Tocilizumab actemra

Study participants were recruited at the First Affiliated Hospital of the University of Science and Technology of China. Eligible patients with severe pre-engraftment syndrome, who are steroid-refractory and had a fever in excess of 38.3 °C for three consecutive days, had to be considered fit in order to receive tocilizumab (Tocilizumab Actemra, Roche). All patients underwent modified myeloablative conditioning chemotherapy or chemoradiotherapy. Patients or their guardians provided written informed consent. Nine patients received fludarabine (30 mg/m² for 4 days), busulfan (a total of 12.8 mg/kg, along with 0.8 mg/kg of intravenous busulfan every 6 h for 4 days) plus 60 mg/kg of cyclophosphamide for 2 days. Two patients received total-body irradiation (a total of 12 cGy; 3 cGy twice a day for two days), cyclophosphamide (a total of 120 mg/kg administered as 60 mg/kg daily for 2 days) and cytarabine (2 g/m² twice a day for 2 days). The clinical trial was registered at www.chictr.org.cn (Reference: ChiCTR1800015472; Title: A single-arm, single-center clinical study of tocilizumab in the treatment of corticosteroid- unresponsive pre-engraftment syndrome patients after unrelated cord blood transplantation). This study was approved by the Chinese Ethics Committee of Registering Clinical Trials (ChiECRCT-20180129).

Alexa Fluor® 647 antihuman TIGIT mAb (clone MBSA43) was obtained from BioLegend® (San Diego, CA, USA). The antibodies to evaluate CD155 (Alexa Fluor® 647, clone SKII.4), CD112 (PE, clone R2.525), PDL-1 (PerCP-Cy5.5, clone 29E.2A3), NKp30 (PE, clone P30-15), NKp44 (PE, clone P44-8), NKp46 (PE, clone 9E2), CD226 (PE, clone DX11), and NKG2D (PE, clone 1D11) were obtained from BioLegend® and MICA (PE, clone 159227), MICB (FITC, clone 236511), ULBP1 (PerCP, clone 170818), ULBP2-5-6 (PE, clone 165903), and B7-H6 (APC, clone 875002) from R&D Systems (Minneapolis, MN, USA). In addition, Ultra-LEAF ™ purified antihuman TIGIT antibody (clone A15153A, mouse IgG2a, BioLegend®) and anti-IL6R antibody (Tocilizumab/Actemra®; Roche, Basil, Switzerland) were used for functional blocking assays at 50 μg/mL and 10 μg/mL, respectively. Stattic (STAT-3 inhibitor; CAS 19983-44-9) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). This reagent was reconstituted in dimethyl sulfoxide (DMSO, Sigma-Merck, Darmstadt, Germany) as 50 mM stock solutions and stored at −20 °C until use.

The non-tumorigenic human breast cell line, MCF-10A, and the human BC cell lines, MCF-7 (ER+, PR+), MDA-MB-453 (HER2+), MDA-MB-231, MDA-MB-157, and BT-459 (TNBC cells) were obtained from the American Type Culture Collection (ATCC). All the BC cell lines were cultured in IMDM (Thermo Fisher Scientific, Dallas, TX, USA) while MCF-10A cells were cultured in DMEM F12 (Thermo Fisher Scientific) supplemented with EGF (20 ng/mL), hydrocortisone (0.5 µg/mL), cholera toxin (100 ng/mL), and insulin (10 µg/mL) (Thermo Fisher Scientific). Fibroblast cells (HFF) were cultured in RPMI (Thermo Fisher Scientific). All media were supplemented with 10% of Fetal Bovine Serum (Thermo Fisher Scientific) and 1× antibiotic-Antimycotic (Thermo Fisher Scientific). All cells were free from mycoplasma and were authenticated through STR analysis.
Cells were treated with the following chemicals and antibodies diluted in serum-free medium: FPR antagonist Cyclosporin H (CsH-Sigma, used at 1 µM), neutralizing anti-IL-6 antibody (Tocilizumab Actemra, Roche, used at 2 µg/mL), STATTIC (STC-Sigma 1 and 5 µM) and recombinant IL-6 (rIL-6, Thermo Fisher Scientific, used at 1 µg/mL).