Legendplex human cd8 nk panel

To evaluate the cytokine release, supernatants from the coculture assays were analyzed at 4 h and 24 h using the Legendplex Human CD8/NK Panel (BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol.

Cytokine concentrations were assessed with commercial kits. Human IFNγ ELISA Set and Human IL-2 ELISA Set (BD, USA) were measured on the Sunrise spectrophotometer and analyzed with Magellan software (Tecan, Switzerland). Interleukin (IL)-2, IL-17a, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), soluble Fas ligand (sFasL), granzyme A, and perforin were measured with the LEGENDplex Human CD8/NK Panel (Biolegend, USA) on the MACSQuant X (Miltenyi Biotec, Germany) and analyzed with Legendplex software (Biolegend, USA).

To determine effector molecule concentrations in co-culture supernatants, the LEGENDplex Human CD8/NK Panel (BioLegend) was performed according to the manufacturer’s instructions. Data were analyzed with LEGENDplex V.8.0 software (BioLegend). Target-induced release of granzyme B, perforin and granulysin was calculated by subtraction of concentrations in the co-cultures by the respective (CAR-)T cells cultured alone.

Human CD8⁺ T cells were stimulated as described above. Cell culture supernatants were collected at day 5 and centrifuged at 10,000 × g for 5 min to remove residual cells. Supernatants were investigated for cytokine release from CD8⁺ T cells using the LEGENDplex™ Human CD8/NK Panel (BioLegend Inc.) according to the manufacturer’s instructions. Samples were diluted 1:100 prior to the analysis. Samples were acquired using a CytoFlex flow cytometer (Beckmann Coulter GmbH, Krefeld, Germany). Data evaluation was performed using the LEGENDplex™ Data Analysis Software (BioLegend Inc.).

Serum was collected from mice from the tail once a week into Monovette 200 Z-Gel (Sarstedt, Cat Nr. 20.1291) tubes, centrifuged as instructed and frozen to −80 °C.
For the serum analysis after OTI transfer, the Legendplex MurineVirusResponse (13-plex, Biolegend Cat. Nr. 740621) was used according to the manufacturer’s instructions and measured on a Fortessa (BD). The Biolegend Legendplex analysis online tool was used to calculate concentrations according to internal standards. TNFα and IFNβ were not detected but can be found in the source data.
For the CAR T cell experiments, the Legendplex Human CD8/NK Panel (13-plex, Biolegend Cat Nr. 741065) was used according to the manufacturer’s instruction and measured on a Fortessa (BD). Analytes that were not detected in fraction of samples were not displayed in figures, but can be found in source data (IL4, IL17, TNFα, sFAS, Granzyme A, Granzyme B). sFasL was not displayed as it only correlated with tumor size (also in source data). Of note, although we used a Legendplex kit for the detection of human proteins (which binds proteins using two anti-human antibodies per analyte), we cannot exclude that this kit also cross-recognizes the murine orthologues.

Culture-expanded iNKT cells were co-incubated at increasing ratios with 4 × 10⁴ Jurkat cells (Clone E6-1, ATCC, Manassas, VA, USA), MOLT-4 cells (ATCC, Manassas, VA, USA), or primary leukemia cells in iNKT-cell culture medium for 16 h at 37°C in a 96-well round bottom plate. Leukemia cell lysis through iNKT cells was measured on a LSR Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) as described previously (14 (link)). To identify tumor cells, PBS57-CD1d tetramer+ iNKT cells were excluded. Only tumor cell samples with a purity ≥90% were used for these experiments. In certain experiments, iNKT cells were culture-expanded from donor cells and co-incubated with primary leukemia cells from patients that received HLA-matched hematopoietic stem cell grafts from their respective donors (10/10-antigen/allele matched unrelated donors). Supernatants were analyzed with a Legendplex Human CD8/NK Panel (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions.