Io test beta mark tcr vbeta repertoire kit

Manufactured by Beckman Coulter

Sourced in United States

The IO Test Beta Mark TCR Vbeta Repertoire Kit is a laboratory equipment product used for the analysis of T-cell receptor (TCR) Vbeta chain repertoire. The kit provides the necessary reagents and protocols to perform this analysis.

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IO Test (3 citations)

11 protocols using io test beta mark tcr vbeta repertoire kit

Characterizing IAV-M1-specific CD8 T-cell TCR Vβ

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Sorted CD8 T-cells from fresh PBMCs directly ex vivo were incubated for 20 min. with IAV-M1-specific tetramer, which was then washed off. An additional 20 min. incubation was performed with 24 TCR Vβ antibodies which cover >70% of commonly used human Vβ (IO Test Beta Mark TCR Vbeta Repertoire Kit, Beckman Coulter, Fullerton, CA). Samples were read on LSRII (Beckman Coulter, Fullerton, CA). IMGT TCR gene nomenclature was used to define TCR Vβ types.

Song I., Gil A., Mishra R., Ghersi D., Selin L.K, & Stern L.J. (2017). Broad TCR Repertoire And Diverse Structural Solutions To Recognition Of An Immunodominant CD8 T Cell Epitope. Nature structural & molecular biology, 24(4), 395-406.

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Profiling Clonal T-cell Populations in PBMC

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Peripheral blood mononuclear cells (PBMC) were isolated via Ficoll centrifugation. Vβ clonal T-cell populations were assessed using IOTest® Beta Mark TCR V beta Repertoire Kit. (Beckman Coulter) according to the manufacturer’s instructions. Based on Vβ clonality assessment, patients with unequivocally identifiable clonal T-cell population in the blood and/or skin were subjected to further analysis.
For flow cytometry anti-human monoclonal antibodies were used as listed: anti-CD3 (clone BW264/56 labeled with PerCP, Miltenyi Biotec #130-096-910), anti-CD4 (clone VIT4 labeled with APC-Vio770, Miltenyi Biotec #130-098-153), anti-CD26 (clone BA5b labeled with PE-Cy7, BioLegend #302714), anti-CD279/PD-1 (clone J105 labeled with APC, ThermoFisher), anti-CD274/PD-L1 (clone MIH1 labeled with PE-Cy7, ThermoFisher), anti-CD273/PD-L2 (clone MIH18 labeled with APC, ThermoFisher), anti- KIR3DL2/CD158 k in Alexa Fluor® 700 (R&D systems, #FAB2878 N), anti CD160 in APC (Biolegend, #341207), and anti-TCR Vβ antibodies specific for the malignant clone of each patient (labeled with PE, Beckman Coulter). Samples were acquired on Becton Dickinson FACSCanto or BD LSR II Fortessa flow cytometer. FCS Express 5 Flow Cytometry RUO, FlowJo V10.0.8, Origin Pro 9.1 G and GraphPad Prism 5.0 Software were used for data analysis.

Saulite I., Ignatova D., Chang Y.T., Fassnacht C., Dimitriou F., Varypataki E., Anzengruber F., Nägeli M., Cozzio A., Dummer R., Scarisbrick J., Pascolo S., Hoetzenecker W., Bobrowicz M, & Guenova E. (2020). Blockade of programmed cell death protein 1 (PD-1) in Sézary syndrome reduces Th2 phenotype of non-tumoral T lymphocytes but may enhance tumor proliferation. Oncoimmunology, 9(1), 1738797.

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Assessing T-cell Clonality in CTCL

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Peripheral blood and skin from the patients with CTCL were collected in the context of the University of Zürich Biobank, funded by the University of Zurich University Research Priority Program (URPP) in translational cancer biology. All patients signed an informed consent agreeing to the use of samples, including the generation of cell cultures according to the Biobank project (EK No. 647, approved on the 25 October 2017). The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of the University of Zurich (KEK-ZH-Nr. 2015–0209). Vβ clonal T-cell populations were assessed using IOTest ® Beta Mark TCR V beta Repertoire Kit. (Beckman Coulter, # IM3497, Chaska, MN, USA) according to the manufacturer’s instructions.

Tusup M., Läuchli S., Jarzebska N.T., French L.E., Chang Y.T., Vonow-Eisenring M., Su A., Kündig T.M., Guenova E, & Pascolo S. (2021). mRNA-Based Anti-TCR CDR3 Tumour Vaccine for T-Cell Lymphoma. Pharmaceutics, 13(7), 1040.

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T-cell Receptor Repertoire Analysis

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5 patient TIL and PBMC and 5 control PBMC were isolated as described above. The repertoire was assessed using the IOTest ® Beta Mark TCR V beta Repertoire Kit (Cat: IM3497 Beckman Coulter, Brea, CA) according to manufacturer’s instructions.

Woroniecka K., Chongsathidkiet P., Rhodin K., Kemeny H., Dechant C., Farber S.H., Elsamadicy A.A., Cui X., Koyama S., Jackson C., Hansen L.J., Johanns T.M., Sanchez-Perez L., Chandramohan V., Yu Y.R., Bigner D.D., Giles A., Healy P., Dranoff G., Weinhold K.J., Dunn G.P, & Fecci P.E. (2018). T Cell Exhaustion Signatures Vary with Tumor Type and are Severe in Glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(17), 4175-4186.

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Characterizing IAV-M1-specific CD8 T-cell TCR Vβ

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Quantifying Cell Surface Antigen Expression

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Surface expression was determined by flow cytometric analysis. A total of 100,000–200,000 cells per sample were stained with primary and secondary antibodies at 4 °C for 20 min. Primary antibodies used were monoclonal antibodies PA2.1 (anti-HLA-A2 [25 (link)]) B1.23.2 (anti-HLA-B/C [26 (link)]), antibodies against HLA-A1 and HLA-B15 (both purchased from One Lambda, West Hills, Los Angeles, CA, USA), as well as those from the IOTest Beta Mark TCR Vbeta Repertoire Kit (Beckman Coulter, Krefeld, Germany). The secondary antibody used was goat anti-mouse IgG conjugated with FITC (Beckman Coulter). Cells were fixed with 2% PFA at 4 °C for 20 min and HLA expression was measured by flow cytometry on a FACSCanto II device (BD Bioscience, Heidelberg, Germany). Data were analyzed using the FlowJo Software (Tree Star Inc., Ashland, OR, USA).

Hoppmann N., Heinig N., Distler U., Kim E., Lennerz V., Krauß Y., Schumann U., Giese A., Tenzer S., Bitar L, & Schmidt M.H. (2022). Gamma Irradiation Triggers Immune Escape in Glioma-Propagating Cells. Cancers, 14(11), 2728.

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Immune Phenotyping of In Vitro Cells

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The immune phenotype of in vitro generated DC, T₁₀ and Tm cells was tested by flow cytometry as previously described [8 (link)]. The TCR Vβ repertoire was determined with the IOTest^® Beta Mark TCR V beta Repertoire Kit (Beckman Coulter, Inc, Brea, CA, USA) following manufacturer’s instructions.
Cells were analyzed with the BD FACS Canto II (Beckton Dickinson, San Jose, CA, USA) within few hours after staining. Data was analyzed using FCS 3.0 (DeNovo Instruments, Los Angeles, CA, USA).

Mfarrej B., Tresoldi E., Stabilini A., Paganelli A., Caldara R., Secchi A, & Battaglia M. (2017). Generation of donor-specific Tr1 cells to be used after kidney transplantation and definition of the timing of their in vivo infusion in the presence of immunosuppression. Journal of Translational Medicine, 15, 40.

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CD4+ TIL Clone Characterization

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The CD4⁺ TIL clone was generated by limiting dilution and confirmed using the IO Test^® Beta Mark TCR V beta Repertoire Kit (Beckman Coulter, Brea, CA) by flow cytometry. A total of 2 × 10⁵ T cells were co-cultured with 4 × 10⁴ autologous tumour cells for 5 h at 37 °C (and 5% CO₂) in a 96-well tissue culture plate containing 200 μl assay medium/well (RPMI 1640 with 10% FBS and penicillin/streptomycin; both from Thermo Fisher Scientific, Waltham, MA). During the incubation period, 1.3 μg/ml of monensin (Merck KGaA, Darmstadt, Germany), and 4 μl of the anti-human CD107a-Alexa Fluor 700 antibody (Clone H4A3; BD Biosciences, Franklin Lakes, NJ) were added. PMA = phorbol 12-myristate 13-acetate (PMA) was used as the positive control and assay medium alone without tumour cells was used as negative control. After 5 h of incubation, the cells were stained with anti-human CD3-PE/Cy7 (Clone HIT3A; BioLegend, San Diego, CA), anti-human CD4-V450 (Clone RPA-T4) and anti-human CD8-APC/Cy7 (Clone SK1) (both from BD Biosciences, Franklin Lakes, NJ), and analysed by flow cytometry.

Meng Q., Valentini D., Rao M., Moro C.F., Paraschoudi G., Jäger E., Dodoo E., Rangelova E., del Chiaro M, & Maeurer M. (2018). Neoepitope targets of tumour-infiltrating lymphocytes from patients with pancreatic cancer. British Journal of Cancer, 120(1), 97-108.

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TCR Vβ Repertoire Profiling in T-LGL Leukemia

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T cell receptor (TCR) Vβ families were analyzed from T-LGL leukemia patients’ whole blood or PB MNC samples by combining 0.5 ul of anti-CD3 APC (Clone: SK7, Cat#: 557851, Lot#: 8037645, BD Biosciences), 4 ul of anti-CD4 PerCP (Clone: SK3, Cat#: 345770, Lot#: 6281605, BD Biosciences), 0.8 ul of anti-CD8 PE-CY7 (Clone: SK1, Cat#: 345774, Lot#: 82152, BD Biosciences) antibodies with the panel of TCR Vβ antibodies (10ul/sample) corresponding to 24 members of variable regions of the TCR β chain (∼70% coverage of normal human TCR Vβ repertoire) (IOTest Beta Mark TCR Vbeta Repertoire Kit, Cat#: IM3497, Lot#: 66, Beckman Coulter). Staining was done in 100 ul of whole blood and stained samples were analyzed with FACSVerse (BD Biosciences) and FlowJo software (Version 10.4.2, Becton Dickinson).

Huuhtanen J., Bhattacharya D., Lönnberg T., Kankainen M., Kerr C., Theodoropoulos J., Rajala H., Gurnari C., Kasanen T., Braun T., Teramo A., Zambello R., Herling M., Ishida F., Kawakami T., Salmi M., Loughran T., Maciejewski J.P., Lähdesmäki H., Kelkka T, & Mustjoki S. (2022). Single-cell characterization of leukemic and non-leukemic immune repertoires in CD8+ T-cell large granular lymphocytic leukemia. Nature Communications, 13, 1981.

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iNKT Cell TCR Vβ Repertoire Analysis

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Human iNKT cell TCR Vβ repertoire was analyzed through flow cytometry using an IOTest® Beta Mark TCR V beta Repertoire Kit (Beckman-Coulter), following the manufacturer’s instructions. Tube G was not used in staining because it included an antibody for Vβ11, which was stained separately. All other combined hTCR Vβs (FITC) and hTCR Vβs (PE) antibodies collectively stained for human TCR Vβ 1, 2, 3, 4, 5.1, 5.2, 5.3, 7.1, 7.2, 8, 9, 12, 13.1, 13.2, 13.6, 16, 17, 18, 20, 21.3, and 23.

Zhu Y., Smith D.J., Zhou Y., Li Y.R., Yu J., Lee D., Wang Y.C., Di Biase S., Wang X., Hardoy C., Ku J., Tsao T., Lin L.J., Pham A.T., Moon H., McLaughlin J., Cheng D., Hollis R.P., Campo-Fernandez B., Urbinati F., Wei L., Pang L., Rezek V., Berent-Maoz B., Macabali M.H., Gjertson D., Wang X., Galic Z., Kitchen S.G., An D.S., Hu-Lieskovan S., Kaplan-Lefko P.J., De Oliveira S.N., Seet C.S., Larson S.M., Forman S.J., Heath J.R., Zack J.A., Crooks G.M., Radu C.G., Ribas A., Kohn D.B., Witte O.N, & Yang L. (2019). Development of Hematopoietic Stem Cell-Engineered Invariant Natural Killer T Cell Therapy for Cancer. Cell stem cell, 25(4), 542-557.e9.

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