Iotest beta mark tcr 5 kit

Peripheral blood samples were stained for lymphocyte clonality analysis with anti-CD3 (SK7), anti-CD4 (SK3), and anti-CD8 (SK-1) (Becton Dickinson) and a panel of T-cell receptor β variable chain (TCR Vβ) antibodies (IOTest Beta Mark TCR V kit, Beckman Coulter Immunotech, cat. no IM3497), which recognize approximately 70% to 80% of the human TCR β V regions. After staining, red blood cells were lysed with BD FACS Lysing Solution (Becton Dickinson Biosciences) and re-suspended to phosphate-buffered saline (PBS) with 2 mM EDTA. Samples were acquired with FACSAria II or FACS Verse (Becton Dickinson) and analyzed with FlowJo software (Becton Dickinson).
Cell sorting was performed either from fresh or from cryopreserved PBMCs using FACSAria II (Becton Dickinson) or Sony SH800 (Sony Biotechnology Inc.). For sorting, MNCs were stained with anti-CD3 (SK7), anti-CD4 (SK3), anti-CD8 (SK-1), and the appropriate anti-Vβ antibody from the IOTest Beta Mark TCR V kit. Cell fractions’ purities were controlled with flow cytometry, and the purities of all sorted fractions were nearly 100%.

Vß diversity of sorted Melan-A and MELOE-1 specific T cell lines was analyzed by labeling with 24 anti-Vß mAbs included in the IOTest Beta Mark TCR V Kit (Beckman-Coulter, IM3497). These cytometric analyses were performed on a Facs Canto II (BD Biosciences). We followed throughout the manuscript the nomenclature of IMGT database [19 (link)].
For TCR sequencing of MELOE-1-specific T cells from P5 patient, total RNA was extracted from 5 × 10⁵ specific-T cells using QIAGEN RNeasy Kit. 25 ng of RNA was used to build libraries with the QIAseq Immune Repertoire T-cell Receptor Panel (Catalog 333705—IMHS-001Z), as previously described [9 (link)]. FASTQ files were analyzed in the QIAGEN GeneGlobe Data Analysis Center using the Immune Repertoire Application. The clonotype calls are generated using the IMSEQ software [20 (link)]. Clonotypes were defined on the basis of unique amino-acid sequences of CDR3 beta regions. In our set of data, the total number of unique TCR sequences was identical to the number of clonotypes.

Peripheral whole-blood samples were stained for lymphocyte clonality analysis with anti-CD3 (SK7, BD, cat. no 345767), anti-CD4 (SK3, BD, cat. no 345770), and anti-CD8 (SK-1, BD, cat. no 335822) and a panel of T-cell receptor β variable chain (TCR Vβ) antibodies (IOTest Beta Mark TCR V kit, Beckman Coulter Immunotech, cat. no IM3497), which recognize ∼70–80% of human TCR β V regions. All fluorochrome-conjugated antibodies were used according to the manufacturer’s instructions. Similarly, all β variable chain (TCR Vβ) antibodies were used according to the manufacturer’s instructions and all antibody information is included in the kit user manual and also in the Supplementary Fig. 13. After staining, red blood cells were lysed with BD FACS Lysing Solution (Becton Dickinson Biosciences, cat. no 349202) and re-suspended to phosphate-buffered saline (PBS) with 2 mM EDTA. Samples were analysed with FACSAria II (Becton Dickinson) and FACSDiva software (Becton Dickinson). The gating strategy for all whole-blood stainings is presented in the Supplementary Fig. 13.

To compare the 24 families of TCR Vβ repertoire in tetramer positive and negative CD8+ T cells, ex vivo PBMCs were stained with HER2/neu_369–377 biotin tetramer labeled with streptavidin Alexa fluor 700 as described above in 8 different tubes, followed by TCR Vβ family label using IOTest Beta Mark TCR V Kit (Beckman Coulter, Pasadena, United States) with each antibody cocktail (vials A to H) and anti-CD8 PE-Texas Red (eBiosciences, San Diego, United States); a minimum of 5×10⁴ CD8+ T cells were acquired, and the percentage of each family in HER2/neu specific CD8+ T cells was analyzed with FlowJo software (Treestar, Ashland, United States). For CDR3 sequences, 2×10⁷ PBMCs collected before and after anti-TTx were used to obtain genomic DNA using Wizard® Genomic DNA Purification Kit (Promega Corp., Madison, United States) following manufacturer’s protocol. For the CDR3 sequencing of TILs, two tumor slices (3 μm thick each) were obtained from tumor resection by surgery of the MCC-002 patient fixed-formalin paraffin embedded (FFPE). The DNA from the FFPE sample (extracted by ImmunoSEQ) and DNA from the two PBMCs samples were verified and then sequenced by ImmunoSEQ service (Adaptive Biotechnologies, Seattle, United States), raw data can be found in Additional file 3: Table S1.

T-cell clonal expansion was detected using TCR Vβ repertoire analysis by means of an IOTest Beta Mark TCR V Kit (Beckman Coulter, Brea, CA, USA). Relative frequencies of 24 Vβ T cell receptor families were analyzed in CD4+ and CD8+ T cells by flow cytometry in the peripheral blood of 17 recipients of ASCT from the LTR cohort. TCR Vβ panels included antibodies against CD3 (clone UCHT1), CD4 (clone 13B8.2), and CD8 (clone T8) (Beckman Coulter). Cells were acquired on a BD FACSCanto II cytometer and data were analyzed with FlowJo Software v.10 (BD Biosciences, San Jose, CA, USA).