biostudies-arrayexpressStephanie HannaHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-17069We used intradermal injection of GAD-Alum to recruit GAD-specific T cells to accessible sites in the skin and skin-draining lymph nodes (LNs), sampled by skin suction blisters and ultrasound-guided LN aspiration. Peripheral blood samples obtained before GAD injection were restimulated with GAD in vitro to detect reactive CD4+ T cells. Sequencing was performed using 10xGenomics 5' kits (V1 and V2). Samples from LN and blisters were run individually. PBMC samples were combined in a singles 10x reaction using hashing antibodies. LNs include GEX, TCRS and BCRs. Blisters include GEX and TCRs. PBMC include GEX, TCRs, hashing and a panel of cell surface antibodies.biostudies-arrayexpressSample Collection - skin blister: Skin suction blisters were raised as described previously. Briefly, suction cups were applied to the deltoid region of participants’ arms, at the site of previous injection. Skin suction blisters were performed by gradually applying negative pressure (up to 60 kPa) from a suction pump machine VP25 (Eschmann, Lancing, UK) through a suction blister cup with a 15-mm hole in the base (UHBristol NHS Foundation Trust Medical Engineering Department, Bristol, UK) for 2–4 hr until a unilocular blister had formed within the cup. The cup was left in place for a further 30-60 minutes to encourage migration of lymphocytes into the blister fluid. The cup was then removed, and the blister fluid aspirated using a needle and syringe. Blister fluid was immediately suspended in 10ml heparinised RPMI (Gibco)+ 5% foetal calf serum. Cells were counted then washed once and resuspended in an appropriate volume for scRNAseq. Samples were used fresh on the day of the collection.Library Construction - standard 10x library preparation was followed, specific for v1 or V2 chemistryNucleic Acid Extraction - Cells were processed using a 10xgenomics chromium controller and a 5' v1 or V2 kitSequencing - libraries were sequenced on Illumina machinesSample Collection - Ultrasound-guided Fine needle aspiration of LNs Ultrasound-guided FNA was performed as previously reported. Under aseptic conditions, the skin and s.c. tissues down to the identified LN were infiltrated by 1–2 ml 1% (w/v) xylocaine with 1:200,000 adrenaline. Under real-time ultrasound visualization, using a 21-gauge needle and a 5-ml syringe, the LN cortex was sampled. One to two passages were used per sample. The LN sample was immediately transferred to tissue culture medium—5% (v/v) FCS (Biosera) in RPMI 1640 medium (Gibco) and transported to the laboratory at ambient temperature. PBMC were isolated using Lymphoprep (StemCell Technologies) and frozen in Cryostor CS10 (Sigma) until required.Sample Collection - PBMCs were isolated on Ficoll–Paque Plus (GE Healthcare Biosciences, Sweden) gradient centrifugation from the peripheral blood of participants immediately prior to injection of GAD-Alum. Samples were cryopreserved in CryoStor (Sigma-Aldrich, Gillingham, UK) on the day of the collection and thawed on the day of the experiment. In vitro stimulation and flow cytometric sorting of CD4+ T cells The Activation-Induced Marker AIM assay was performed similarly to previously published. Briefly PBMC from donors pre or post in vivo injection of GAD Alum, were thawed and stimulated for 18 hours in one of four conditions in X-VIVO media with 10% human AB serum at 4x106 cells/ml(Merck): 1. Unstimulated 2. Whole GAD (Diamyd) (no Alum) in buffer (final concentration 10μg/ml). 3.Control-using the GAD dilution buffer only 4. Staphylococcal Enterotoxin B (SEB) (final concentration 50ng/ml). Following this, cells were stained with a cocktail of flow cytometry, cell hashing and CITE-seq antibodies. CD4+ T cells were then sorted as follows : SEB, GAD and buffer control- Effector T cells: CD154+ and either CD69+ or CD137+ or both; Tregs: CD154- CD137+ and either CD69+ or GARP+ or both. For GAD and buffer control conditions - all cells were sorted from the positive gate. For SEB-stimulated - 100 activated Tregs and 1000 activated effectors were sorted. From the unstimulated condition, 1000 cells negative for all four activation markers were sorted. All cells were processed for scRNAseq on the day of sorting.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesUnknownTranscriptomicsGenomicsProteomics10x genomic Chromium controllerNextSeq 500Identifying and monitoring autoreactive T cells that drive beta cell destruction remains a major obstacle to developing effective immunotherapies for type 1 diabetes (T1D). These cells are extremely rare in peripheral blood and cannot be accessed directly from the pancreas. We used intradermal injection of GAD-Alum to recruit GAD-specific T cells to accessible sites in the skin and skin-draining lymph nodes (LNs), sampled by skin suction blisters and ultrasound-guided LN aspiration. Peripheral blood samples obtained before GAD injection were restimulated with GAD in vitro to detect reactive CD4+ T cells. Re-expression of selected T cell receptors (TCRs) confirmed antigen specificity. Up to 70% of T cells at the skin injection site were clonally-expanded and 4 of 14 (28%) re-expressed TCRs were GAD-reactive. In draining LNs 1 of 14 (4%) clonally-expanded TCRs was GAD-reactive, representing ∼0.08% of all T-cells. GAD-reactive cells across compartments displayed Th1 and Th17-associated transcription signatures. These results demonstrate the intradermal autoantigen challenge, coupled with scRNAseq, enables direct identification and molecular profiling of autoreactive T cells in vivo . This minimally invasive approach provides a powerful platform for tracking antigen-specific specific T cells to monitor disease activity and evaluate immune interventions in T1D.RNA-seq of coding RNA from single cellsHomo sapiensTracking GAD-specific T-cell expansions in Type 1 diabetes by intradermal GAD-Alum challengeSJ Hanna Stephanie HannafalseTracking GAD-specific T-cell expansions in Type 1 diabetes by intradermal GAD-Alum challengeWe used intradermal injection of GAD-Alum to recruit GAD-specific T cells to accessible sites in the skin and skin-draining lymph nodes (LNs), sampled by skin suction blisters and ultrasound-guided LN aspiration. Peripheral blood samples obtained before GAD injection were restimulated with GAD in vitro to detect reactive CD4+ T cells. Sequencing was performed using 10xGenomics 5' kits (V1 and V2). Samples from LN and blisters were run individually. PBMC samples were combined in a singles 10x reaction using hashing antibodies. LNs include GEX, TCRS and BCRs. Blisters include GEX and TCRs. PBMC include GEX, TCRs, hashing and a panel of cell surface antibodies.2026-05-26T00:00:00Z2026-05-26T12:17:24.486Z2026-05-22T14:39:09.675ZE-MTAB-17069ERP193708EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_000418410.64898/2025.12.02.690944