Rantes

Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Lonza (Verviers, Belgium), fetal bovine serum (FBS) from Biological Industries (Beit Haemek, Israel). Charcoal-stripped (steroid-free) FBS (csFBS), trypsin-EDTA, penicillin, streptomycin, HEPES, RT-PCR and immunofluorescence and Western Blot reagents, small interfering RNAs (siRNA), and all other transfection reagents were purchased from Life Technologies (Paisley, UK). Dexamethasone (Fortecortin) was obtained from Merck (Darmstadt, Germany) and amphotericin B and LPS (from Escherichia coli 0111:B4) from Sigma-Aldrich (St Louis, Missouri, USA). The RNeasy Mini kit was purchased from Qiagen Inc. (Valencia, California, USA). All antibodies were from Santa Cruz Biotechnology (Santa Cruz, California, USA), unless otherwise specified. RANTES, eotaxin, GM-CSF, and CXCL8 BD Cytometric Bead Array Flex Sets were from BD Biosciences (San Diego, California, USA), and IL-6 and CXCL8 DuoSet commercial enzyme-linked immunosorbent assay (ELISA) kits were from R&D Systems (Abingdon, UK).

Similar to the evaluation of NK cell activation, supernatants of co-cultured cells were collected, however they were obtained from independent wells and stored at − 80 °C until they were used. Supernatants were thawed at 4 °C right before running the CBA assay. The panel for the CBA flex set included: TNF-α, Granzyme, IFN-γ, MIP-1α and RANTES (BD Biosciences, San Jose, CA, USA). The CBA assay was done according to the manufacturer´s instructions. The beads complex was acquired using LS Fortessa (BD Biosciences, San Jose, CA, USA). Obtained data were normalized with NK cells co-cultured with uninfected CD4⁺ T cells and analyzed using FlowJo version 10.5.3 (FlowJo, LLC, Oregon, USA).