IL-6 receptor blockade does not slow β cell loss in new-onset type 1 diabetes

Abstract

BackgroundIL-6 receptor (IL-6R) signaling drives development of T cell populations important to type 1 diabetes pathogenesis. We evaluated whether blockade of IL-6R with monoclonal antibody tocilizumab would slow loss of residual β cell function in newly diagnosed type 1 diabetes patients.MethodsWe conducted a multicenter, randomized, placebo-controlled, double-blind trial with tocilizumab in new-onset type 1 diabetes. Participants were screened within 100 days of diagnosis. Eligible participants were randomized 2:1 to receive 7 monthly doses of tocilizumab or placebo. The primary outcome was the change from screening in the mean AUC of C-peptide collected during the first 2 hours of a mixed meal tolerance test at week 52 in pediatric participants (ages 6-17 years).ResultsThere was no statistical difference in the primary outcome between tocilizumab and placebo. Immunophenotyping showed reductions in downstream signaling of the IL-6R in T cells but no changes in CD4 memory subsets, Th17 cells, Tregs, or CD4+ T effector cell resistance to Treg suppression. A DC subset decreased during therapy but regressed to baseline once therapy stopped. Tocilizumab was well tolerated.ConclusionTocilizumab reduced T cell IL-6R signaling but did not modulate CD4+ T cell phenotypes or slow loss of residual β cell function in newly diagnosed individuals with type 1 diabetes.Trial RegistrationClinicalTrials.gov NCT02293837.FundingNIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and National Institute of Allergy and Infectious Diseases (NIAID) UM1AI109565, UL1TR000004 from NIH/National Center for Research Resources (NCRR) Clinical and Translational Science Award (CTSA), NIH/NIDDK P30DK036836, NIH/NIDDK U01DK103266, NIH/NIDDK U01DK103266, 1UL1TR000064 from NIH/NCRR CTSA, NIH/National Center for Advancing Translational Sciences (NCATS) UL1TR001878, UL1TR002537 from NIH/CTSA; National Health and Medical Research Council Practitioner Fellowship (APP1136735), NIH/NIDDK U01-DK085476, NIH/CTSA UL1-TR002494, Indiana Clinical and Translational Science Institute Award UL1TR002529, Vanderbilt Institute for Clinical and Translational Research UL1TR000445. NIH/NCATS UL1TR003142, NIH/CTSA program UL1-TR002494, Veteran Affairs Administration, and 1R01AI132774.

Keywords: Beta cells; Diabetes; Endocrinology; Immunology; T cells.

Conflict of interest statement

Conflict of interest: CJG and SL have research support for their clinical trial site from Pfizer. DMW received support from Tolerion. SEG has served on advisory boards for Avotres, Biolojic, Caladrius Biosciences, ImmunoMolecular Therapeutics, Provention Bio, SAB Biotherapeutics, and Tolerion and participated in clinical trials with Caladrius Biosciences, Intrexon, Janssen, Provention Bio, and Tolerion. He led a Data and Safety Monitoring Board (DSMB) for Novo Nordisk (pediatric type 2 diabetes). JLG is a consultant to Vertex Pharmaceuticals and receives research support from Avotres. CLS’s husband works for FUJIFILM Irvine Scientific. JHB is a scientific cofounder and Scientific Advisory Board member of GentiBio, is a consultant for Bristol Myers Squibb and HotSpot Therapeutics, and has past and current research projects sponsored by Amgen, Bristol Myers Squibb, Janssen, Novo Nordisk, and Pfizer. She is a member of the Type 1 Diabetes TrialNet, a partner of the Allen Institute for Immunology, and a member of the scientific advisory boards for the La Jolla Institute for Immunology and Bristol Myers

Figures

Figure 1. CONSORT diagrams for both cohorts.

(A) Pediatric participants and (B) adult participants. ANC, absolute neutrophil count; BILI, bilirubin; AST, aspartate aminotransferase.

Figure 2. Tocilizumab does not affect 2-hour C-peptide mAUC.

Markers represent the means, lines connect the medians, and error bars represent the 25th and 75th percentiles of C-peptide mAUC collected during the first 2 hours of the mixed meal tolerance test (MMTT) shown over the first year for (A) the pediatric cohort and (B) the adult cohort. ANCOVA models and mixed model analysis did not detect any statistically significant differences between the treatment groups at key time points.

Figure 3. Tocilizumab does not affect insulin usage and glucose control.

Markers represent the means, lines connect the medians, and error bars represent the 25th and 75th percentiles. HbA1c values for (A) the pediatric cohort and (B) the adult cohort. Average daily insulin usage expressed as total daily units/kg for (C) the pediatric cohort and (D) the adult cohort. ANCOVA models and mixed model analysis did not detect any statistically significant differences between the treatment groups at key time points.

Figure 4. Tocilizumab impairs IL-6R signaling in T cells.

(A) Gating strategy of CD4+ Tregs and memory Teff cells and phosphorylated STAT3 (p-STAT3) expression after in vitro IL-6 stimulation, at baseline and week 12 of treatment of a representative treated patient. Longitudinal fold changes from baseline for (B) percentage of CD4+ Tem cells expressing p-STAT3, (C) MFI of p-STAT3 in CD4+ Tem cells, (D) percentage of Tregs expressing p-STAT3, (E) MFI of IL-6R in total CD4. Y axis scales are log transformed. Mean fold changes from baseline are presented at each visit. Error bars display SEM. P values were calculated using repeated measure 2-way ANOVA model. Statistically significant comparisons are shown with asterisks (***P < 0.0001; **P ≤ 0.001; *P ≤ 0.05). DN, double negative.

Figure 5. Tocilizumab does not alter frequency of CD4+ T cell subsets.

(A) Percentage of Tregs in total CD4. Percentage of (B) memory, (C) central memory, (D) effector memory, (E) Th17 (IL-17a+), (F) Tfh (IL-21+) in CD4+ Teff cells. Y axis scales are log transformed. Mean values are presented at each visit. Error bars display SEM. Repeated measure 2-way ANOVA did not detect any statistically significant differences between the treatment groups at key time points.

Figure 6. Tocilizumab does not alter Teff cell response to Treg suppression.

Percentage suppression of CD25 expression on Teff cells by Tregs. For all time points, Teff cells were cocultured with EF670-labeled Tregs and anti-CD3/anti-CD28 Dynabeads (1:28 beads/Teff) for 48 hours. Four Treg/Teff ratios were tested: 1:4 (top left), 1:8 (top right), 1:16 (bottom left), and 1:32 (bottom right). For flow cytometry, Teff cells were in the EF670– gate and stained with anti-CD25 PE-Cy7. Percentage suppression was calculated as follows: s = ([a – b]/a) × 100, where a is the percentage CD25+ in the absence of Tregs and b is the percentage of CD25+ in the presence of Tregs. Data are represented as mean ± SEM. There were no statistical differences between placebo and tocilizumab at any Treg/Teff ratio or any time point. Repeated measures 2-way ANOVA did not detect any statistically significant differences between the treatment groups at key time points.

Figure 7. Tocilizumab changes frequencies of DC populations.

(A) Gating strategy for DC subsets (plasmacytoid DCs, pDCs; conventional type 1 DCs, cDC1s; and conventional type 2 DCs, cDC2s) and classical, nonclassical, and intermediate monocytes. (B) Percentage of classical monocytes in HLA-DR+ cells. (C) Percentage of pDCs (CD123+HLA-DR+) in CD14loCD1c– cells. (D) Percentage of cDC1s (CD141hiCD1c–) and (E) cDC2s (CD141loCD1c+) in CD14–CD16–cells. Y axis scales are log transformed and presented in actual scale. Mean values are presented at each visit. Error bars display SEM. P values were calculated using repeated measure 2-way ANOVA model. Statistically significant comparisons are shown with asterisks (***P < 0.0001; *P ≤ 0.05).

Figure 8. Increased serum IL-6 and IL-6R with tocilizumab therapy.

(A) C-reactive protein, (B) IL-6, and (C) soluble IL-6R. Y axis scales are log transformed and presented in actual scale for IL-6 and IL-6R analytes. C-reactive protein is plotted in actual scale. Mean values are presented at each visit. Error bars display SEM. P values were calculated using repeated measure 2-way ANOVA model. (***P < 0.0001.)