunosuppressive function of the IL-2 treated CD3 +PD1 +TIM3 + lymphocytes, we investigated expressions of other co-inhibitory surface markers such as LAG3 and TIGIT. Amongst the cultured CD3 +PD1 +TIM3 + lymphocytes, 96.2±3.3% of the CD3 +PD1 +TIM3 + lymphocytes were LAG3 +, 28.9±5.4% of the CD3 +PD1 +TIM3 + lymphocytes were TIGIT +, and 63.7±7.8% of the cells were TIGIT -. Interestingly, CD3 +PD1 +TIM3 +LAG3 +TIGIT - lymphocytes demonstrated significantly enhanced levels of immunosuppression compared to CD3 +PD1 +TIM3 +LAG3 +TIGIT + lymphocytes (% of unstimulated T cells in CD3 +PD1 +TIM3 +LAG3 +TIGIT - lymphocytes vs. in CD3 +PD1 +TIM3 +LAG3 +TIGIT + lymphocytes, 53.8±5.5% vs. 23.2±7.4%).Conclusion.We demonstrate that treating CD3 + G-PBSCs with low-dose IL-2 markedly increases the percentage of CD3 +PD1 +TIM3 + lymphocytes, which is known to exert strong immunosuppression. Also, we suggest that CD3 +PD1 +TIM3 +LAG3 +TIGIT - lymphocytes are the subpopulation within CD3 +PD1 +TIM3 + cells that demonstrate the most potent immunosuppression compared to other subpopulations of CD3 +PD1 +TIM3 + lymphocytes. Taken together, such findings suggest that the low-dose IL-2 therapy has therapeutic potential to treat patients with GVHD. DisclosuresNo relevant conflicts of interest to declare.

Low-dose IL-2 therapy invigorates CD8+ T cells for viral control in systemic lupus erythematosus.

Autoimmune diseases are often treated by glucocorticoids and immunosuppressive drugs that could increase the risk for infection, which in turn deteriorate disease and cause mortality. Low-dose IL-2 (Ld-IL2) therapy emerges as a new treatment for a wide range of autoimmune diseases. To examine its influence on infection, we retrospectively studied 665 patients with systemic lupus erythematosus (SLE) including about one third receiving Ld-IL2 therapy, where Ld-IL2 therapy was found beneficial in reducing the incidence of infections. In line with this clinical observation, IL-2 treatment accelerated viral clearance in mice infected with influenza A virus or lymphocytic choriomeningitis virus (LCMV). Noticeably, despite enhancing anti-viral immunity in LCMV infection, IL-2 treatment exacerbated CD8+ T cell-mediated immunopathology. In summary, Ld-IL2 therapy reduced the risk of infections in SLE patients and enhanced the control of viral infection, but caution should be taken to avoid potential CD8+ T cell-mediated immunopathology.

Cohort study of off-label use of low-dose IL-2 therapy for systemic autoimmune diseases

Cohort study of off-label use of low-dose IL-2 therapy for systemic autoimmune diseases

T Follicular Regulatory (TFR) Cell Deficiency during Chronic Graft-Versus-Host Disease Is Improved By Low-Dose IL-2 Therapy

Background: Chronic Graft versus Host Disease (cGVHD) reflects the inability to achieve immune tolerance after allogeneic stem cell transplantation (allo-SCT) resulting in the persistence of allo and auto-reactive T cells and B cells. We previously showed that regulatory T cells (Treg) are decreased in patients with cGVHD and may be rescued by low dose IL-2 therapy resulting in improved Treg number and survival and clinical improvement of cGVHD symptoms. More recently, we showed that circulating T follicular helper cells (cTFH) have increased function during cGVHD, thus stimulating B cells to produce auto and allo-antibodies involved in cGVHD. T follicular regulatory cells (TFR) regulate the germinal center (GC) reaction and suppress B cell differentiation and immunoglobulin secretion. TFR reside primarily in lymphoid tissues but also circulate in small numbers. In the present study we characterized the numbers and function of blood CD3+CD4+CD45RA-CD25hiFoxP3+CXCR5+ TFR in healthy donors (HD; N=18) and allo-SCT patients (N=64).Results: Patients undergoing allo-SCT showed a significant reduction of circulating TFR (cTFR) compared to HD (median: 0.08% vs 0.34% of CD4+T respectively, p<10-4). Moreover, patients with active cGVHD (N=37) had significantly lower cTFR frequency compared to patients with no cGVHD (N=15) (median: 0.034 vs 0.18% of CD4+, p=0.0012) and resolved cGVHD (N=12) (median: 0.034 vs 0.14, p=0.02). Interestingly, cTFR values tended to be negatively associated with cGVHD grade (median cTFR %: 0.11 vs 0.029, p=0.004 for mild vs moderate; 0.11 vs 0.012, p=0.005 for mild vs severe; 0.029 vs 0.012, p=0.2 for moderate vs severe). The numeric reduction of cTFR in cGVHD patients appears to be a consequence of decreased survival as attested by lower BCL-2 expression (median: 1831 vs 1939, p=0.037 in active cGVHD and without cGVHD, respectively). This favors the survival of TFH effector cells (CD4+CD45RA-CXCR5+ excluding CD25hiFoxP3+) as measured by the ratio of expression of BCL-2 in effectors/regulators in active compared to no cGVHD (BCL-2 E/R ratio: 1.26 vs 1.14, p=0.0013 respectively). Similar to tonsil TFR, blood TFR exhibit suppressive functions in in vitro assays. Purified TFR were co-cultured with TFH and memory B cells at different ratios. The addition of TFR was associated with a significant reduction of plasmablast (PB) generation (CD27hiCD38hiCD19+ cells) (Figure 1A, t-test: p=0.0087) and IgG production (Figure 1B, t-test: p=0.0087). Human cTFR in vivo display similar suppressive function toward B cells, as analysis of active cGVHD patients revealed an inverse correlation between TFR and PB frequency in the peripheral blood (Spearman test: r = -0.49, p=0.003) (Figure 1C). As IL-2 has been shown to enhance regulatory T cells, we assessed its impact on the GC reaction in vitro . Purified TFH and memory B cells from HD were co-cultured at different doses of IL-2 from 0 to 100 IU/mL, simulating a germinal center reaction in vitro . Low dose IL-2 starting at 10 IU/mL was associated with a suppressive effect on PB generation and IgG production that peaked at 50 IU/mL reflecting the high expression level of IL-2 high affinity receptor (CD25). We also evaluated the capacity of IL-2 to restore in vivo cTFR:cTFH ratio using mass cytometry (CyTOF) in a cohort of patients receiving IL-2 (1x106 IU/M2/day) for 12 weeks as previously described (Koreth et al, Blood 2016). We observed an improvement of blood cTFR:cTFH ratio over the course of IL-2 therapy, with a maximum ratio observed at 2 weeks, and subsequently decreasing to reach baseline values after stopping IL-2 (Figure 2).Conclusion: Our results confirm that blood TFR exert a suppressive function as evidence by an inverse correlation with PB and in vitro suppression of memory B cell Ig production. This suppressive subset is deficient in patients with cGVHD due to altered TFR survival, thus favoring imbalance toward TFH effectors which may promote a GC reaction in vivo resulting in PB and memory B cell pathogenic Ig production. By enhancing TFR, low-dose IL-2 therapy promotes effective control of the GC reaction. [Display omitted] DisclosuresForcade:Neovii: Other: Travel grant. Koreth:Millennium Pharmaceuticals: Research Funding; Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Roche: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Sequenta/Adaptive: Research Funding; Affimed: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Sigma Tau: Research Funding; Otsuka: Research Funding; Pfizer: Consultancy, Research Funding; Infinity: Consultancy; Tensha: Research Funding; Genmab: Consultancy. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Therapeutic potential of targeting Tfr/Tfh cell balance by low-dose-IL-2 in active SLE: a post hoc analysis from a double-blind RCT study

ObjectiveTo investigate the regulation of T follicular regulatory (Tfr) and T follicular (Tfh) cell subtypes by low-dose IL-2 in systemic lupus erythematosus (SLE) in a randomized, double-blind, placebo-controlled clinical trial.MethodsA post hoc analysis was performed in a randomized cohort of SLE patients (n=60) receiving low-dose IL-2 therapy (n=30) or placebo (n=30), along with the standard of care treatment. The primary endpoint was the attainment of SLE responder index-4 (SRI-4) at week 12 in the trial. Twenty-three healthy controls were enrolled for T cell subset detection at the same time as the trial. The t-stochastic neighbor embedding (tSNE) analysis of CD4 T subsets based on immune cells flow cytometry markers was performed to distinguish Tfh, Tfh1, Tfh2, Tfh17, and Tfr cell subsets.ResultsCompared with HC, the frequency of Tfr (CXCR5+PD-1low Treg and CXCR5+PD-1high Treg) cells was significantly reduced, while the pro-inflammatory Tfh cells were increased in patients with SLE. The imbalanced Tfh cell was associated with several pathogenic factors (anti-dsDNA antibodies (r=0.309, P=0.027) and serum IL-17 (r=0.328, P=0.021)) and SLE Disease Activity Index (SLEDAI) score (r=0.273, P=0.052). Decreased CXCR5+PD-1low Treg/Tfh and CXCR5+PD-1low Treg/Tfh17 were both associated with increased immunoglobulin M (IgM) (r=−0.448, P=0.002 and r=−0.336, P=0.024, respectively). Efficacy of low-dose IL-2 therapy was associated with a restored Tfr/Tfh cell balance.ConclusionThese data support the hypothesis that promotion of Tfr is associated with decreased disease activities and that low-dose IL-2 therapy can recover Tfr/Tfh immune balance.Trial registration numberClinicalTrials.gov Registries (NCT02465580).