Study design and participants
In DIAGNODE-1 as described earlier , 12 individuals (4 females and 8 males), aged 12–24 years with type 1 diabetes < 6 months from diagnosis, received a primary injection of 4 μg of GAD-alum (Diamyd Medical, Stockholm) into an inguinal lymph node administrated by help of ultrasound technique, followed by two booster injections of 4 μg each with one-month interval. In parallel, patients also received vitamin D (calciferol) in oral solution (2000 U/d) for 4 months, starting 1 month prior to first GAD-alum injection. The participants were eligible if fasting C-peptide ≥ 0.12 nmol/L and GAD65 antibody levels (GADA) > 63.2 RU .
In the first-in-human DIAGNODE Extension study, the Research Ethics Board and the Swedish Medical Product Agency allowed the inclusion of three pilot adult patients who just ended the DIAGNODE-1 trial. They received a fourth injection of GAD-alum (4 μg) into an inguinal lymph node 1.5 months after the 30-month follow-up of DIAGNODE 1, that is, 31.5 months after the start of their first treatment in DIAGNODE-1, in combination with oral vitamin D intake one month before and one month after the fourth intra-lymphatic GAD-alum injection. The patients were followed with mixed meal tolerance tests (MMTTs)  and immunological studies for 12 months (NCT02352974).
Laboratory analyses were performed at Linköping University, Sweden. Blood and serum samples from all the DIAGNODE-1 participants were collected at baseline and after 15, 30 months. Additionally, samples from the three patients in DIAGNODE Extension were collected at 31.5, 33.5, 36.5 and 42.5 months. Samples were drawn during the morning hours, and peripheral blood mononuclear cells (PBMCs) were isolated within 24 h using Leucosep (Greiner Bio One) according to the manufacturer’s instructions.
Analysis of serum C-peptide was performed using a solid-phase two-sided enzyme immunoassay (Mercodia, Uppsala). Results for each assay were validated with the inclusion of a Diabetes Antigen Control Human (Low/High) (Mercodia, Uppsala, Sweden). The assay is calibrated against the international reference reagent for C-peptide IRR C-peptide 84/510. Inter- and intra-assay variations were 6.6% and 3.5%, respectively.
Serum antibodies and IgG subclasses
Serum GAD autoantibodies (GADA) were estimated in duplicate by means of a radio-binding assay, using 35S-labeled recombinant human GAD65 (rhGAD65) as previously described . Sepharose protein A was used to separate free from antibody-bound labeled GAD65.
GADA IgG1, IgG2, IgG3 and IgG4 subclasses were measured by radio-binding assays  using IgG subclass-specific biotin-labeled mouse–antihuman monoclonal antibodies bound on streptavidin sepharose high-performance beads (GE Healthcare Life Sciences, Freiburg, Germany) . Results were expressed as delta cpm (IgG subclass-specific cpm—anti-rat IgM cpm) and converted to arbitrary units (AUs) proportional to the GADA IgG subclass-specific delta cpm of a local standard serum.
Lymphocyte proliferation assay
Proliferative responses were analyzed in PBMCs cultured in triplicates in medium alone (AIM-V medium with β-mercaptoethanol), in the presence of 5 μg/mL rhGAD65 (Diamyd Medical, Stockholm, Sweden) or with CD3/CD28 beads (Gibco, Life Technologies AS, Oslo, Norway). After 3 days, cells were incubated with 0.2 µCi of [3H] thymidine/well (PerkinElmer) for 18 h and thereafter harvested. Proliferation was expressed as stimulation index (SI), calculated as the mean cpm of cells cultured triplicates in the presence of stimulus divided by the mean cpm of cells with medium alone.
Cytokine secretion assay
PBMCs were cultured for 7 days in the presence of 5 μg/mL rhGAD65 or in medium alone at 37 °C in 5% CO2, as previously described . The cytokines IL-2, IL-5, IL-10, IL-13, IL-17, tumor necrosis factor (TNF-α) and interferon (IFN-γ) were measured in cell culture supernatants using Bio-Plex Pro Cytokine Panel (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Data were collected using the Luminex 200 ™ (Luminex xMAP™ Corporation, Austin, TX USA). The antigen-induced cytokine secretion level was calculated by subtracting the spontaneous secretion (i.e. secretion from PBMCs cultured in medium alone) from the one following stimulation with GAD65.
PBMCs were washed in PBS containing 0.1% BSA and subsequently stained with Alexa-700-conjugated anti-CD3 (clone UCHT1, BD Biosciences), Pacific Blue-conjugated anti-CD4 (clone RPA-T4, BD Biosciences), allophycocyanin (APC)-H7-conjugated anti-CD8 (clone SK1, BD Biosciences), PerCP-Cy5.5-conjugated anti-CD45RA (clone HI100, BD Biosciences), phycoerythrin (PE)-conjugated anti-CCR7 (clone G043H7, Biolegend), FITC-conjugated anti-CD127 (clone eBioRDR5, eBioscience) and PE-Cy7-conjugated anti-CD25 (clone BC96, eBioscience). Then, cells were fixed and permeabilized using FOXP3 staining buffer set (eBioscience), according to the manufacturer’s instructions. Cells were then stained with APC-conjugated anti-FOXP3 (clone PCH101, eBiosciences) and acquired on a FACS Aria III (BD Biosciences) running FACS Diva version 8 software (Becton Dickinson). Data were analyzed using Kaluza version 1.3 (Beckman Coulter).
T cell differentiation was determined according to the expression of CD45RA and CCR7 as naive (TN, CD45RA + CCR7 +), central memory (TCM, CD45RA−CCR7 +) and effector memory (TEM, CD45RA−CCR7- and CD45RA + CCR7−).
Regulatory T cells were defined as CD25 + CD127-FOXP3 + CD4 + T cells. Further analysis of regulatory T cell subpopulations was based on the expression of FOXP3 and CD45RA as resting (rTreg, FOXP3low + CD45RA +), activated (aTreg, FOXP3high CD45RA−) and non-suppressive (nsTregFOXP3lowCD45RA−) regulatory T cells.
Induction of activated T cells was calculated as the difference between the percentage of CD25 + CD127 + T cells in GAD65-stimulated cultures and the percentage of CD25 + CD127 + T cells in medium alone.
In addition to C-peptide measurements during 2-h mixed meal tolerance test , we also used insulin dose U/kg body weight, 24 h as well as HbA1c. Insulin dose and HbA1c were combined to calculate insulin dose-adjusted A1c according to Mortensen et al.  whose results suggested the definition of an insulin dose-adjusted A1C (IDAA1C) as A1C (percent) + [4 × insulin dose (units per kilogram per 24 h)]. A calculated IDAA1C ≤ 9 corresponding to a predicted stimulated C-peptide > 300 pmol/l was used to define partial remission. To investigate immune response in relation to C-peptide preservation, patients were stratified into good responders (GR, n = 8; no loss of fasting C-peptide and loss of C-peptide AUC < 30%) and poor responders (PR, n = 4; decreasing fasting C-peptide and loss of C-peptide AUC ≥ 30%) according to C-peptide preservation and clinical response at 15 months, with cutoff for AUC 55% at 30 months.
Variables that followed a normal distribution (clinical data) were presented as mean. Differences between groups were calculated using Student t-test, and differences within groups were calculated by paired t test. Variables that follow a non-normal distribution (immune data), were presented as median and nonparametric tests were applied. Differences between groups were calculated using Mann–Whitney test, and for the calculation of differences within groups, Wilcoxon test was applied. A probability level of < 0.05 was considered statistically significant. Calculations were performed using GraphPad Prism 8.0.1 for Windows (GraphPad Software, La Jolla, CA, USA).