InVivoMAb anti-human HLA-A2
Product Details
The BB7.2 monoclonal antibody reacts with the alpha subunit of the human leukocyte antigen-A2 (HLA-A2), also known as HLAA, HLA class I histocompatibility antigen A Ī± chain, and major histocompatibility complex, class I, A (MHC-IA). With 31 known alleles, the HLA-A2 family represents the largest and most diverse allele family at the HLA-A locus. Unlike many other HLA allele families, HLA-A2 is found more frequently in all ethnic groups; however, the frequencies of the alleles within the A2 family vary among ethnic groups. MHC class I antigens are expressed on the cell surface of all human nucleated cells, and as complexed with beta 2 microglobulin (Ī²2M), the HLA-A2 molecule displays viral as well as tumor-derived peptides on antigen-presenting cells (APCs) for recognition by the Ī±Ī² TCR on HLA-A-restricted CD8+ T cells, initiating antigen-specific T cell immune response to eliminate infected cells or transformed cells. HLA-A2 can also be involved in presenting self-peptides derived from the signal sequence of secreted or membrane proteins, but the T cells specific for these self-peptides are usually inactivated to prevent autoreactivity. HLA-A2 typically presents intracellular peptide antigens of 8 to 13 amino acids that arise from cytosolic proteolysis via IFNĪ³-induced immunoproteasome or via endopeptidase IDE/insulin-degrading enzyme. Immunopeptidomics, functional experiments and various immunoassays often utilize the BB7.2 antibody as HLA-A*02-specific antibody with a pan HLA class I antibody (W6/32 antibody) in pre-clinical immunology studies. Mutation at position 107 of the Ī±2 domain of the HLA-A2 molecule results in a partial loss of BB7.2 binding with its epitope. Because of overlapping epitopes, the BB7.2 antibody cannot be used in combination with the PA2.1 antibody. The BB7.2 antibody is also reported to cross-react with the HLA-A2 antigen of various non-human primate species.Specifications
| Isotype | Mouse IgG2b, Īŗ |
|---|---|
| Recommended Isotype Control(s) | InVivoMAb mouse IgG2b isotype control, unknown specificity |
| Recommended Dilution Buffer | InVivoPure pH 7.0 Dilution Buffer |
| Immunogen | Priess human B cell line |
| Reported Applications |
in vitro functional assay Immunopeptidomics Immunoprecipitation Flow cytometry Immunofluorescence |
| Formulation |
PBS, pH 7.0 Contains no stabilizers or preservatives |
| Endotoxin |
<2EU/mg (<0.002EU/Ī¼g) Determined by LAL gel clotting assay |
| Purity |
>95% Determined by SDS-PAGE |
| Sterility | 0.2 Āµm filtration |
| Production | Purified from cell culture supernatant in an animal-free facility |
| Purification | Protein A |
| Molecular Weight | 150 kDa |
| Storage | The antibody solution should be stored at the stock concentration at 4Ā°C. Do not freeze. |
Recommended Products
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Recommended Isotype Control(s)
InVivoMAb mouse IgG2b isotype control, unknown specificity
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Recommended Dilution Buffer
InVivoPure pH 7.0 Dilution Buffer
MHC-I Immunopeptidomics
Hunt DF, Henderson RA, Shabanowitz J, Sakaguchi K, Michel H, Sevilir N, Cox AL, Appella E, Engelhard VH. (Science). "Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry" Science 255(5049):1261-3. PubMed
Antigens recognized by T cells are expressed as peptides bound to major histocompatibility complex (MHC) molecules. Microcapillary high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to fractionate and sequence subpicomolar amounts of peptides isolated from the MHC molecule HLA-A2.1. Of 200 different species quantitated, eight were sequenced and four were found in cellular proteins. All were nine residues long and shared a distinct structural motif. The sensitivity and speed of this approach should enhance the analysis of peptides from small quantities of virally infected and transformed cells as well as those associated with autoimmune disease states.
MHC-I Immunopeptidomics
Tokita S, Fusagawa M, Matsumoto S, Mariya T, Umemoto M, Hirohashi Y, Hata F, Saito T, Kanaseki T, Torigoe T. (2024). "Identification of immunogenic HLA class I and II neoantigens using surrogate immunopeptidomes" Sci Adv 10(38):eado6491. PubMed
Neoantigens arising from somatic mutations are tumor specific and induce antitumor host T cell responses. However, their sequences are individual specific and need to be identified for each patient for therapeutic applications. Here, we present a proteogenomic approach for neoantigen identification, named Neoantigen Selection using a Surrogate Immunopeptidome (NESSIE). This approach uses an autologous wild-type immunopeptidome as a surrogate for the tumor immunopeptidome and allows human leukocyte antigen (HLA)-agnostic identification of both HLA class I (HLA-I) and HLA class II (HLA-II) neoantigens. We demonstrate the direct identification of highly immunogenic HLA-I and HLA-II neoantigens using NESSIE in patients with colorectal cancer and endometrial cancer. Fresh or frozen tumor samples are not required for analysis, making it applicable to many patients in clinical settings. We also demonstrate tumor prevention by vaccination with selected neoantigens in a preclinical mouse model. This approach may benefit personalized T cell-mediated immunotherapies.
MHC-I Immunopeptidomics
Arieta CM, Xie YJ, Rothenberg DA, Diao H, Harjanto D, Meda S, Marquart K, Koenitzer B, Sciuto TE, Lobo A, Zuiani A, Krumm SA, Cadima Couto CI, Hein S, Heinen AP, Ziegenhals T, Liu-Lupo Y, Vogel AB, Srouji JR, Fesser S, Thanki K, Walzer K, Addona TA, TĆ¼reci Ć, Åahin U, Gaynor RB, Poran A. (2023). "The T-cell-directed vaccine BNT162b4 encoding conserved non-spike antigens protects animals from severe SARS-CoV-2 infection" Cell 186(11):2392-2409.e21. PubMed
T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).
MHC-I Immunopeptidomics, Flow Cytometry
Pandey K, Wang SS, Mifsud NA, Faridi P, Davenport AJ, Webb AI, Sandow JJ, Ayala R, Monje M, Cross RS, Ramarathinam SH, Jenkins MR, Purcell AW. (2023). "A combined immunopeptidomics, proteomics, and cell surface proteomics approach to identify immunotherapy targets for diffuse intrinsic pontine glioma" Front Oncol . PubMed
Introduction: Diffuse intrinsic pontine glioma (DIPG), recently reclassified as a subtype of diffuse midline glioma, is a highly aggressive brainstem tumor affecting children and young adults, with no cure and a median survival of only 9 months. Conventional treatments are ineffective, highlighting the need for alternative therapeutic strategies such as cellular immunotherapy. However, identifying unique and tumor-specific cell surface antigens to target with chimeric antigen receptor (CAR) or T-cell receptor (TCR) therapies is challenging. Methods: In this study, a multi-omics approach was used to interrogate patient-derived DIPG cell lines and to identify potential targets for immunotherapy. Results: Through immunopeptidomics, a range of targetable peptide antigens from cancer testis and tumor-associated antigens as well as peptides derived from human endogenous retroviral elements were identified. Proteomics analysis also revealed upregulation of potential drug targets and cell surface proteins such as Cluster of differentiation 27 (CD276) B7 homolog 3 protein (B7H3), Interleukin 13 alpha receptor 2 (IL-13RĪ±2), Human Epidermal Growth Factor Receptor 3 (HER2), Ephrin Type-A Receptor 2 (EphA2), and Ephrin Type-A Receptor 3 (EphA3). Discussion: The results of this study provide a valuable resource for the scientific community to accelerate immunotherapeutic approaches for DIPG. Identifying potential targets for CAR and TCR therapies could open up new avenues for treating this devastating disease.
Immunoprecipitation, MHC-I Immunopeptidomics
Shahbazy M, Ramarathinam SH, Illing PT, Jappe EC, Faridi P, Croft NP, Purcell AW. (2023). "Benchmarking Bioinformatics Pipelines in Data-Independent Acquisition Mass Spectrometry for Immunopeptidomics" Mol Cell Proteomics 22(4):100515. PubMed
Immunopeptidomes are the peptide repertoires bound by the molecules encoded by the major histocompatibility complex [human leukocyte antigen (HLA) in humans]. These HLA-peptide complexes are presented on the cell surface for immune T-cell recognition. Immunopeptidomics denotes the utilization of tandem mass spectrometry to identify and quantify peptides bound to HLA molecules. Data-independent acquisition (DIA) has emerged as a powerful strategy for quantitative proteomics and deep proteome-wide identification; however, DIA application to immunopeptidomics analyses has so far seen limited use. Further, of the many DIA data processing tools currently available, there is no consensus in the immunopeptidomics community on the most appropriate pipeline(s) for in-depth and accurate HLA peptide identification. Herein, we benchmarked four commonly used spectral library-based DIA pipelines developed for proteomics applications (Skyline, Spectronaut, DIA-NN, and PEAKS) for their ability to perform immunopeptidome quantification. We validated and assessed the capability of each tool to identify and quantify HLA-bound peptides. Generally, DIA-NN and PEAKS provided higher immunopeptidome coverage with more reproducible results. Skyline and Spectronaut conferred more accurate peptide identification with lower experimental false-positive rates. All tools demonstrated reasonable correlations in quantifying precursors of HLA-bound peptides. Our benchmarking study suggests a combined strategy of applying at least two complementary DIA software tools to achieve the greatest degree of confidence and in-depth coverage of immunopeptidome data.
in vitro functional assay
Bharadwaj P, Shrestha S, Pongracz T, Concetta C, Sharma S, Le Moine A, de Haan N, Murakami N, Riella LV, Holovska V, Wuhrer M, Marchant A, Ackerman ME. (2022). "Afucosylation of HLA-specific IgG1 as a potential predictor of antibody pathogenicity in kidney transplantation" Cell Rep Med 3(11):100818. PubMed
Antibody-mediated rejection (AMR) is the leading cause of graft failure. While donor-specific antibodies (DSAs) are associated with a higher risk of AMR, not all patients with DSAs develop rejection, suggesting that the characteristics of alloantibodies determining their pathogenicity remain undefined. Using human leukocyte antigen (HLA)-A2-specific antibodies as a model, we apply systems serology tools to investigate qualitative features of immunoglobulin G (IgG) alloantibodies including Fc-glycosylation patterns and FcĪ³R-binding properties. Levels of afucosylated anti-A2 antibodies are elevated in seropositive patients, especially those with AMR, suggesting potential cytotoxicity via FcĪ³RIII-mediated mechanisms. Afucosylation of both glycoengineered monoclonal and naturally glycovariant polyclonal serum IgG specific to HLA-A2 drives potentiated binding to, slower dissociation from, and enhanced signaling through FcĪ³RIII, a receptor widely expressed on innate effector cells, and greater cytotoxicity against HLA-A2+ cells mediated by natural killer (NK) cells. Collectively, these results suggest that afucosylated DSA may be a biomarker of AMR and contribute to pathogenesis.
MHC-I Immunopeptidomics
Vadakekolathu J, Boocock DJ, Pandey K, Guinn BA, Legrand A, Miles AK, Coveney C, Ayala R, Purcell AW, McArdle SE. (2022). "Multi-Omic Analysis of Two Common P53 Mutations: Proteins Regulated by Mutated P53 as Potential Targets for Immunotherapy" Cancers (Basel) 14(16):3975. PubMed
The p53 protein is mutated in more than 50% of human cancers. Mutated p53 proteins not only lose their normal function but often acquire novel oncogenic functions, a phenomenon termed mutant p53 gain-of-function. Mutant p53 has been shown to affect the transcription of a range of genes, as well as protein-protein interactions with transcription factors and other effectors; however, no one has intensively investigated and identified these proteins, or their MHC presented epitopes, from the viewpoint of their ability to act as targets for immunotherapeutic interventions. We investigated the molecular changes that occurred after the TP53 null osteosarcoma cells, SaOS-2, were transfected with one of two conformational p53-mutants, either R175H or R273H. We then examined the phenotypic and functional changes using macroscopic observations, proliferation, gene expression and proteomics alongside immunopeptidome profiling of peptide antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. We identified several candidate proteins in both TP53 mutant cell lines with differential expression when compared to the TP53 null vector control, SaOS-V. Quantitative SWATH proteomics combined with immune-peptidome analysis of the class-I eluted peptides identified several epitopes presented on pMHC and in silico analysis shortlisted which antigens were expressed in a range of cancerous but not adjacent healthy tissues. Out of all the candidates, KLC1 and TOP2A showed high levels of expression in every tumor type examined. From these proteins, three A2 and four pan HLA-A epitopes were identified in both R175H and R273H from TOP2A. We have now provided a short list of future immunotherapy targets for the treatment of cancers harboring mutated TP53.
MHC-I Immunopeptidomics
Pandey K, Ramarathinam SH, Purcell AW. (2021). "Isolation of HLA Bound Peptides by Immunoaffinity Capture and Identification by Mass Spectrometry" Curr Protoc 1(3):e92. PubMed
This article describes the purification of HLA-bound peptides and their subsequent sequencing by mass spectrometry. These methods can be used for both HLA class I and class II molecules and can be adapted to different species depending on the availability of specific antibodies. Peptides can be successfully isolated from a variety of sample types, including in vitro cultured cells and primary tissues. The method involves the affinity capture of HLA-peptide complexes and separation of peptides from HLA heavy chains, followed by tailored interrogation by mass spectrometry to take into account the non-tryptic nature of endogenously derived HLA-bound peptides. Ā© 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of immunoaffinity column Alternate Protocol 1: Preparation of microscale immunoaffinity column Basic Protocol 2: Generation of cell lysate and HLA immunoaffinity purification Alternate Protocol 2: Microscale immunoaffinity purification Basic Protocol 3: Separation of HLA peptides by reverse-phase high-performance liquid chromatography (RP-HPLC) Alternate Protocol 3: Isolation of HLA peptides using molecular weight cutoff (MWCO) filter Basic Protocol 4: Mass spectrometry and data analysis.
MHC-I Immunopeptidomics
Pandey K, Mifsud NA, Lim Kam Sian TCC, Ayala R, Ternette N, Ramarathinam SH, Purcell AW. (2020). "In-depth mining of the immunopeptidome of an acute myeloid leukemia cell line using complementary ligand enrichment and data acquisition strategies" Mol Immunol . PubMed
The identification of T cell epitopes derived from tumour specific antigens remains a significant challenge for the development of peptide-based vaccines and immunotherapies. The use of mass spectrometry-based approaches (immunopeptidomics) can provide powerful new avenues for the identification of such epitopes. In this study we report the use of complementary peptide antigen enrichment methods and a comprehensive mass spectrometric acquisition strategy to provide in-depth immunopeptidome data for the THP-1 cell line, a cell line used widely as a model of human leukaemia. To accomplish this, we combined robust experimental workflows that incorporated ultrafiltration or off-line reversed phase chromatography to enrich peptide ligand as well as a multifaceted data acquisition strategy using an Orbitrap Fusion LC-MS instrument. Using the combined datasets from the two ligand enrichment methods we gained significant depth in immunopeptidome coverage by identifying a total of 41,816 HLA class I peptides from THP-1 cells, including a significant number of peptides derived from different oncogenes or over expressed proteins associated with cancer. The physicochemical properties of the HLA-bound peptides dictated their recovery using the two ligand enrichment approaches and their distribution across the different precursor charge states considered in the data acquisition strategy. The data highlight the complementarity of the two enrichment procedures, and in cases where sample is not limiting, suggest that the combination of both approaches will yield the most comprehensive immunopeptidome information.
MHC-I Immunopeptidomics
Jappe EC, Garde C, Ramarathinam SH, Passantino E, Illing PT, Mifsud NA, Trolle T, Kringelum JV, Croft NP, Purcell AW. (2020). "Thermostability profiling of MHC-bound peptides: a new dimension in immunopeptidomics and aid for immunotherapy design" Nat Commun 11(1):6305. PubMed
The features of peptide antigens that contribute to their immunogenicity are not well understood. Although the stability of peptide-MHC (pMHC) is known to be important, current assays assess this interaction only for peptides in isolation and not in the context of natural antigen processing and presentation. Here, we present a method that provides a comprehensive and unbiased measure of pMHC stability for thousands of individual ligands detected simultaneously by mass spectrometry (MS). The method allows rapid assessment of intra-allelic and inter-allelic differences in pMHC stability and reveals profiles of stability that are broader than previously appreciated. The additional dimensionality of the data facilitated the training of a model which improves the prediction of peptide immunogenicity, specifically of cancer neoepitopes. This assay can be applied to any cells bearing MHC or MHC-like molecules, offering insight into not only the endogenous immunopeptidome, but also that of neoepitopes and pathogen-derived sequences.
MHC-I Immunopeptidomics
Komov L, Kadosh DM, Barnea E, Milner E, Hendler A, Admon A. (2018). "Cell Surface MHC Class I Expression Is Limited by the Availability of Peptide-Receptive "Empty" Molecules Rather than by the Supply of Peptide Ligands" Proteomics 18(12):e1700248. PubMed
While antigen processing and presentation (APP) by the major histocompatibility complex class I (MHC-I) molecules have been extensively studied, a question arises as to whether the level of MHC-I expression is limited by the supply of peptide-receptive (empty) MHC molecules, or by the availability of peptide ligands for loading. To this end, the effect of interferons (IFNs) on the MHC peptidomes of human breast cancer cells (MCF-7) were evaluated. Although all four HLA allotypes of the MCF-7 cells (HLA-A*02:01, B*18, B*44, and C*5) present peptides of similar lengths and C-termini, which should be processed similarly by the proteasome and by the APP chaperones, the IFNs induced differential modulation of the HLA-A, B, and C peptidomes. In addition, overexpression of recombinant soluble HLA-A*02:01, introduced to compete with the identical endogenous membrane-bound HLA-A*02:01 for peptides of the MCF-7 cells, did not alter the expression level or the presented peptidome of the membrane-bound HLA-A*02:01. Taken together, these results indicate that a surplus supply of peptides is available inside the ER for loading onto the MHC-I peptide-receptive molecules, and that cell surface MHC-I expression is likely limited by the availability of empty MHC molecules.
Flow Cytometry
Gonzalez-Duque S, Azoury ME, Colli ML, Afonso G, Turatsinze JV, Nigi L, Lalanne AI, Sebastiani G, CarrĆ© A, Pinto S, Culina S, Corcos N, Bugliani M, Marchetti P, Armanet M, Diedisheim M, Kyewski B, Steinmetz LM, Buus S, You S, Dubois-Laforgue D, Larger E, Beressi JP, Bruno G, Dotta F, Scharfmann R, Eizirik DL, Verdier Y, Vinh J, Mallone R. (2018). "Conventional and Neo-antigenic Peptides Presented by Ī² Cells Are Targeted by Circulating NaĆÆve CD8+ T Cells in Type 1 Diabetic and Healthy Donors" Cell Metab 28(6):946-960.e6. PubMed
Although CD8+ T-cell-mediated autoimmune Ī² cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by Ī² cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known Ī² cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by Ī² cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.
in vitro functional assay, Flow Cytometry
Lowe DB, Bivens CK, Mobley AS, Herrera CE, McCormick AL, Wichner T, Sabnani MK, Wood LM, Weidanz JA. (2017). "TCR-like antibody drug conjugates mediate killing of tumor cells with low peptide/HLA targets" MAbs 9(4):603-614. PubMed
The currently marketed antibody-drug conjugates (ADC) destabilize microtubule assembly in cancer cells and initiate apoptosis in patients. However, few tumor antigens (TA) are expressed at high densities on cancer lesions, potentially minimizing the therapeutic index of current ADC regimens. The peptide/human leukocyte antigen (HLA) complex can be specifically targeted by therapeutic antibodies (designated T cell receptor [TCR]-like antibodies) and adequately distinguish malignant cells, but has not been the focus of ADC development. We analyzed the killing potential of TCR-like ADCs when cross-linked to the DNA alkylating compound duocarmycin. Our data comprise proof-of-principle results that TCR-like ADCs mediate potent tumor cytotoxicity, particularly under common scenarios of low TA/HLA density, and support their continued development alongside agents that disrupt DNA replication. Additionally, TCR-like antibody ligand binding appears to play an important role in ADC functionality and should be addressed during therapy development to avoid binding patterns that negate ADC killing efficacy.
in vitro functional assay
Chawla A, Alatrash G, Philips AV, Qiao N, Sukhumalchandra P, Kerros C, Diaconu I, Gall V, Neal S, Peters HL, Clise-Dwyer K, Molldrem JJ, Mittendorf EA. (2016). "Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells" Cancer Immunol Immunother 65(6):741-51. PubMed
Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response.
Immunoprecipitation
Cho S, Kim BY, Ahn K, Jun Y. (2013). "The C-terminal amino acid of the MHC-I heavy chain is critical for binding to Derlin-1 in human cytomegalovirus US11-induced MHC-I degradation" PLoS One 8(8):e72356. PubMed
Derlin-1 plays a critical role in endoplasmic reticulum-associated protein degradation (ERAD) of a particular subset of proteins. Although it is generally accepted that Derlin-1 mediates the export of ERAD substrates from the ER to the cytosol, little is known about how Derlin-1 interacts with these substrates. Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules and evade immune surveillance. US11 requires the cytosolic tail of the MHC-I heavy chain to divert MHC-I molecules into the ERAD pathway for degradation; however, the underlying mechanisms remain unknown. Here, we show that the cytosolic tail of the MHC-I heavy chain, although not required for interaction with US11, is required for tight binding to Derlin-1 and thus for US11-induced dislocation of the MHC-I heavy chain to the cytosol for proteasomal degradation. Surprisingly, deletion of a single C-terminal amino acid from the cytosolic tail disrupted the interaction between MHC-I molecules and Derlin-1, rendering mutant MHC-I molecules resistant to US11-induced degradation. Consistently, deleting the C-terminal cytosolic region of Derlin-1 prevented it from binding to MHC-I molecules. Taken together, these results suggest that the cytosolic region of Derlin-1 is involved in ERAD substrate binding and that this interaction is critical for the Derlin-1-mediated dislocation of the MHC-I heavy chain to the cytosol during US11-induced MHC-I degradation.
in vitro functional assay
Zhao X, Bose A, Komita H, Taylor JL, Chi N, Lowe DB, Okada H, Cao Y, Mukhopadhyay D, Cohen PA, Storkus WJ. (2012). "Vaccines targeting tumor blood vessel antigens promote CD8(+) T cell-dependent tumor eradication or dormancy in HLA-A2 transgenic mice" J Immunol 188(4):1782-8. PubMed
We have recently shown that effective cytokine gene therapy of solid tumors in HLA-A2 transgenic (HHD) mice lacking murine MHC class I molecule expression results in the generation of HLA-A2-restricted CD8(+) T effector cells selectively recognizing tumor blood vessel-associated pericytes and/or vascular endothelial cells. Using an HHD model in which HLA-A2(neg) tumor (MC38 colon carcinoma or B16 melanoma) cells are not recognized by the CD8(+) T cell repertoire, we now show that vaccines on the basis of tumor-associated blood vessel Ags (TBVA) elicit protective Tc1-dependent immunity capable of mediating tumor regression or extending overall survival. Vaccine efficacy was not observed if (HLA-A2(neg)) wild-type C57BL/6 mice were instead used as recipient animals. In the HHD model, effective vaccination resulted in profound infiltration of tumor lesions by CD8(+) (but not CD4(+)) T cells, in a coordinate reduction of CD31(+) blood vessels in the tumor microenvironment, and in the "spreading" of CD8(+) T cell responses to alternate TBVA that were not intrinsic to the vaccine. Protective Tc1-mediated immunity was durable and directly recognized pericytes and/or vascular endothelial cells flow-sorted from tumor tissue but not from tumor-uninvolved normal kidneys harvested from these same animals. Strikingly, the depletion of CD8(+), but not CD4(+), T cells at late time points after effective therapy frequently resulted in the recurrence of disease at the site of the regressed primary lesion. This suggests that the vaccine-induced anti-TBVA T cell repertoire can mediate the clinically preferred outcomes of either effectively eradicating tumors or policing a state of (occult) tumor dormancy.
Immunofluorescence, in vitro functional assay, Flow Cytometry
Sergeeva A, Alatrash G, He H, Ruisaard K, Lu S, Wygant J, McIntyre BW, Ma Q, Li D, St John L, Clise-Dwyer K, Molldrem JJ. (2011). "An anti-PR1/HLA-A2 T-cell receptor-like antibody mediates complement-dependent cytotoxicity against acute myeloid leukemia progenitor cells" Blood 117(16):4262-72. PubMed
PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)-restricted leukemia-associated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)-like immunoglobulin G2a (IgG2a) antibody (8F4) with high specific binding affinity (dissociation constant [K(D)] = 9.9nM) for a combined epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes (P = .046). 8F4 mediated complement-dependent cytolysis of AML blasts and Lin(-)CD34(+)CD38(-) leukemia stem cells (LSCs) but not normal leukocytes (P < .005). Although PR1 expression was similar on LSCs and hematopoietic stem cells, 8F4 inhibited AML progenitor cell growth, but not normal colony-forming units from healthy donors (P < .05). This study shows that 8F4, a novel TCR-like antibody, binds to a conformational epitope of the PR1/HLA-A2 complex on the cell surface and mediates specific lysis of AML, including LSCs. Therefore, this antibody warrants further study as a novel approach to targeting leukemia-initiating cells in patients with AML.
in vitro functional assay
Verma B, Neethling FA, Caseltine S, Fabrizio G, Largo S, Duty JA, Tabaczewski P, Weidanz JA. (2010). "TCR mimic monoclonal antibody targets a specific peptide/HLA class I complex and significantly impedes tumor growth in vivo using breast cancer models" J Immunol 184(4):2156-65. PubMed
Our laboratory has developed a process for generating mAbs with selectivity to unique peptides in the context of MHC molecules. Recently, we reported that RL4B, an mAb that we have called a TCR mimic (TCRm) because it recognizes peptide in the context of MHC, has cytotoxic activity in vitro and prevented growth of tumor cells in a prophylactic setting. When presented in the context of HLA-A2, RL4B TCRm recognizes the peptide GVLPALPQV derived from human chorionic gonadotropin (hCG)-beta. In this study, we show that RL4B TCRm has strong binding affinity for the GVLPALPQV peptide/HLA-A2 epitope and fine binding specificity for cells that express endogenous hCGbeta Ag and HLA-A2. In addition, suppression of tumor growth with RL4B TCRm was observed in orthotopic models for breast cancer. Using two aggressive human tumor cell lines, MDA-MB-231 and MCF-7, we provide evidence that RL4B TCRm significantly retards tumor growth, supporting a possible role for TCRm agents in therapeutic settings. Moreover, tumors in mice responded to RL4B TCRm therapy in a dose-dependent manner, eliminating tumors at the highest dose. RL4B TCRm strongly detects the hCGbeta peptide/HLA-A2 epitope in human primary breast tumor tissue, but does not react or reacts weakly with normal breast tissue from the same patient. These results further illustrate the selective nature of TCRm Abs and the clinical relevance of the GVLPALPQV peptide/HLA-A2 epitope expression in tumor cells, because they provide the first evidence that Abs that mimic the TCR can be used to markedly reduce and suppress tumor growth.
Immunofluorescence, Immunoprecipitation, Flow Cytometry
Schaefer MR, Williams M, Kulpa DA, Blakely PK, Yaffee AQ, Collins KL. (2008). "A novel trafficking signal within the HLA-C cytoplasmic tail allows regulated expression upon differentiation of macrophages" J Immunol 180(12):7804-17. PubMed
MHC class I molecules (MHC-I) present peptides to CTLs. In addition, HLA-C allotypes are recognized by killer cell Ig-like receptors (KIR) found on NK cells and effector CTLs. Compared with other classical MHC-I allotypes, HLA-C has low cell surface expression and an altered intracellular trafficking pattern. We present evidence that this results from effects of both the extracellular domain and the cytoplasmic tail. Notably, we demonstrate that the cytoplasmic tail contains a dihydrophobic (LI) internalization and lysosomal targeting signal that is partially attenuated by an aspartic acid residue (DXSLI). In addition, we provide evidence that this signal is specifically inhibited by hypophosphorylation of the adjacent serine residue upon macrophage differentiation and that this allows high HLA-C expression in this cell type. We propose that tightly regulated HLA-C surface expression facilitates immune surveillance and allows HLA-C to serve a specialized role in macrophages.
Flow Cytometry
Sprengers D, van der Molen RG, Kusters JG, De Man RA, Niesters HG, Schalm SW, Janssen HL. (2006). "Analysis of intrahepatic HBV-specific cytotoxic T-cells during and after acute HBV infection in humans" J Hepatol 45(2):182-9. PubMed
Background/aims: Characteristics of the intrahepatic virus-specific T-cell response in patients with acute hepatitis B virus (HBV) infection have not been studied due to the risk of complications associated with standard liver biopsies. In this study we aimed to characterize the virus-specific CD8 + T-cell response in the liver of patients with acute HBV infection using fine-needle aspiration-biopsy (FNAB). Methods: In HLA-A2 positive patients with acute HBV infection a FNAB was performed at first presentation, at the time of HBsAg-seroconversion and 3 months after HBsAg-seroconversion. HLA-A2 tetramers were used to identify HBV-specific CD8 + T-cells in FNAB-cytology and peripheral blood (PB). Results: At first presentation there was a correlation between the frequency of intrahepatic CD8 + T-cells and the degree of liver damage. At all time points there was sequestering of HBV-specific CD8 + T-cells in the liver, and the percentage of intrahepatic HLA-DR expressing HBV-specific CD8 + T-cells was higher than in PB. Three months after HBsAg-seroconversion the frequency of intrahepatic HBV-specific CD8 + T-cells remained high. Conclusions: HBV-specific CD8 + T-cells are compartmentalized in the liver during acute HBV infection. Their presence in the liver may suggest a role in the resolution of the infection. Intrahepatic HBV-specific CD8 + T cells remain detectable at high frequencies after HBsAg-seroconversion.
Flow Cytometry
Sprengers D, van der Molen RG, Kusters JG, Kwekkeboom J, van der Laan LJ, Niesters HG, Kuipers EJ, De Man RA, Schalm SW, Janssen HL. (2005). "Flow cytometry of fine-needle-aspiration biopsies: a new method to monitor the intrahepatic immunological environment in chronic viral hepatitis" J Viral Hepat 12(5):507-12. PubMed
Information about the character and grade of the intrahepatic immune response in viral hepatitis is important for the evaluation of disease stage and effect of therapy. Complications like haemorrhage limit the frequent performance of tissue-needle biopsies (TB), and the cells of peripheral blood have to be used as surrogate markers instead. Fine-needle-aspiration biopsy (FNAB) of the liver represents a safe and atraumatic method that allows frequent cytological sampling. Our aim was to investigate whether flow cytometry of FNAB specimens allows co-analysis of phenotype, function and specificity of key populations of liver-infiltrating lymphocytes (LIL). In 20 consecutive patients with chronic viral hepatitis [10 hepatitis B virus (HBV), 10 hepatitis C virus (HCV)], flow cytometry was performed on FNAB cytology, and simultaneously on lymphocytes isolated from a TB and peripheral blood mononuclear cells (PBMC). The ratio of CD8+/CD4+ lymphocytes in FNAB correlated well with LIL from TB (r =0.78, P < 0.05) but differed from PBMC (mean ratio: 2.6, 2.1 and 0.7, respectively). Similarly, a correlation was observed for percentage CD56+ natural killer (NK) cells (mean %: 29.9, 32.3 and 14.5, respectively; r = 0.69, P < 0.05). The percentage of interferon (IFN)-gamma-producing CD3+ lymphocytes in both FNAB and TB was higher than in PBMC (mean %: 41, 44 and 22, respectively; P < 0.05). Furthermore, tetrameric complexes allowed analysis of HBV-specific T cells in FNAB specimens. In conclusion, flow cytometry of FNAB allows easy, atraumatic and reliable analysis of lymphocytes obtained from the intrahepatic compartment. Therefore, the FNAB is a valuable tool in the study of the immunopathology of viral hepatitis, and it may contribute to the improved clinical evaluation of chronic viral liver disease.
Flow Cytometry
Peshwa MV, Shi JD, Ruegg C, Laus R, van Schooten WC. (1998). "Induction of prostate tumor-specific CD8+ cytotoxic T-lymphocytes in vitro using antigen-presenting cells pulsed with prostatic acid phosphatase peptide" Prostate 36(2):129-38. PubMed
Background: Most strategies in cancer immunotherapy are aimed at the induction of a strong cellular immune response against the tumor. Particularly, CD8+ T lymphocytes have been proven in multiple animal models to be critical for the eradication of solid tumors. Methods: We used a population of peripheral blood-derived antigen-presenting cells (APC), containing dendritic cells (DC), to generate prostate tumor-specific CD8+ T cells. Selected peptides from prostatic acid phosphatase (PAP), a prostate tissue-specific antigen, were shown to bind HLA-A2. A high-affinity peptide was used to generate peptide-specific CD8+ cytolytic T lymphocytes (CTL) from the peripheral blood of healthy donors. Results: The obtained PAP-peptide-specific CTL lysed peptide-coated target cells, vaccinia-infected target cells, and HLA-A2-positive prostate-tumor cells in vitro in an antigen-specific manner. Conclusions: Our results indicate that CTL precursors to the PAP gene product exist and could be potentially recruited to elicit an antitumor response. Thus, PAP is a suitable antigen for inclusion in prostate cancer vaccines.
Immunoprecipitation, Flow Cytometry
Forero L, Zwirner NW, Fink CW, FernƔndez-ViƱa MA, Stastny P. (1998). "Juvenile arthritis, HLA-A2 and binding of DEK oncogene-peptides" Hum Immunol 59(7):443-50. PubMed
Previous studies have shown that susceptibility to Pauciarticular Juvenile Arthritis is associated with HLA-A*0201. Recently, autoantibodies against the protein of the DEK oncogene have been found in sera of patients with this disease. If T cells are involved in the pathogenesis of joint lesions, it is possible that they target autoantigens presented by HLA-A*0201. Therefore, we investigated whether DEK-derived peptides can bind efficiently to HLA-A*0201. Nonameric peptides selected considering anchor positions 2 and 9, and preferred amino acids at other positions, were incubated either with the human TAP-deficient cell line 174CEM.T2 (T2) or with the homozygous B cell line JESTHOM (A*0201, B*2705, Cw1), previously depleted of endogenous peptides. Binding was measured as the increase of detection of fully assembled, HLA-A*0201 molecules by flow cytometry with the anti-HLA-A2 monoclonal antibody (mAb) BB7.2. Three out of ten selected DEK-derived peptides showed binding to HLA-A*0201, which was peptide concentration-dependent (1 microM to 100 microM). DEK155-163 (AMLKSICEV), which also has two preferred amino acid residues at positions 6 and 8, yielded the highest binding. DEK163-171 (VLDLERSGV) and DEK72-80 (SLQREPFTI), which also has one preferred amino acid residue at position 8, also were able to bind to HLA-A*0201. Furthermore, peptide-induced, fully assembled, HLA-A*0201 molecules were immunoprecipitated with the BB7.2 mAb from metabolically-labeled T2 cells incubated with DEK72-80, DEK155-163, and DEK163-171. A faint band was observed in the immunoprecipitates of cells incubated with DEK65-73 (it carries a preferred amino acid residue at position 6), suggesting that this peptide interacts weakly with HLA-A*0201. These results indicate that several nonameric peptides derived from the DEK protein can bind to HLA-A 0201 and suggest that the complexes formed may be able to stimulate CD8+ T cells in patients with Pauciarticular Juvenile Arthritis.
Flow Cytometry
Panina-Bordignon P, Lang R, van Endert PM, Benazzi E, Felix AM, Pastore RM, Spinas GA, Sinigaglia F. (1995). "Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes" J Exp Med 181(5):1923-7. PubMed
Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that results in the destruction of the pancreatic islet beta cells. Glutamic acid decarboxylase (GAD) has been recently indicated as a key autoantigen in the induction of IDDM in nonobese diabetic mice. In human diabetes, the mechanism by which the beta cells are destroyed is still unknown. Here we report the first evidence for the presence of GAD-specific cytotoxic T cells in asymptomatic and recent diabetic patients. GAD65 peptides displaying the human histocompatibility leukocyte antigen (HLA)-A*0201 binding motif have been synthesized. One of these peptides, GAD114-123, binds to HLA-A*0201 molecules in an HLA assembly assay. Peripheral blood mononuclear cells from individuals with preclinical IDDM, recent-onset IDDM, and from healthy controls were stimulated in vitro with the selected peptide in the presence of autologous antigen-presenting cells. In three cases (one preclinical IDDM and two recent-onset IDDM), we detected specific killing of autologous antigen-presenting cells when incubated with GAD114-123 peptide or when infected with a recombinant vaccinia virus expressing GAD65. These patients were the only three carrying the HLA-A*0201 allele among the subjects studied. Our finding suggests that GAD-specific cytotoxic T lymphocytes may play a critical role in the initial events of IDDM.
in vitro functional assay
Parham P, Brodsky FM. (1981). "Partial purification and some properties of BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28" Hum Immunol 3(4):277-99. PubMed
The purification and properties of a cytotoxic mouse monoclonal anti-HLA-A2 antibody are described. This antibody, BB7.2, can be used as an HLA-A2 typing reagent with little modification of current typing techniques. It also recognizes a low frequency variant of HLA-A28. BB7.2 provides an example of an antibody which requires bivalent attachment to a polymeric antigen, e.g., a cell, to produce a readily detectable complex. This is due to a high rate of dissociation of the complex formed between a single BB7.2 combining sites and HLA-A2. The consequences of this property were investigated and some complications for potential uses of such monoclonal antibodies are discussed.