Iotest beta mark tcr vβ repertoire kit

Both within total Tαβ-cells and their major (TαβCD4⁺, TαβCD8⁺, TαβDP and TαβDN cells) subsets, the TRBC1⁺/TRBC1⁻ ratio within each cell population defined by the expression of the different TCRVβ families was analyzed in a group of 27 PB samples (12 HD, 10 patients with reactive lymphocytosis and five HDc whose clonal T-cell populations were excluded from the analysis), stained with the IOTest^® Beta Mark TCRVβ Repertoire Kit (Beckman Coulter), following the CD3-10 min-TRBC1 and the TRBC1-10 min-TCRVβ conditions described in Supplementary Material (Table S1F).
In turn, the TRBC1⁺/TRBC1⁻ ratio for the distinct maturation-associated compartments of normal Tαβ-cells (i.e., naïve, central memory, transitional memory, effector memory, early effector and terminal effector T-cells and regulatory T-cells), identified according to the phenotypic profile shown in Table S3, was investigated in PB samples from 10 HD (Table S1G).

As a first approximation, the TCR Vβ usage of neoantigen-specific T cell clones was analyzed with the IOTest Beta Mark TCR Vβ repertoire kit (Beckman Coulter, Krefeld, Germany). Their TCR Vβ sequences were determined by TCR Vβ-RT-PCR using primers published in [57 (link)] and Sanger sequencing of PCR products (Supplementary Table 3). TCR Vβ deep sequencing was performed on PBMCs collected in 05/2002 and in 08/2004 (Supplementary Figure 1) using the ImmunoSEQ assay platform (Adaptive Biotechnologies, Seattle) and the frequencies of the neoantigen-specific TCRβ clonotypes were calculated (Supplementary Table 3).

Ten SS patients, eight females and two males, with a median age of 69.5 years (range: 52–93), were recruited for this study. The diagnosis was established in accordance with the criteria of the WHO‐EORTC (World Health Organization and the European Organization for Research and Treatment of Cancer) [1 (link)]. All of them presented a B2 stage; eight, a T4 stage; one, a T3 stage; and one, a T2b stage. Samples from patients 1 to 4 were used to establish PDC as mentioned above. Samples from patients 5 to 10 were used to obtain fresh SS cells. Peripheral blood mononuclear cells (PBMCs) were isolated using Pancoll (Pan Biotech, Aidenbach, Germany). Clonal TCRvβ was determined using IOTest® Beta Mark TCRVβ Repertoire Kit (Beckman Coulter, Villepinte, France). Tumor cells were sorted either according to the TCRvβ using a BD FACSAria™ II Cell Sorter (BD Biosciences, Le Pont de Claix, France) or according to the CD4⁺, as mentioned in Ref. [21 (link)] (Fig. S1 shows the evaluation of tumor cells' proportion before and after cell sorting). This study was approved by the local ethics committee and was carried out in accordance with the standards set by the Declaration of Helsinki. All SS patients included in this study signed an informed consent.

Immune phenotypic analysis of PB and/or bone marrow (BM) samples were determined using specific antibodies against CD2 (Leu-5b), CD3 (SK7), CD4 (Leu-3a), CD5 (Leu-1), CD7 (3A1), CD8 (Leu-2a), CD16 (3G8), CD56 (NCAM16.2), CD57 (HNK-1), TCRαβ (WT31) and TCRγδ (11F2). All antibody conjugates were from BD Biosciences (San Jose, CA, USA). A 4-color flow cytometric immune phenotype strategy, antibodies to CD3, CD8 and the IO Test Beta Mark TCRVβ Repertoire kit (Beckman Coulter, Marseille, France) were used. Analyses were done as described [11 (link)].

The TCR-Vβ repertoire of vaccine-activated CD4+ T cells was determined using the IO Test Beta Mark TCR-Vβ Repertoire kit (Beckmann Coulter) according to the manufacturer’s instructions. This kit consists of mAbs to 24 distinct TCR-Vβ families (approximately 70% coverage of the normal human TCR-Vβ repertoire). CD3+CD4+ cells were stained and analyzed for TCR-Vβ usage according to the manufacturer's instructions.

The IOTest Beta Mark TCR-Vβ Repertoire kit (Beckman Coulter, Miami, FL, USA) was used for the assessment of the following TCR-Vβ regions: Vβ1, Vβ2, Vβ3, Vβ4, Vβ5.1, Vβ5.2, Vβ5.3, Vβ7.1, Vβ7.2, Vβ8, Vβ9, Vβ11, Vβ12, Vβ13.1, Vβ13.2, Vβ13.6, Vβ14, Vβ16, Vβ17, Vβ18, Vβ20, Vβ21.3, Vβ22, and Vβ23. This kit includes 8 cocktails, each containing antibodies against 3 different TCR-Vβ regions covering a total of 24 TCR-Vβ regions and approximately 70% of the normal human TCR-Vβ repertoire. In performing the TCR-Vβ repertoire assay, anti-IFN-γ APC, anti-CD3 BV510, anti-CD4 PercPCy5.5, anti-CD45RA BV450, and anti-CCR7 PECy7 were added to the 8 Vβ cocktails. TCR-Vβ antigen usage was analyzed by using general gating strategies based on amine-reactive LIVE/DEAD fixable NIR dead cells exclusion. Selected T-cell populations per each gating strategy were analyzed for TCR-Vβ expression and quantitation. The values were compared to published normal ranges provided by the manufacturer.