Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Abdel Rahim A. Hamad ( ahamad@jhmi.edu ).

The bacterial strains used in this study are listed in the Key Resources Table. LB agar or LB broth (Invitrogen) were used for bacterial growth at 37°C. Supplements, 50 mM (20 mg/mL) X-Gal (fisher scientific) was applied directly to the top of the agar plates for blue/white screening. Antibiotics were added to media at the following concentrations: 50 μg/mL of ampicillin. In all procedure, 37°C shaking (225 rpm) and non-shaking incubator have been applied. Glycerol stocks of each culture have been provided by transferring 300 mL of 1:1 sterile LB/glycerol and 700 mL of the confluent culture to a 2 mL tube, mix well and freeze at −80°C.

HEK293 (female) were obtained from Thermo Fisher Scientific and cultured in basal media (An aliquot of 250 mL each of sterile RPMI and DMEM; 3.75 mL of antibiotic/antimycotic and 5 mL each of L-glutamine (200 mM), 100 × Nutridoma and sodium pyruvate (100 mM) was used. Basal media must be made fresh every 7 d. L-Glutamine can be stored at −20°C for up to 1 year, Nutridoma can be stored at room temperature (20–25°C) for up to 1 year and sodium pyruvate can be stored for up to 6 months at 4°C. Cells were incubated at 37°C in humidified air containing 5% CO 2 and transfections were performed using polyplus jet-prime transfection reagent (Polypus transfection) according to the manufacturer’s protocols.

Peripheral blood samples were obtained from donors using protocols approved by the Johns Hopkins Institutional Review Board. All donors provided written informed consent and sample size estimation was not employed. All T1D subjects met the American Diabetes Association criteria for classification and were recruited at Johns Hopkins Comprehensive Diabetes Center. Donors with no T1D are classified as healthy controls (HCs) and were recruited from normal volunteers. Patients were males and females. The clinical characteristics of donors are summarized in ( Table S1 A). The study was conducted in accordance with the declaration of Helsinki principles. Peripheral blood mononuclear cells (PBMCs) were freshly isolated using Ficoll-paque density centrifugation (GE Healthcare) gradient. Islet autoantibodies profiles and HLA genotypes of subjects ( Tables S1 B and S1C), whose repertoires were analyzed by high-throughput was performed at Autoantibody/HLA Core Service Center at the Barbara Davis Center for Childhood Diabetes.

Method Details

Flow cytometric analysis Dai et al., 2015 Dai H.

Rahman A.

Saxena A.

Jaiswal A.K.

Mohamood A.

Ramirez L.

Noel S.

Rabb H.

Jie C.

Hamad A.R. Syndecan-1 identifies and controls the frequency of IL-17-producing naïve natural killer T (NKT17) cells in mice. Martina et al., 2016 Martina M.N.

Noel S.

Saxena A.

Bandapalle S.

Majithia R.

Jie C.

Arend L.J.

Allaf M.E.

Rabb H.

Hamad A.R. Double-Negative αβ T Cells Are Early Responders to AKI and Are Found in Human Kidney. (Key Resources Table). Acquired samples (5 × 105 to 1 × 106 live events) were properly compensated using single color stains. Data analysis, gating, and graphical presentation were done using FlowJo software (TreeStar). Doublets were excluded from analysis using FSC-Height versus FSC-Width and SSC-Height versus SSC-Width plots. Multiple specificity controls were used. These included human FcR blocking reagent (Miltenyi Biotec), Fluorescence-Minus One (FMO) for CD5, CD19, TCR, IgD, dump gating, and isotype controls. In addition, when applicable, irrelevant cell types were used as internal biological controls and in the case of in vitro stimulation, we used unstimulated cultures as negative controls. Cell phenotypes were analyzed by flow cytometry (). Briefly, single cell suspensions were surface-stained for 20 min on ice with predetermined optimal concentrations of indicated fluorochrome-conjugated antibodies. Acquired samples (5 × 10to 1 × 10live events) were properly compensated using single color stains. Data analysis, gating, and graphical presentation were done using FlowJo software (TreeStar). Doublets were excluded from analysis using FSC-Height versus FSC-Width and SSC-Height versus SSC-Width plots. Multiple specificity controls were used. These included human FcR blocking reagent (Miltenyi Biotec), Fluorescence-Minus One (FMO) for CD5, CD19, TCR, IgD, dump gating, and isotype controls. In addition, when applicable, irrelevant cell types were used as internal biological controls and in the case of in vitro stimulation, we used unstimulated cultures as negative controls.

Imaging flow cytometry (AMNIS) Freshly isolated PBMCs were stained with FITC-conjugated anti-TCRαβ, PE-conjugated anti-IgD, APC-conjugated anti-CD5, and BV421-conjugated anti-CD19 and analyzed at X60 magnification on an Image Stream flow cytometer (Amnis corporation) with low flow rate/high sensitivity using INSPIRE software. For each sample, 10,000 events were acquired. Single color controls were used for creation of a compensation matrix, to set the optimal laser power for each fluorochrome and to avoid saturation of the camera. The compensation matrix was applied to all files to correct for spectral cross-talk. Positive cutoff values were calculated on the basis of the bright detail similarity (BDS) background of TCRαβ and an irrelevant signal (for example, side scatter). Image analysis was performed with the IDEAS 6.2 software package using bright field images to set cell boundary and gating on internalized events. Compensated data files were analyzed using a gating strategy that involved selecting focused cells on the basis of gradient RMS and an aspect ratio that was consistent with single events and devoid of debris or multi-cellular events (doublets). T cell and B cell singlets were successfully identified using this strategy and the selection of good quality, focused singlets within the viewing window allowed refining of final gating. After the gating of T con and B con cells, individual IgD+ DE cells were identified based on their surface profile (CD19+CD5+TCR+IgD+) and analyzed for the indicated markers. Bright field imagery was collected with an LED-based bright field illuminator. Each plot was manually adjusted so that the machine noise generated at the beginning of acquisition was set to zero.

Single cell RNA-seq data generation and processing Picelli et al., 2014 Picelli S.

Faridani O.R.

Björklund A.K.

Winberg G.

Sagasser S.

Sandberg R. Full-length RNA-seq from single cells using Smart-seq2. FACS sorted single cells (see Figures S5 A and S5B for sorting strategy) were processed with the Smart-seq2 protocol () with the following modifications. RNA purification was performed prior to reverse transcription using RNAClean XP beads (Beckman Coulter). cDNA was amplified with 21 PCR cycles followed by DNA cleanup with AMPure XP beads. Libraries were prepared using the Nextera XT Library Prep kit (Illumina) using custom barcode adapters. Uniquely barcoded Libraries were sequenced together on a NextSeq 500 sequencer (Illumina).

Bioinformatic analysis of scRNA-seq Data Lun et al., 2016 Lun A.T.

McCarthy D.J.

Marioni J.C. A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor. con cells and 1/24 T con cells. All the 19 cells had library sizes lower than or comparable to the empty wells. 64 out of the 74 good quality samples have a sequencing depth of 1-3 million reads and are deemed to reach saturation while the other 10 samples have a depth between 0.7-1 million reads, good for the detection of large majority of genes ( Michel et al., 2012 Michel M.L.

Pang D.J.

Haque S.F.

Potocnik A.J.

Pennington D.J.

Hayday A.C. Interleukin 7 (IL-7) selectively promotes mouse and human IL-17-producing γδ cells. Wu et al., 2014 Wu A.R.

Neff N.F.

Kalisky T.

Dalerba P.

Treutlein B.

Rothenberg M.E.

Mburu F.M.

Mantalas G.L.

Sim S.

Clarke M.F.

Quake S.R. Quantitative assessment of single-cell RNA-sequencing methods. Ziegenhain et al., 2017 Ziegenhain C.

Vieth B.

Parekh S.

Reinius B.

Guillaumet-Adkins A.

Smets M.

Leonhardt H.

Heyn H.

Hellmann I.

Enard W. Comparative Analysis of Single-Cell RNA Sequencing Methods. QC checks were performed on the scRNA-seq data with R bioconductor package scater following the methods described by Lun et al. (). The QC metrics included library size, number of features expressed, proportions of ERCC spike-in controls, and three empty wells that were included in the experimental design as negative controls. In addition to the three empty wells, 18 out of 93 biological (B, T and DE) cells had either log-library sizes and/or log-transformed number of expressed transcripts blow the respective medians by more than 3 median absolute deviations (MADs) and were filtered out as low quality outlier samples. Another DE-cell D07 had a library size below the maximum of the three empty wells and was viewed as a low quality sample. Among the 19 low-quality biological cell samples, 12/45 are DE cells, 6/24 Bcells and 1/24 Tcells. All the 19 cells had library sizes lower than or comparable to the empty wells. 64 out of the 74 good quality samples have a sequencing depth of 1-3 million reads and are deemed to reach saturation while the other 10 samples have a depth between 0.7-1 million reads, good for the detection of large majority of genes (). The sequencing assay kit also included 12 ERCC spike in controls. The 19 low quality cells had a pattern of spike-in ERCC proportions similar to the good quality ones above and did not show any increase. Assuming the majority of cells are of high quality, it suggests there is little loss of endogenous RNA in all the cells. Taken together, the analyses above suggest good overall quality of the scRNA-seq experiment. con cells, T con cells, or DE cells were identified using the Template Matching method, which tests for an association between each profile and an artificial profile that represents an ideal, cluster- or condition-specific, profile using the Pearson’s product moment correlation coefficient ( Pavlidis and Noble, 2001 Pavlidis P.

Noble W.S. Analysis of strain and regional variation in gene expression in mouse brain. Holm, 1979 Holm S. Simple Sequentially Rejective Multiple Test Procedure. Canzar et al., 2017 Canzar S.

Neu K.E.

Tang Q.

Wilson P.C.

Khan A.A. BASIC: BCR assembly from single cells. Following a biology-guided strategy, we limited downstream analysis of the scRNA-Seq data to cells in which at least two of three housekeeping genes (PPIA, ACTB, and UBB) were detected as expressed, defined as having log2 (RSEM value + 1) > 0. This resulted in 77 high quality cells. Genes preferentially expressed in either Bcells, Tcells, or DE cells were identified using the Template Matching method, which tests for an association between each profile and an artificial profile that represents an ideal, cluster- or condition-specific, profile using the Pearson’s product moment correlation coefficient (). Multiple testing corrections were performed using Holm’s method (). To identify BCR and TCR transcripts expressed in the single cells, we used BASIC (). We annotated each cell according to whether BASIC was able to assemble BCR or TCR transcripts.

Polyclonal TCR stimulation 6 cells in 1 mL complete culture medium) in the presence or absence of anti-CD3/CD28 beads (106 bead/well) and incubated at 37°C and 5% CO 2 . Alternatively, plates coated with anti-CD3 (10 μg/mL) and anti-CD28 (10 μg/mL) were used ( Yoneshiro et al., 2017 Yoneshiro T.

Matsushita M.

Hibi M.

Tone H.

Takeshita M.

Yasunaga K.

Katsuragi Y.

Kameya T.

Sugie H.

Saito M. Tea catechin and caffeine activate brown adipose tissue and increase cold-induced thermogenic capacity in humans. Freshly isolated PBMCs were placed onto the wells of 24-well tissue culture plates (10cells in 1 mL complete culture medium) in the presence or absence of anti-CD3/CD28 beads (10bead/well) and incubated at 37°C and 5% CO. Alternatively, plates coated with anti-CD3 (10 μg/mL) and anti-CD28 (10 μg/mL) were used (). After 7 days in culture, viable cells were harvested, counted using trypan blue, and analyzed for the expression of indicated molecules using a BD LSRII flow cytometer. Absolute cell numbers were determined by multiplying the frequency of the indicated subset by the viable cell count.

CFSE proliferation assay Freshly isolated PBMCs were washed twice with warm (37°C) 1x PBS to remove serum that affect staining and the cells were resuspended in warm (37°C) 1x PBS at a density of 1.5-2.0 × 106 cells/mL. Cells were labeled with 1 μM CFSE (eBioscience) for 1-2 min at 37°C with continuous vortexing. The labeling reaction was quenched by adding chilled complete culture media. CFSE-labeled cells were washed in 1x PBS, resuspended in complete media, and plated into 24-well tissue culture plates (1.5-2.0 × 106 cells/well in 1 mL complete culture medium). To evaluate functionality of HLA-DQ8 molecules, we immobilized DQ8 molecules loaded with indicated peptides (x-Id, TP-Id, mimotope, native insulin and CDR3 peptide from IgD+ DE from HC#1 (referred to as h-Id) into wells of 24-well plates (10 μM) and examined their ability to stimulate CFSE-labeled CD4 T cells from among PBMCs. In parallel experiments, we activated cultures in the presence (20 uM) of mouse anti-HLA-DQ (SPV-L3; Abcam) and anti-HLA-DR (L243;Abcam) to assess MHC restriction. In similar manner, CFSE-labeled cells were also stimulated in the presence or absence of the above-indicated peptides (10 μM) as soluble antigen. Alternatively, in a separate experiment, to evaluate the mAb-specific proliferative response, purified mAbR and mAbN (described later in the method) concentration of 2.5 and 5ug, immobilized into the wells of 24-well plates, and used to stimulate CFSE-labeled PBMCs. CFSE labeled cells without stimulation and with CD3-28 stimulation were taken as specific negative and positive controls respectively. After 7 days of incubation, cultures were stained as indicated in Figure legends and proliferation assessed by determining frequency of CFSElow CD4 T cells.

Intracellular Cytokine analysis 2 with phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) and ionomycin (500 ng/mL) in the presence of Golgi-Plug ( Saxena et al., 2017 Saxena A.

Yagita H.

Donner T.W.

Hamad A.R.A. Expansion of FasL-Expressing CD5+ B Cells in Type 1 Diabetes Patients. Xiao et al., 2011 Xiao Z.

Mohamood A.S.

Uddin S.

Gutfreund R.

Nakata C.

Marshall A.

Kimura H.

Caturegli P.

Womer K.L.

Huang Y.

et al. Inhibition of Fas ligand in NOD mice unmasks a protective role for IL-10 against insulitis development. Single cell suspensions were stimulated for 4 h at 37°C in 5% COwith phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) and ionomycin (500 ng/mL) in the presence of Golgi-Plug (). Intracellular cytokine analysis was performed using the manufacturers’ instructions. Briefly, surface-stained samples were fixed, permeabilized and incubated with mAbs against indicated intracellular cytokines for 30 min on ice, washed, acquired and analyzed using the above described strategy.

Anti-IgM stimulation 6) in RPMI-1640 medium were allowed to rest at 37°C in CO 2 incubator for 30 min before stimulation. BCR signaling was induced by crosslinking the BCR ( Wang et al., 2014 Wang J.

Sohn H.

Sun G.

Milner J.D.

Pierce S.K. The autoinhibitory C-terminal SH2 domain of phospholipase C-γ2 stabilizes B cell receptor signalosome assembly. 2 anti-IgM (Jackson ImmunoResearch) at 37°C in CO 2 incubator for indicated time points. Time course analysis was achieved by adding anti-IgM to each sample in reverse time points and fixing all samples in unison. To determine basal levels of phosphorylation, parallel cultures of unstimulated PBMCs were fixed at time zero. To detect phosphorylated CD79a (pIgα), cells were fixed (1.5% paraformaldehyde, 5 min, room temperature), permeabilized (90% methanol, 10 min, 4°C), and stained with rabbit antibodies specific for pCD79A (Igα, Tyr82) followed by PE–conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Freshly isolated PBMCs (1X10) in RPMI-1640 medium were allowed to rest at 37°C in COincubator for 30 min before stimulation. BCR signaling was induced by crosslinking the BCR () with 10 μg/mL goat F(abʹ)anti-IgM (Jackson ImmunoResearch) at 37°C in COincubator for indicated time points. Time course analysis was achieved by adding anti-IgM to each sample in reverse time points and fixing all samples in unison. To determine basal levels of phosphorylation, parallel cultures of unstimulated PBMCs were fixed at time zero. To detect phosphorylated CD79a (pIgα), cells were fixed (1.5% paraformaldehyde, 5 min, room temperature), permeabilized (90% methanol, 10 min, 4°C), and stained with rabbit antibodies specific for pCD79A (Igα, Tyr) followed by PE–conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories).

Cell sorting and DNA extraction for high-throughput sequencing of IGHV and TRB + and IgD− DE cells, B con , and T con cells were sorted from freshly isolated PBMCs using a FACSAria II (BD Biosciences, Bedford, MA). The sorting strategy and purity of isolated cell populations are shown in (( ). Autologous B con cells were used as controls for IGVH analysis and T con cells for TRB analysis. Briefly, freshly isolated PBMCs were stained for CD19, CD5, IgD, and TCRαβ for 30 min on ice, washed thoroughly, and suspended in a pre-sort buffer (BD Biosciences). Propidium iodide (PI) was added immediately prior to sorting to exclude non-viable cells. Sorted cells were collected in RPMI medium supplemented with 50% FBS on ice. IgD+ DE cells were identified as CD19+CD5+IgD+TCRβ+ (800-1000 cells per sort) and IgD− DE cells as CD19+CD5+IgD−TCRβ+ cells (100-200 cells per sort). B con cells were identified as CD19+CD5−TCRβ− and T con cells as CD19−CD5+TCRβ+ cells. Total DNA was directly extracted from sorted cells using QIAmp DNA blood mini Kit (QIAGEN) according to the manufacturer’s instructions. DNA from sorted IgD+ and IgD− DE cells, B con cells and T con cells were used for BCR or TCRBV sequencing as described in text. For repertoire analysis, IgDand IgDDE cells, B, and Tcells were sorted from freshly isolated PBMCs using a FACSAria II (BD Biosciences, Bedford, MA). The sorting strategy and purity of isolated cell populations are shown in ( Figures S5 A and S5B). Two sorts were performed from each donor and (except HC#2) were performed at different time points with one sort used for IGHV and the second for TRB analysis. Donor characteristics, including islet autoantibodies and HLA genotypes, are shown in Table S1 ). Autologous Bcells were used as controls for IGVH analysis and Tcells for TRB analysis. Briefly, freshly isolated PBMCs were stained for CD19, CD5, IgD, and TCRαβ for 30 min on ice, washed thoroughly, and suspended in a pre-sort buffer (BD Biosciences). Propidium iodide (PI) was added immediately prior to sorting to exclude non-viable cells. Sorted cells were collected in RPMI medium supplemented with 50% FBS on ice. IgDDE cells were identified as CD19CD5IgDTCRβ(800-1000 cells per sort) and IgDDE cells as CD19CD5IgDTCRβcells (100-200 cells per sort). Bcells were identified as CD19CD5TCRβand Tcells as CD19CD5TCRβcells. Total DNA was directly extracted from sorted cells using QIAmp DNA blood mini Kit (QIAGEN) according to the manufacturer’s instructions. DNA from sorted IgDand IgDDE cells, Bcells and Tcells were used for BCR or TCRBV sequencing as described in text.

High-throughput immune SEQ and data analysis Carlson et al., 2013 Carlson C.S.

Emerson R.O.

Sherwood A.M.

Desmarais C.

Chung M.W.

Parsons J.M.

Steen M.S.

LaMadrid-Herrmannsfeldt M.A.

Williamson D.W.

Livingston R.J.

et al. Using synthetic templates to design an unbiased multiplex PCR assay. DeWitt et al., 2016 DeWitt W.S.

Lindau P.

Snyder T.M.

Sherwood A.M.

Vignali M.

Carlson C.S.

Greenberg P.D.

Duerkopp N.

Emerson R.O.

Robins H.S. A Public Database of Memory and Naive B-Cell Receptor Sequences. Robins et al., 2009 Robins H.S.

Campregher P.V.

Srivastava S.K.

Wacher A.

Turtle C.J.

Kahsai O.

Riddell S.R.

Warren E.H.

Carlson C.S. Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells. − from T1D#1 and IgD+ cells from T1D#2 were unsuccessful. TCRβ and IGH sequences are available at Adaptive Biotechnologies (DOI: Analyses of IGHV and TRBV clonotypes were performed on genomic DNA from each sorted cell type using the immunoSEQ platform at survey level resolution (Adaptive Biotechnologies). The immunoSEQ assay combines multiplex PCR with high-throughput sequencing and sophisticated bioinformatics pipeline for CDR3 region analysis (). Samples were amplified from 40 ng to 100 ng genomic DNA per sample. Attempts to sequence IgDfrom T1D#1 and IgDcells from T1D#2 were unsuccessful. TCRβ and IGH sequences are available at Adaptive Biotechnologies (DOI: https://doi.org/10.21417/RA042019 ). Raw ImmunoSeq data from individual samples were processed with ImmunoSeq Analyzer 2.0 software (Adaptive Biotechnologies). Measurement metrics of processed data were exported in the tsv file format and analyzed using the R platform. Clones of uncertain vGene identity or out-of-frame were excluded from downstream analysis. For vGene in each cell type in individual samples, counts of distinct cell clones were obtained by summing the metric of the “estimated number of cell genomes present in the sample,” upon which the corresponding percentages were calculated. The percentage quantification provides a uniform basis for the vGene (VH and Vβ) usages to be fairly and consistently compared across the different cell types and samples, minimizing any effects that could result from sequencing different and very tiny numbers of DE subset cells. Percentages were visualized with bar plots to make straight comparisons of vGene usages between different cell types. The presence or absence of vGenes in the different cell subsets was determined on the basis of the vGene usages. Unique and shared vGenes among different cell subsets were identified and displayed in Venn diagrams using the functions of R Limma package. The vGene mutations are identified based on alignment with the IMGT database, upon which the differences from germlines are marked, counted and recorded in the column of “vAlignSubstitutionCount” of the Raw ImmunoSeq data tsv spreadsheet. The vGene mutation values were further summed per gene and displayed with a combination of boxplot and scatterplot using R. con cells, which was downloaded from String searches with the amino acid sequences of the selected clones were performed to determine their presence in individual subjects. A string search of the amino acid sequence of the invariant clonotype, “CARQEDTAMVYYFDYW” was also performed with an R script against a public ImmunoSEQ database of 37 million unique BCR sequences of naive and memory Bcells, which was downloaded from https://clients.adaptivebiotech.com/immuneaccess and that of IBC examined from nPOD Adaptive Immune Repertoire ( https://public.tableau.com/profile/npod.adaptive.immune.repertoire#!/ ).

PCR probes for detection of x-clonotype in peripheral blood - GCTGGAGTGGATTGGGAGTA-3′) paired with antisense primer (5′- CCCAGTAGTCAAAGTAGTAAACCATA-3′) complementary to the entire CDR3 region (see diagram, To determine whether the x-Id clonotype can be detected in peripheral blood, we designed and used two PCR probes for analysis of PBMCs. In the first probe we used a VH04-b-specific sense primer (5′GCTGGAGTGGATTGGGAGTA-3′) paired with antisense primer (5′- CCCAGTAGTCAAAGTAGTAAACCATA-3′) complementary to the entire CDR3 region (see diagram, Figure 3 J). In the second probe, the VH04-b-specific primer was paired with a reverse primer (5′-TCCCTGGCCCCAGTAGTCAAAGTAGTA-3′) that span JH04 and ended at the N2 region (see diagram, Figure S7 ). Briefly, RNA was extracted from fresh PBMCs using the RNeasy mini kit (Quigen), and analyzed by NanoDrop (ND-1000 spectrophotometer) to assess purity and measure concentration. Reverse transcription (RT) PCR was performed on approximately 1 μg of purified RNA to prepare cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermofisher) according to the kit protocol: RNA was incubated with 5X reaction mix, random hexamer primer, and RevertAid M-MuLV RT (200 U/μL) enzyme mix in a final volume of 20 μL at 25°C for 10 min, followed by 42°C for 60 min and inactivation at 70°C for 5 min. Positive (GAPDH-specific primers) and negative (reaction mixture without RT enzymes) control reactions were used to verify specificity of cDNA synthesis. PCR reaction (2 μL cDNA in a total volume of 25 μL prepared using 2X QIAGEN HotStarTaq master mix) was performed under the following conditions: initial denaturation at 95°C for 3 min, 95°C for 30 s, 54°C for 30 s, 40 cycles at 72°C for 1 min followed by a final extension step at 72°C for 10 min using a thermocycler (BioRad T100). PCR product was visualized as a band size of 200 bp on 1.2% agarose gel and the band was excised, purified using PCR purification kit (Quigen) and Sanger sequenced at the Johns Hopkins Medical Institute GRCF sequencing core. Sequences were analyzed using the Immunogenetics IMGT/V-QUEST software: http://imgt.org/IMGT_vquest/share/textes/

Molecular dynamics simulations Sharp, 2012 Sharp K.A. Statistical thermodynamics of binding and molecular recognition models. Wang et al., 2018 Wang Y.

Sosinowski T.

Novikov A.

Crawford F.

Neau D.B.

Yang J.

Kwok W.W.

Marrack P.

Kappler J.W.

Dai S. C-terminal modification of the insulin B:11-23 peptide creates superagonists in mouse and human type 1 diabetes. α and β were mutated to match the sequence of the HLA in the insulin crystal structure (PDB: α was mutated to Isoleucine to match the 1JK8 HLA sequence. Each system was solvated in a TIP3P water box and then charged, neutralized, and ionized with 100mM concentration using Na+ and Cl-. The new peptide system was built from a crystal structure of an insulin B chain epitope bound to HLA-DQ8 (PDB: 1JK8 ) (). The insulin epitope sequence was mutated to the new peptide epitope sequence using the Mutator plugin from VMD, ensuring the new peptide epitope was in the desired register. The CDR3 epitope from HC#1 was also built from the insulin-bound epitope structure (PDB: 1JK8 ), following the same protocol as the new peptide system. The super-agonist system was built from the crystal structure of an insulin mimotope bound to HLA-DQ8 (PDB: 5UJT ) (). For this system, in addition to mutating the epitope to match the super-agonist, both HLA chainsandwere mutated to match the sequence of the HLA in the insulin crystal structure (PDB: 1JK8 ). More specifically, besides distal residues, residue 72C of HLA-was mutated to Isoleucine to match the 1JK8 HLA sequence. Each system was solvated in a TIP3P water box and then charged, neutralized, and ionized with 100mM concentration using Naand Cl Best et al., 2012 Best R.B.

Zhu X.

Shim J.

Lopes P.E.

Mittal J.

Feig M.

Mackerell Jr., A.D. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ(1) and χ(2) dihedral angles. Following system creation, each system underwent at least 20,000 steps of conjugate-gradient minimization to hold protein atoms fixed, followed by at least 10,000 steps of minimization allowing all the atoms to move. The systems were subsequently equilibrated for 1ns at 310K using a 2fs timestep. Production MD simulations were run for 500ns using a 2fs timestep. A Langevin thermostat maintained the temperature at 310K. The CHARMM36 force field () was used for protein parameters. The Particle Mesh Ewald (PME) method was used to compute long-range electrostatics with the electrostatics and van der Waals cutoff of 12Å. All simulations were run using NAMD2.11. For the MD simulations, only the last 250ns were used for analysis, dividing the trajectory into 5 parts. The contact area was computed using solvent accessible surface area (SASA) calculation in Gromacs tools with a water radius of 1.4Å. Van der Waals interaction energy was computed using NAMDEnergy. The electrostatics energy was biased due to the absence of solvent screening and was left out of the interaction energy. The RMSD and RMSF were computed using Gromacs tools. Averages and error bars for contact area, interaction energy, and RMSF were computed by taking the last half (250ns) of the MD simulations and dividing them into 5 sections with 50ns each and taking the average of each section as a measurement in the sample. Error bars shown are standard error.

Free energy perturbation Binding affinity was calculated via the free energy perturbation (FEP) method. The final structures of the production MD simulations were selected for FEP computation. We computed free energy perturbation calculations for the bound (HLA + epitope) and free states (epitope only) with 6 replicas for each calculation. Due to the extensive sequence differences between epitopes, we mutated the epitopes to a neutral, intermediate sequence of polyglycine the length of the epitope. The dual topology was implemented using the Mutator plugin from VMD. Each system was slowly mutated from the epitope to polyglycine using λ increments of maximum 0.04 with smaller increments toward the ends, totaling at least 34 FEP windows for each system. Each FEP window was run for 1ns, leading to well over 800ns simulation (6 replicas x 34 windows x 2 states (complex + free) x 2 epitopes). Electrostatics was switched on starting at λ = 0.1. Convergence at each window was assessed by comparing values across replicas. NAMD2.11 with the CHARMM36 protein force field and TIP3P water model were used for FEP calculations, matching the MD work. From observing the FEP trajectories, the polyglycines do not shift registers but maintain the starting register of the epitope. Free energy error bars are standard errors.

Analysis of x-Id peptide binding to DQ8 using gentle SDS-PAGE assay Kim et al., 2013 Kim A.

Ishizuka I.

Hartman I.

Poluektov Y.

Narayan K.

Sadegh-Nasseri S. Studying MHC class II peptide loading and editing in vitro. Sadegh-Nasseri and Germain, 1991 Sadegh-Nasseri S.

Germain R.N. A role for peptide in determining MHC class II structure. 3 . Reactions were neutralized, mixed with equal volumes of SDS-PAGE sample buffer containing 0.1% SDS (final concentration) and placed for 15 min at room temperature and run on 10% PAGE gels and silver-stained using a standard protocol. To assess stability some samples were boiled for 3 min, which resulted in degradation of complexes (data not shown). Gentle SDS-PAGE was used to assess formation of stable complexes between peptides and HLA-DQ8 molecules as previously described (). Briefly, 0.5 μM of HLA-DQ8 monomers (provided by NIH tetramer core facility) were treated with thrombin to cleave and remove CLIP peptide. Empty monomers were incubated in the absence or presence of 100 μM of indicated peptides (x-Id, TP-Id, mimotope (R22E), native insulin B:9-23, and h-Id) at 37°C for 72 h in citrate phosphate buffer, pH 5.5 with 1 mM PMSF and 0.025% NaN. Reactions were neutralized, mixed with equal volumes of SDS-PAGE sample buffer containing 0.1% SDS (final concentration) and placed for 15 min at room temperature and run on 10% PAGE gels and silver-stained using a standard protocol. To assess stability some samples were boiled for 3 min, which resulted in degradation of complexes (data not shown).

Generation of EBV-immortalized DE x1.1 clone + DE cells from freshly isolated PBMCs using a FACSAria II using described strategy (® 55-X). Cultures were pulsed with 2.5 μg/mL CpG ODN 2006 (ODN7909) and EBV supernatant stock (ATCC® VR-1492) from B95-8 cells ( Caputo and Flytzanis, 1991 Caputo J.G.

Flytzanis N. Kink-antikink collisions in sine-Gordon and phi4 models: Problems in the variational approach. Hui-Yuen et al., 2011 Hui-Yuen J.

McAllister S.

Koganti S.

Hill E.

Bhaduri-McIntosh S. Establishment of Epstein-Barr virus growth-transformed lymphoblastoid cell lines. Hamad et al., 1994 Hamad A.R.

Herman A.

Marrack P.

Kappler J.W. Monoclonal antibodies defining functional sites on the toxin superantigen staphylococcal enterotoxin B. To generate immortalized DE cells, we sorted IgDDE cells from freshly isolated PBMCs using a FACSAria II using described strategy ( Figure 6 A). Sorted cells were seeded at 10, 25, 50 or 100 cells per well of 96-well microplates that had been coated 24 h earlier with irradiated fibroblasts (ATCC55-X). Cultures were pulsed with 2.5 μg/mL CpG ODN 2006 (ODN7909) and EBV supernatant stock (ATCCVR-1492) from B95-8 cells (). Cultures were maintained by replacing half of culture medium with fresh medium every 5 to 7 days. Immortalized cells were visible after 8 days in cultures seeded with 50 or 100 DE cells. We selected on lymphoblastoid cell line (hereafter referred to as x-LCL) for subsequent analysis. In one set of experiments, we sorted single cells from x-LCL and examined for expression of Ig heavy and light chains. In a second set, we used x-LCL to generate x1.1 clone by limiting dilution (0.3 cell/well) as described (). Cells of the x1.1 clone were used for analysis of BCR and TCR and spontaneous antibody production.

Analysis of x1.1 clone for coexpression of BCR and TCRαβ Smith et al., 2009 Smith K.

Garman L.

Wrammert J.

Zheng N.Y.

Capra J.D.

Ahmed R.

Wilson P.C. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. We used two approaches to ensure single cell clonality of immmortalized DE cell populations, outgrown monoclonal DE cells were sorted on 96 well microplates using a FACSAria II (BD Biosciences, Bedford, MA) as described above containing RNA catch buffer ().

Cloning and expression of BCR from x1.1 clone and fresh single DE cells Smith et al., 2009 Smith K.

Garman L.

Wrammert J.

Zheng N.Y.

Capra J.D.

Ahmed R.

Wilson P.C. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. We used the same protocol that was developed by Smith et al. () for analysis of BCR expression from single cells of the x1.1 clone and freshly sorted single DE cells. Briefly, individual cells were sorted into wells of 96-well PCR plate loaded with catch buffer containing RNase inhibitor to perform RT-PCR using OneStep RT-PCR Kit (QIAGEN). Two primers were utilized to amplify all VH4 gene family members and 8 primers for amplification of genes encoding lamda chain. Cloning PCR of the heavy chain was performed using primers that incorporate the cloning restriction sites and place VDJ heavy chain and constant region genes in frame within the cloning vector (AbVec-hIgG1). Cloning PCR products were purified using Monarch PCR & DNA Cleanup Kit (New England, BioLabs) and visualization as a band of approximately 400 bp in 1.5% agarose gel. Insert and vector were digested with AgeI and SalI and purified as described above. A three-fold molar excess of insert to vector were used to transform DH5α cells. Positive colonies were picked, cultured and plasmid extracted by QIAprep Spin Miniprep Kit (QIAGEN) followed by sequencing using the AbVec primer. The lambda chain was cloned using the same procedure except that insert and vector were digested with AgeI and XhoI and cloned into AbVec- Igλ. Complete sequences of the variable regions were used to identify VDJ usage and CDR3 by IMGT/V-QUEST software.

Expression and analysis of recombinant x-mAbR from single DE cells Smith et al., 2009 Smith K.

Garman L.

Wrammert J.

Zheng N.Y.

Capra J.D.

Ahmed R.

Wilson P.C. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. R was purified using immobilized protein A columns (Pierce). Antibody expression and purity was verified by SDS-PAGE, and purified antibody concentrations were determined using the EZQ Protein Quantitation Kit (Invitrogen). Following purification and digestion, amplified cDNAs of the antibody variable genes from single cells were cloned into expression vectors containing human IgG (AbVec-hIgG1) and Igλ (AbVec- Igλ) constant regions (). Briefly, AbVec-hIgG1 containing the heavy- and AbVec- Igλ containing light-chain Ig genes were co-transfected into the HEK293A cell line using polyplus jet-prime transfection (Polypus transfection) and manufacturer’s instruction. Transfected 293A cells were allowed to secrete antibodies in serum-free basal media for 4 to 5 days and mAbwas purified using immobilized protein A columns (Pierce). Antibody expression and purity was verified by SDS-PAGE, and purified antibody concentrations were determined using the EZQ Protein Quantitation Kit (Invitrogen).

Cloning of α and β chains of TCR from the x1.1 clone Eugster et al., 2013 Eugster A.

Lindner A.

Heninger A.K.

Wilhelm C.

Dietz S.

Catani M.

Ziegler A.G.

Bonifacio E. Measuring T cell receptor and T cell gene expression diversity in antigen-responsive human CD4+ T cells. Eugster et al., 2013 Eugster A.

Lindner A.

Heninger A.K.

Wilhelm C.

Dietz S.

Catani M.

Ziegler A.G.

Bonifacio E. Measuring T cell receptor and T cell gene expression diversity in antigen-responsive human CD4+ T cells. The genes for TCRα and TCRβ chains were cloned using a modified version of the method described by Eugster et al. (). Briefly, total RNA was isolated from cells of the x1.1 clone using RNA extraction kit (Biolabs). cDNA was prepared and mixed with degenerate primers for the α and β chains using OneStep RT-PCR Kit (QIAGEN) and used to amplify the α and β chains by nested-PCR using specific primers for the α chain and β chains, separately. Amplified products were visualized on 1.5% agarose gel and cloned into pGEM-T Easy vector (Promega). DNA was extracted using plasmid extraction kit (QIAGEN) and Sanger-sequenced using the M1 primer (). Complete sequences of the variable regions were used to identify VDJ usage and CDR3 by the IMGT/V-QUEST software.

Characterization of the x-mAbN produced by x1.1 clone Cells of x1.1 clones were expanded in complete medium for 3-4 days, washed with PBS and cultured in basal media (Key Resource Table) for five days. Secreted mAbN was detected in supernatants by using SDS-PAGE. Isotype of secreted mAb was determined as IgM using Pro-Detect Rapid Antibody Isotyping Assay human Kit (Thermo Fisher). Antibody concentration was determined using the EZQ Protein Quantitation Kit (Invitrogen).