Im2287

The previously published primary T cell lines A25Salmonella, which contains the clones PG10 and PG90 (33 (link)), LDN5 (26 (link)), BC24A, BC24B, and BC24C (34 (link)) were grown by stimulation with 30 ng/mL anti-CD3 antibody and 25 × 10⁶ irradiated PBMC and 5 × 10⁶ irradiated Epstein-Barr virus-transformed B cells, and 1 ng/mL IL-2, which was added on day 2 of the culture. To study tetramer binding to the GEM42 TCR, we used a TCR-transduced 5KC-78.3.20 hybridoma (14 (link), 15 (link)). To generate primary TRBV4–1⁺ and TRBV4–1⁻ T cell lines, PBMC from random blood bank donor D43 were stained with anti-CD3 (555342, BD) and anti-TRBV4–1 (IM2287, Beckman Coulter). Cells were sorted on a BD FACSAria (BD Biosciences), and lymphocytes were selected based on forward scatter and side scatter. 1×10⁶ CD3⁺TRBV4–1⁺ and 1×10⁶ CD3⁺TRBV4–1⁻ cells were sorted. After 2 weeks of stimulation as described above both cells were stained with anti-CD3 and anti-TRBV4–1 antibodies to check purity.

PBMCs from subject HD1 were sorted based on their binding to anti-CD3 (clone SK7, Becton Dickinson) and CD1c tetramers treated with a mixture of MAG and fatty acid. Population expansion of sorted cells was performed using anti-CD3 (clone OKT3, produced in-house), irradiated feeder cells and IL-2. After 2 weeks, the sorting and expansion procedure was repeated, and the resulting cell line was named ‘HD1’. IFN-γ ELISpot assays were performed using anti-1D1K and GB-11-biotin according to the manufacturer’s instructions (Mabtech). After an initial screen using V_β-specific antibodies (V_β2: IM1484; V_β4: IM3602; V_β5.1: IM1552; V_β9: IM2003; V_β13.6: IM1330 and V_β7.1: IM2287 (Beckman Coulter), Vβ13.1 (clone H31, Ebioscience), Vβ8 (clone JR2, Biolegend), V_β21.3 (Catalog: 1483, Immunotech), three subsets of HD1 were sorted using anti-Vβ5.1 and cultured in vitro. The TCR β-chain sequences of HD1CD4⁺ and HD1CD4^– were determined from RNA isolated with an RNeasy Kit (Qiagen), with cDNA synthesized using a Quantitect Reverse Transcription Kit (Qiagen). V-segment usage was determined by PCR using primer set IPS000030, as described online (https://www.imgt.org/) and via a multiplex approach⁴⁶ .

T cells (3×10⁶) and tetramers (0.2 μg per 50 μl staining volume) were incubated for 10 min at room temperature in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA). Subsequently anti-CD3 (clone SK7, BD Biosciences) antibody was added an incubated for 10 min at room temperature. Subsequently, additional antibodies (anti-TRBV4–1, IM2287, Beckman Coulter) were added and incubated for 20 min at 4°C. Cells were washed with PBS with 1% BSA and analyzed on an LSRFortessa (BD Biosciences) flow cytometer. For display in a matrix, percentage positive cell or mean fluorescence intensity of the positive cells were normalized to Z score and a heat map was created using the heatmap.2 function of R (https://cran.r-project.org/web/packages/gplots/index.html).