Low-dose IL-2 reduces IL-21+ T cell frequency and induces anti-inflammatory gene expression in type 1 diabetes.

Despite early clinical successes, the mechanisms of action of low-dose interleukin-2 (LD-IL-2) immunotherapy remain only partly understood. Here we examine the effects of interval administration of low-dose recombinant IL-2 (iLD-IL-2) in type 1 diabetes using high-resolution single-cell multiomics and flow cytometry on longitudinally-collected peripheral blood samples. Our results confirm that iLD-IL-2 selectively expands thymic-derived FOXP3+HELIOS+ regulatory T cells and CD56bright NK cells, and show that the treatment reduces the frequency of IL-21-producing CD4+ T cells and of two innate-like mucosal-associated invariant T and Vγ9Vδ2 CD8+ T cell subsets. The cellular changes induced by iLD-IL-2 associate with an anti-inflammatory gene expression signature, which remains detectable in all T and NK cell subsets analysed one month after treatment. These findings warrant investigations into the potential longer-term clinical benefits of iLD-IL-2 in immunotherapy.

Amyotrophic lateral sclerosis transcriptomics reveals immunological effects of low-dose interleukin-2.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease causing upper and lower motor neuron loss and currently no effective disease-modifying treatment is available. A pathological feature of this disease is neuroinflammation, a mechanism which involves both CNS-resident and peripheral immune system cells. Regulatory T-cells are immune-suppressive agents known to be dramatically and progressively decreased in patients with amyotrophic lateral sclerosis. Low-dose interleukin-2 promotes regulatory T-cell expansion and was proposed as an immune-modulatory strategy for this disease. A randomized placebo-controlled pilot phase-II clinical trial called Immuno-Modulation in Amyotrophic Lateral Sclerosis was carried out to test safety and activity of low-dose interleukin-2 in 36 amyotrophic lateral sclerosis patients (NCT02059759). Participants were randomized to 1MIU, 2MIU-low-dose interleukin-2 or placebo and underwent one injection daily for 5 days every 28 days for three cycles. In this report, we describe the results of microarray gene expression profiling of trial participants' leukocyte population. We identified a dose-dependent increase in regulatory T-cell markers at the end of the treatment period. Longitudinal analysis revealed an alteration and inhibition of inflammatory pathways occurring promptly at the end of the first treatment cycle. These responses are less pronounced following the end of the third treatment cycle, although an activation of immune-regulatory pathways, involving regulatory T-cells and T helper 2 cells, was evident only after the last cycle. This indicates a cumulative effect of repeated low-dose interleukin-2 administration on regulatory T-cells. Our analysis suggested the existence of inter-individual variation amongst trial participants and we therefore classified patients into low, moderate and high-regulatory T-cell-responders. NanoString profiling revealed substantial baseline differences between participant immunological transcript expression profiles with the least responsive patients showing a more inflammatory-prone phenotype at the beginning of the trial. Finally, we identified two genes in which pre-treatment expression levels correlated with the magnitude of drug responsiveness. Therefore, we proposed a two-biomarker based regression model able to predict patient regulatory T-cell-response to low-dose interleukin-2. These findings and the application of this methodology could be particularly relevant for future precision medicine approaches to treat amyotrophic lateral sclerosis.

Repeated 5-day cycles of low dose aldesleukin in amyotrophic lateral sclerosis (IMODALS): A phase 2a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Low-dose interleukin-2 (ld-IL-2) enhances regulatory T-cell (Treg) function in auto-inflammatory conditions. Neuroinflammation being a pathogenic feature of amyotrophic lateral sclerosis (ALS), we evaluated the pharmacodynamics and safety of ld-IL-2 in ALS subjects. METHODS: We performed a single centre, parallel three-arm, randomised, double-blind, placebo-controlled study. Eligibility criteria included age < 75 years, disease duration < 5 years, riluzole treatment > 3 months, and a slow vital capacity ≥ 70% of normal. Patients were randomised (1:1:1) to aldesleukin 2 MIU, 1 MIU, or placebo once daily for 5 days every 4 weeks for 3 cycles. Primary outcome was change from baseline in Treg percentage of CD4+ T cells (%Tregs) following a first cycle. Secondary laboratory outcomes included: %Treg and Treg number following repeated cycles, and plasma CCL2 and neurofilament light chain protein (NFL) concentrations as surrogate markers of efficacy. Safety outcomes included motor-function (ALSFRS-R), slow vital capacity (SVC), and adverse event reports. This trial is registered with ClinicalTrials.gov, NCT02059759. FINDINGS: All randomised patients (12 per group), recruited from October 2015 to December 2015, were alive at the end of follow-up and included in the intent-to-treat (ITT) analysis. No drug-related serious adverse event was observed. Non-serious adverse events occurred more frequently with the 1 and 2 MIU IL-2 doses compared to placebo, including injection site reactions and flu-like symptoms. Primary outcome analysis showed a significant increase (p < 0·0001) in %Tregs in the 2 MIU and 1 MIU arms (mean [SD]: 2 MIU: +6·2% [2·2]; 1 MIU: +3·9% [1·2]) as compared to placebo (mean [SD]: -0·49% [1·3]). Effect sizes (ES) were large in treated groups: 2 MIU ES=3·7 (IC95%: 2·3-4·9) and 1 MIU ES=3·5 (IC95%: 2·1-4·6). Secondary outcomes showed a significant increase in %Tregs following repeated cycles (p < 0·0001) as compared to placebo, and a dose-dependent decrease in plasma CCL2 (p = 0·0049). There were no significant differences amongst the three groups on plasma NFL levels. INTERPRETATION: Ld-IL-2 is well tolerated and immunologically effective in subjects with ALS. These results warrant further investigation into their eventual therapeutic impact on slowing ALS disease progression. FUNDING: The French Health Ministry (PHRC-I-14-056), EU H2020 (grant #633413), and the Association pour la Recherche sur la SLA (ARSLA).

Phenotypic Analysis of Human Lymph Nodes in Subjects With New-Onset Type 1 Diabetes and Healthy Individuals by Flow Cytometry.

Background: Ultrasound guided sampling of human lymph node (LN) combined with advanced flow cytometry allows phenotypic analysis of multiple immune cell subsets. These may provide insights into immune processes and responses to immunotherapies not apparent from analysis of the blood. Methods: Ultrasound guided inguinal LN samples were obtained by both fine needle aspiration (FNA) and core needle biopsy in 10 adults within 8 weeks of diagnosis of type 1 diabetes (T1D) and 12 age-matched healthy controls at two study centers. Peripheral blood mononuclear cells (PBMC) were obtained on the same occasion. Samples were transported same day to the central laboratory and analyzed by multicolour flow cytometry. Results: LN sampling was well-tolerated and yielded sufficient cells for analysis in 95% of cases. We confirmed the segregation of CD69+ cells into LN and the predominance of CD8+ Temra cells in blood previously reported. In addition, we demonstrated clear enrichment of CD8+ naïve, FOXP3+ Treg, class-switched B cells, CD56bright NK cells and plasmacytoid dendritic cells (DC) in LNs as well as CD4+ T cells of the Th2 phenotype and those expressing Helios and Ki67. Conventional NK cells were virtually absent from LNs as were Th22 and Th1Th17 cells. Paired correlation analysis of blood and LN in the same individuals indicated that for many cell subsets, especially those associated with activation: such as CD25+ and proliferating (Ki67+) T cells, activated follicular helper T cells and class-switched B cells, levels in the LN compartment could not be predicted by analysis of blood. We also observed an increase in Th1-like Treg and less proliferating (Ki67+) CD4+ T cells in LN from T1D compared to control LNs, changes which were not reflected in the blood. Conclusions: LN sampling in humans is well-tolerated. We provide the first detailed "roadmap" comparing immune subsets in LN vs. blood emphasizing a role for differentiated effector T cells in the blood and T cell regulation, B cell activation and memory in the LN. For many subsets, frequencies in blood, did not correlate with LN, suggesting that LN sampling would be valuable for monitoring immuno-therapies where these subsets may be impacted.

Untreated relapsing remitting multiple sclerosis patients show antibody production against latent Epstein Barr Virus (EBV) antigens mainly in the periphery and innate immune IL-8 responses preferentially in the CNS.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory and neurodegenerative disorder of the central nervous system (CNS). Reliable biomarkers are urgently needed for its diagnosis and management, and as clues to its pathogenesis, in which EBV is implicated. OBJECTIVE: To measure IgG antibodies against EBV nuclear antigen-1 (EBNA-1) and innate inflammation status in paired serum and cerebrospinal fluid (CSF) samples from untreated relapsing-remitting MS (RRMS) patients. MATERIALS AND METHODS: Anti-EBNA-1 IgG titers and IL-8, IL-1ß, IL-6, IL-10, TNF-α and IL-12p70 cytokine levels were measured in 20 untreated RRMS-patients and 17 healthy controls. RESULTS: We found higher serum anti-EBNA-1 IgG and IL-8 levels in RRMS-patients than in healthy controls. Interestingly, levels of IL-8 - relative to total protein - were much higher in the CSF, whereas the anti-EBNA-1 antibodies were significantly higher in the sera. More detailed analysis showed that anti-EBNA-1 antibodies relative to total IgG were also higher in the serum in the majority of RRMS patients compared to CSF. Levels of anti-EBNA-1 IgG and IL-8 showed a strong correlation between serum and CSF. CONCLUSION: These findings in newly diagnosed RRMS-patients imply anti-EBNA-1 antibody production mainly in the periphery and innate immune responses preferentially in the CNS. Both their potential as disease biomarkers and their implications for the pathogenesis of MS warrant further investigation.