04/09/2023
EBioMedicine
Redirector of Vaccine-induced Effector Responses (RoVER) for specific killing of cellular targets
To detect various HLA types, ten different biotin-conjugated HLA class I molecules carrying immunogenic YF peptides (ImmunAware Cat# 1001-03, 1002-09, 1016-04, Save;ed from YF-17D vaccinated study participants were quantified by tetramer staining. To detect various HLA types, ten different biotin-conjugated HLA class I molecules carrying immunogenic YF peptides (ImmunAware Cat# 1001-03, 1002-09, 1016-04, 1020-04, 1048-05, 1058-02, 1072-02, 1088-02, 1105-02 or custom design) were analyzed: NS5286-295(KSEYMTSWFY)-A_01:01, NS4B214-222 (LLWNGPMAV)-A_02:01, NS
01/09/2023
The Journal of allergy and clinical immunology
Dupilumab strengthens herpes simplex virus type 1-specific immune responses in atopic dermatitis
Background Impaired viral clearance in a subgroup of atopic dermatitis (AD) patients can lead to severe herpes simplex virus (HSV) infections named eczema herpeticum ( Save;d Monensin (BD Biosciences). Cells were stained with a phycoerythrin (PE)-conjugated HSV-1 UL25367-375 MHC class I tetramer (EasYmer; ImmunAware, Horsholm, Denmark) in 40 _L PBS for 20 minutes at room temperature followed by 30 minutesÕ st
21/08/2023
bioRxiv : the preprint server for biology
Coxsackievirus infection induces direct pancreatic _-cell killing but poor anti-viral CD8+ T-cell responses
DB #27205). HLA-I binding assays Peptides predicted to bind HLA-A2 or -A3 (NetMHCStabPan 1.0) were synthesized (>95% pure, Synpeptide) and binding tested using biotin-tagged HLA-A2 and -A3 monomers (ImmunAware), as described18. Briefly, biotinylated monomers were folded (1.2 nM final concentration) and captured on streptavidin-coated beads (6-8 _m, Spherotech), followed by incubation with anti
21/07/2023
papers.ssrn.com
Circulating Cancer-Specific CD8 T Cell Frequency Associates with Response to Pd-1 Blockade in Merkel Cell Carcinoma
HLA multimers used for scRNAseq were created 987 using HLA-I easYmers (Immunaware) and PE or APC and DNA barcode-labeled streptavidins 988 (Biolegend). DNA barcode- and fluorophore-labeled HLA-I dextramers were prepared by 989 Immudex. These multimers were used for staining expanded T cells as directed. A full list of all 990 epitopes used is provided in Supplemental Tables 1 and 3
01/05/2023
Journal of hepatology
Boosting compromised SARS-CoV-2-specific immunity with mRNA vaccination in liver transplant recipients
This study investigates the immune response of liver transplant recipients (LTRs) to COVID-19 mRNA vaccines, focusing on both humoral and cellular immunity after up to four doses. The research involved 24 LTRs and 19 healthy controls, examining their responses after three to four vaccine doses. Results showed that most LTRs required a third dose to develop high antibody levels, while their spike-specific CD8+ T-cell response, although robust, was less diverse and frequent compared to healthy controls. The study highlights that three vaccine doses generally induce a strong humoral and cellular response in LTRs. However, due to their reduced response, some LTRs might benefit from additional booster doses, especially considering new COVID-19 variants. These findings are crucial for guiding booster vaccination strategies in LTRs.
14/03/2023
Research Square
T cell immunity of the nonadjuvanted HLA-restricted peptide COVID-19 vaccine
The easYmer kit (ImmunAware Aps, Copenhagen, Denmark) was used to determine the binding ability of the peptide to the HLA-A201 molecule. The detailed methods were carried out according to the kitÕs manual instruction protocol. Briefly, the peptide was incubated with the _2m-microglobulin (_2m) light chain subunit and biotin-tagged recombinant HLA-A201 heavy chain at 18¡C for 48 h to form a 500 nM folded HLA complex. The folded complexes were diluted with dilution phosphate-buffered saline (PBS) buffer containing 5% glycerol to serial concentrations of 8.9 nM, 3.0 nM and 1.0 nM. Then, 20 µL of 45-fol
08/02/2023
JCI insight
Slow progression of pediatric HIV associates with early CD8+ T cell PD-1 expression and a stem-like phenotype
te fluorochrome-conjugated antibody list is in Supplemental Table 3. Tetramer and staining of virus-specific CD8+ T cells. Four sets of peptide-HLA tetramers conjugated with BV421 were obtained from ImmunAware ApS: HLA-B*42:01/Gag-TL9 (TPQDLNTML), HLA-B*42:01/pp65-RV10 (RPHERNGFTV), HLA-B*81:01/Gag-TL9 (TPQDLNTML), and HLA-B*81:01/pp65-GL8 (GPISGHVL). Two million cryopreserved PBMCs were thawed a
03/02/2023
Science immunology
Noncanonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent neo-antigens in patients with lung cancer
Recombinant biotinylated HLA-A*02:01 monomers were purchased from ImmunAware (Copenhagen, Denmark) as easYmers (catalog no. 1002-01), and peptides were synthesized with a purity of 98% (GeneCust). Efficiency of peptide-HLA-A*02:01 complex formation was assessed by flow cytometry following the manufacturerÕs instructions
13/01/2023
bioRxiv
Multimodal HLA-I genotype regulation by human cytomegalovirus US10 and resulting surface patterning
supplemented RPMI with 20 IU/ml IL-2 (Stemcell) every three days. After 14 533 days, specificity of the cells was tested by staining with tetramers generated from HLA-I easYmer and 534 streptavidin-PE (BD Biosciences) according to the manufacturerÕs instructions (immunAware). For analysis, 5×104 535 cells were incubated with the tetramer, further stained with anti-CD8 (BD 536 Biosciences) and viability dye (EBioscience),
15/12/2022
STAR protocols
Screening self-peptides for recognition by mouse alloreactive CD8+ TÊcells using direct exÊvivo multimer staining
tetramers in order to screen an array of >100 pMHC epitopes. Several other commercially-available reagents enable peptide MHC exchange or loading (BioLegend Flex-TÊ- UV-mediated exchange monomer, ImmunAware easYmer, TetramerShop Empty Loadable Tetramer, etc). In a preliminary evaluation, comparable results were obtained with different peptide-exchange methods (FigureÊ6). Chaperone-mediated pe
15/11/2022
Nature communications
Selective retention of virus-specific tissue-resident T cells in healed skin after recovery from herpes zoster
II and images were analyzed using QuPath version 0.2.363. In situ tetramer staining was performed using VZV ORF9-specific APC-conjugated HLA-A*03:01 AVYENPLSVEK and HLA-A*33:01 QTTGRITNR tetramers (Immunaware), as described64. Cryosections (8 µm-thick) were fixed in 4% paraformaldehyde solution in PBS (4% PFA), washed twice with PBS, blocked for 30_min at RT using 5% goat serum diluted in PBS
18/10/2022
Frontiers in immunology
HIV specific CD8+ TRM-like cells in tonsils express exhaustive signatures in the absence of natural HIV control
Peptide-MHC tetramers were acquired from ImmunAware, Copenhagen, Denmark and stained during cell surface antibody labelling. Blocking with 20% goat serum for 20 min was Save
12/10/2022
medRxiv : the preprint server for health sciences
A common allele of HLA mediates asymptomatic SARS-CoV-2 infection
upplementary Table S2) using the PURExpress in vitro protein synthesis kit (New England BioLabs, Ipswich, USA) as previously described.33 Each peptide was loaded on biotinylated HLA-B*15:01 easYmers (immunAware, Copenhagen, Denmark) according to manufacturerÕs instructions. Peptide-loaded HLA-B*15:01 tetramers were generated using streptavidin conjugated to phycoerythrin (PE), allophycocyanin (A
23/09/2022
Nature communications
Accumulation of mutations in antibody and CD8 T cell epitopes in a B cell depleted lymphoma patient with chronic SARS-CoV-2 infection
A-binding and T-cell recognition of the two predicted epitope candidates ÒTPSGTWLTYÓ and ÒCTDDNALAY(Y)Ó, HLA-peptide tetramers were produced from HLA-A*01:01 or HLA-B*35:01 monomers (easYmer, ImmunAware, Copenhagen, Denmark) by incubation with peptide for two days at room temperature and subsequent tetramerization with streptavidin-PE (BD Biosciences, Heidelberg, Germany), according to the
15/09/2022
The Journal of clinical investigation
Herpes simplex virus lymphadenitis is associated with tumor reduction in a patient with chronic lymphocytic leukemia
bed previously (40). Of the peptides tested, one (DRLDNRLQL) elicited a positive IFN-_ response and was used for monomer generation. HLA-B*14:01/UL25405-413 (DRLDNRLQL) monomers were obtained from ImmunAware and tetramerized in-house according to the manufacturerÕs instructions. Cytokine measurement. Plasma cytokine levels were measured using a custom electrochemiluminescence multiarray assa
10/09/2022
Methods in molecular biology (Clifton, N.J.)
Identification of Human Antigen-Specific T Cells Using Class II MHC Tetramer Staining and Enrichment
Class II tetramers and higher-order oligomers can also be purchased from several companies, including Beckman Coulter, ProImmune, Immunaware, and MBL International Corporation (MBLI)
05/09/2022
The Journal of experimental medicine
A CMV-induced adaptive human V_1+ __ T cell clone recognizes HLA-DR
Biotinylated HLA-DRB1*03 containing the CMV-specific peptides (sequence IE1) and (sequence IE2) were obtained from immunAware. Tetramerization was performed according to manufacturerÕs
01/09/2022
Journal of hepatology
SARS-CoV-2 vaccination can elicit a CD8 T-cell dominant hepatitis
flow cytometry. Analysis of spike-specific CD8 T cells SARS-CoV-2 spike peptide (S378-386: KCYGVSPTK) was synthesized with a purity of >70% (Genaxxon Bioscience), loaded on HLA-A_03:01 easYmers (immunAware) and subsequently conjugated with phyocerythrin (PE)-streptavidin (Agilent) according to manufacturer’s instructions. Spike(A_03/S378)-specific CD8 T cells were analyzed after magnetic be
08/08/2022
Nature communications
COVID-19 mRNA booster vaccine induces transient CD8+ T effector cell responses while conserving the memory pool for subsequent reactivation
oscience). All peptides showed a purity of >70%. To generate tetramers, SARS-CoV-2 spike peptides (A*01/S865: LTDEMIAQY, A*02/S269: YLQPRTFLL) were loaded on biotinylated HLA class I (HLA-I) easYmer (immunAware) according to manufacturerÕs instructions. Subsequently, peptide-loaded-HLA class I monomers were tetramerized with phycoerythrin (PE)-conjugated streptavidin according to the manufacture
11/05/2022
Viruses
HLA-B*57:01 Complexed to a CD8 T-Cell Epitope from the HSV-2 ICP22 Protein Binds NK and T Cells through KIR3DL1
NK-Cell Binding by B*57:01 Tetramer Containing HSV2-US1 (228-236) To confirm the HLA-restriction of the HSV2-US1 (228-236) RTRLGPRTW peptide, a custom PE-conjugated peptide-HLA-B*57:01 tetramer (ImmunAware, H¿rsholm, Denmark), termed B57-RTR, was tested for binding to HSV-2-specific CD8 T-cell lines and PBMC. The specificity of B57-RTR tetramer for binding KIR3DL1 was assessed using COS-7 ce
11/05/2022
Research Square
Short-lived booster effect and stable CD8+ T cell memory after 3rd COVID-19 vaccine dose
Peptides were manufactured with an unmodified N-terminus and an amidated C-terminus with standard Fmoc chemistry (Genaxxon Bioscience). All peptides showed a purity of 70%. To generate tetramers, SARS-CoV-2 spike peptides (A*01/S865 : LTDEMIAQY, A*02/S269 : YLQPRTFLL) were loaded on biotinylated HLA class I (HLA-I) easYmer¨ (immunAware) according to manufacturerÕs instructions. Subsequently
01/05/2022
Nature immunology
SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8+ T cells
easYmers® used to study the impact of repeated exposures to SARS-CoV-2, including vaccination and infections, on memory T cells. Using advanced techniques, the research profiles T cells in individuals with varying exposure histories to the virus. It was found that the sequence of exposure (vaccination or infection first) influences the balance between spike-specific and non-spike-specific T cell responses. Vaccination following infection tends to boost spike-specific T cells, while breakthrough infections after vaccination lead to stronger non-spike-specific responses. Analysis of over 4,000 T cell receptor sequences revealed a diverse range of responses with shared motifs, regardless of exposure type, and no evidence of narrowed repertoire due to repeated exposure. The study indicates that breakthrough infections can diversify T cell memory, and current vaccination strategies continue to enhance spike-specific memory responses.
04/02/2022
Methods in molecular biology (Clifton, N.J.)
Analysis of the Cellular Immune Responses to Vaccines
Tetramers (can be procured through immunAware, Denmark, or any other biological supplies company). 2. Dasatinib (Selleckchem, cat # S1021). Stock solution is made in DMSO, e.g. 10 mM. 3. Anti-IgG1-FITC (SouthernBiotech cat #1070-02)
01/12/2021
Diabetes
CD8+ T Cells Variably Recognize Native Versus Citrullinated GRP78 Epitopes in Type 1 Diabetes
Experimental binding of peptides (90% purity; SynPeptide) to HLA-A2 was measured by flow cytometry using biotin-tagged HLA-A2 monomers (immunAware) assembled as previously described (14), captured on streptavidin-coated beads (Spherotech), and revealed by an anti-_2m monoclonal antibody (Research Resource Identifier [RRID] AB_626748) and an Alexa Fluor (AF)488-conjugated goat anti-mouse IgG (RRID AB_2728715)
05/11/2021
bioRxiv
HIV-specific CD8+ T-cells in tonsils express exhaustive TRM-like signatures
Cells were stained with fluorochrome-conjugated monoclonal antibodies 432 and subsequently fixed, permeabilized and stained by BD Cytofix/cytoperm kit (BD Biosciences). 433 Peptide-MHC tetramers were acquired from ImmunAware, Copenhagen, Denmark and stained during 434 cell surface antibody labelling. Blocking with 20% goat serum for 20 min was done prior to intracellular 435 antibody staining. After staining cells were washed and fixed in 2% paraformaldehyde before acquisition 436 on a 4 laser, 17 parameter BD FACSAria Fusion flow cytometer
01/09/2021
Nature
Rapid and stable mobilization of CD8+ T cells by SARS-CoV-2 mRNA vaccine
Peptide was loaded on HLA class I easYmers (immunAware) according to manufacturer’s instructions (A*01/S 865 LTDEMIAQY, A*02/S 269 YLQPRTFLL and A*03/S 378 KCYGVSPTK) Save
01/09/2021
Nature
Functional HPV-specific PD-1+ stem-like CD8 T cells in head and neck cancer
Peptides of identified HLA-peptide pairs were synthesized at greater than 90% purity (A&A Labs) and the binding affinity to the respective HLA class I alleles was determined by in vitro binding assays31. Ten HLA-monomers were generated by the NIH Tetramer Core facility, one by Immunaware (HLA-A*29:02 E565-73 (peptide sequence IFVYIPLFL)) and one in our laboratory (HLA-A*02:01-E546-54 (peptide sequence VLLLWITAA))
01/08/2021
Nature immunology
Epigenetic scars of CD8+ T cell exhaustion persist after cure of chronic infection in humans
For the HIV cohort, the following fluorochrome-conjugated multimers from ImmunAware were used: HIV A*02:01 RLRPGGKKK, HIV B*07:02 GPGHKARVL, HIV B*57:01 KAFSPEVIPMF Save
01/08/2021
Cancer discovery
Splicing Patterns in SF3B1-Mutated Uveal Melanoma Generate Shared Immunogenic Tumor-Specific Neoepitopes
Recombinant HLA-A*02:01 molecules (24) were purchased from immunAware (Copenhagen, Denmark) as easYmers (catalog #1002-1). All peptides were synthesized at 95% purity (Synpeptide) and tested for HLA-A2 monomer avidity following immunAware bead-based recommended assay and ELISA (45)
15/07/2021
Journal of immunology (Baltimore, Md. : 1950)
HLA-DR Marks Recently Divided Antigen-Specific Effector CD4 T Cells in Active Tuberculosis Patients
and then stained with surface antibodies as described above. Tetramer is CFP1052-66-DRB5*01:01, an MHC class II tetramer specific for an epitope contained within the CFP10 protein of Mtb (CFP1052-66; QAAVVRFQEAANKQK) (24, 25) and was obtained from ImmunAware (Copenhagen, Denmark)
04/06/2021
Frontiers in immunology
Epigenetic Features of HIV-Induced T-Cell Exhaustion Persist Despite Early Antiretroviral Therapy
l (Los Alamos National Laboratory). Multimer Staining and Cell Sorting Biotinylated monomers of MHC Class I/peptide complexes were custom produced for HIV peptides of interest by Anette Stryhn Buus (immunAware, Laboratory of Experimental Immunology, University of Copenhagen, Denmark), using previously published methods (71). Tetramerisation was performed by the addition of Streptavidin-APC (Invit
18/05/2021
Frontiers in cellular and infection microbiology
Persistent Herpes Simplex Virus Type 1 Infection of Enteric Neurons Triggers CD8+ T Cell Response and Gastrointestinal Neuromuscular Dysfunction
d sham infected mice were stained with anti-CD3 antibody; FITC-conjugated anti-CD8_ antibody (clone 53-6.7, Invitrogen); PE-conjugated MHC class I tetramer complexed with the gB (LMWYELSKI) peptide (immunAware Aps, Denmark). Staining was performed in 2% BSA for 60Êmin at 4¡C. CellsÊwere washed and analyzed by flow cytometry, as described above cultured (18Êh at 37¡C) in RMPI 1640 medium sup
11/05/2021
Research Square
Rapid and stable mobilization of fully functional spike-specific CD8+ T cells preceding a mature humoral response after SARS-CoV-2 mRNA vaccination
Peptide was loaded on HLA class I easYmers¨ (immunAware, Denmark) 373 according to manufacturerÕs instructions (A*01/ S865 LTDEMIAQY and A*02/ S269 374 YLQPRTFLL). SARS-CoV-2 peptide-loaded HLA class I tetramers were produced by 375 conjugation of biotinylated peptide-loaded HLA class I easYmers¨ with phycoerythrin 376 (PE)-conjugated streptavidin (Agilent, USA) according to the manufacturerÕs 377 instructions
06/02/2021
International journal of molecular sciences
Complementary Effects of Carbamylated and Citrullinated LL37 in Autoimmunity and Inflammation in Systemic Lupus Erythematosus
thy donorsÕ plasma plus two standard deviations (+2SD), calculated on the HD group values, as described [6,11]. 4.5. Peptide-MHC Class II Binding Assays Peptide-MHC-binding assays was performed by ImmunAware (Horsholm, Denmark). Binding was measured using a Luminescent Oxygen Channeling Immunoassay (LOCI), marketed by Perkin Elmer (Hopkinton, MA, USA) as AlphaScreen [6,38]. This is a nonradioac
01/01/2021
Thesis
Charakterisierung von CMV-assoziierten NKG2C+ NK-Zellen und deren Einfluss auf antivirale Immunitt
CellTraceª Far Red Cell Proliferation Kit Thermo Fisher Scientific CellTraceª Violet Cell Proliferation Kit Thermo Fisher Scientific easYmer¨ Kit ImmunAware LEGENDScreenª Human PE Kit BioLegend
01/01/2021
Nature medicine
Characterization of pre-existing and induced SARS-CoV-2-specific CD8+ T cells
HLA class I easYmers (immunAware) were loaded with peptide according to the manufacturer’s instructions (A*01/ORF3a 207-215 , A*01/ORF1ab 4163-4172 , A*02/ORF3a 139-147 , Save
01/12/2020
Diabetes
Peptides Derived From Insulin Granule Proteins Are Targeted by CD8+ T Cells Across MHC Class I Restrictions in Humans and NOD Mice
Peptides (90% pure; Synpeptide Co. Ltd.) were tested for binding to HLA-A3 by flow cytometry using biotin-tagged HLA-A3 monomers (immunAware), per the manufacturerÕs protocol. Briefly, biotinylated monomers (final concentration 1.2 nmol/L) were folded as described (3) and captured on 6-8-µm streptavidin-coated beads (Spherotech Inc.)
15/08/2020
bioRxiv
Ex vivo detection of SARS-CoV-2-specific CD8+ T cells: rapid induction, prolonged contraction, and formation of functional memory
Peptides were synthesized with an unmodified N-terminus and an amidated C-terminus with 19 standard Fmoc chemistry and a purity of 70% (Genaxxon Bioscience, Ulm, Germany). HLA 20 class I easYmers¨ (immunAware, Copenhagen, Denmark) were loaded with peptide 21 according to manufacturerÕs instructions (A*01/ORF3a207-215, A*01/ORF1ab4163-4172, 22 A*02/ORF3a139-147, B*07/N105-113) or ordered as peptide-loaded monomers (B*44:03/N322-330, 23 B*44:03/ORF1ab3946-3954). S
01/07/2020
Journal of immunology (Baltimore, Md. : 1950)
HLA Class II Specificity Assessed by High-Density Peptide Microarray Interactions
HLA-DR molecules (DRA1*01:01, DRB1*01:01, DRB1*03:01; immunAware, Copenhagen, Denmark) dissolved in storage buffer (8 M urea, 25 mM Tris [pH 8], 25 mM NaCl) were diluted in 12 ml of refolding buffer (PBS [pH 7.4] supplemented with 0.01% Lutrol F68, glycerol 20% [v/v], TPCK, TLCK, PMSF, TCEP) to a final concentration of 500 nM HLA-DRA and HLA-DRB and transferred to the incubation jar containing the hydrated peptide array and allowed to (de novo) refold at 18¡C for 48 h
01/05/2020
Cancer immunology research
Prevalent and Diverse Intratumoral Oncoprotein-Specific CD8+ T Cells within Polyomavirus-Driven Merkel Cell Carcinomas
Monomeric complexes of HLA B*44:02 and peptide (ImmunAware) were tetramerized with a 4:1 molar ratio of PE- or APC-labeled streptavidin (Thermo Fisher Scientific). TIL cells were Save
03/04/2020
Scientific reports
Native/citrullinated LL37-specific T-cells help autoantibody production in Systemic Lupus Erythematosus
1 were expressed in E. coli and purified in a series of liquid chromatography based steps71. Peptide-MHC class II binding assays We performed peptide-MHC binding assays by two different protocols by ImmunAware, Denmark and Benaroya Institute (USA). In the first protocol, binding was measured using a Luminescent Oxygen Channelling Immunoassay (LOCI), marketed by Perkin Elmer as AlphaScreen72. This
15/03/2019
Molecular therapy. Methods & clinical development
Efficient Induction of T Cells against Conserved HIV-1 Regions by Mosaic Vaccines Delivered as Self-Amplifying mRNA
kinson). The frequencies of the subtypes in CD8+ and CD4+ TÊcells represent the differences in stimulated and unstimulated immune cells. MHC Class I Tetramer Staining Refolded MHC-peptide monomers (immunAware, Denmark) were tetramerized by adding streptavidin-conjugate-APC (Life Technologies) at 4¡C. Splenocytes were stained with 30Ê_L of the optimal tetramer concentration for 20Êmin at room
01/01/2019
Thesis
Epigenetic Determinants of CD8+ T Cell Exhaustion
For the HIV cohort, the following fluorochrome-conjugated multimers from ImmunAware were used: HIV A*02:01 RLRPGGKKK, HIV B*07:02 GPGHKARVL, HIV B*57:01 KAFSPEVIPMF, HIV B*57:01 TSTLQEQIAW, HIV B*53:01 YPLTFGWCF. Cell isolation and processing was performed as previously described
06/11/2017
Scientific reports
Butyrate and propionate inhibit antigen-specific CD8+ T cell activation by suppressing IL-12 production by antigen-presenting cells
production by anti-human IFN-_ (clone B27) and TNF-_ (MAb11) (both BD Bioscience). For analysis of antigen-specificity, CD8+ T cells stained with PE-conjugated peptide-HLA-A2 MHC-tetramer (30_nM, immunAware, Denmark) for 30_min at 37_¡C followed by surface markers staining of CD8 and CD3 for 30_min at 4_¡C. Data acquisition and flow cytometric analysis were done on by LSRFortessa