Anti tim 3 clone f382e2

Blocking monoclonal antibodies were used to analyse the impact of PD-1 and TIM-3 signalling on the anti-tumour response of activated NK cells. Considering the direct effect of ICIs on NK cell receptors, purified NK cells were incubated with 20 µg/ml of anti-PD1 (pembrolizumab) and/or 20 µg/ml of anti-TIM-3 (clone F382E2; BioLegend, San Diego, CA, USA) for 20 min at 4°C, before the co-culture with target cells. Human IgG1 isotype (Enzo, Farmingdale, NY, USA) measuring 5 µg/ml was used as control.
The efficacy of blockade was assessed by flow cytometry. Total PBMCs were incubated with the blocking antibodies before staining with the labelled flow cytometry antibody targeting PD-1 or TIM-3.

CD4⁺T cells from HCs were stimulated with different concentrations of ox-LDL (0, 1, 10, 50, and 100 µg/ml) for 48 h. Some CD4⁺T cells were also cultured with a plate-bound anti-CD3 antibody (OKT-3; 5 μg/ml) plus anti-CD28 antibody (28.2; 1 μg/ml) to get activated.
For Tim-3- and PD-1-targeting experiments, arterial CD4⁺T cells were cultured (5 × 10⁵ per well) in the presence of 10 μg/ml anti-Tim-3 (clone F38-2E2; BioLegend San Diego, CA, USA) and anti-PD-1 (clone EH12.2H7, BioLegend San Diego, CA, USA), or isotype control. After 48 h, the culture supernatants were collected and further analyzed by flow cytometry (FCM).
For intracellular cytokine analysis, brefeldin A (a Golgi inhibitor) (10 mg/ml, Biolegend, San Diego, CA, USA) was used for 4 h (at the end of the culture) to block the secretion of cytokines into the media after the activation of cells by phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, 12 h) and ionomycin (1 μg/ml, 12 h). Cells were harvested and analyzed by FCM for intracellular cytokine production.

CD8⁺ T lymphocytes were activated for 12 hours in 96-well plates coated with anti-CD3 Ab (OKT3 clone, CRL-8001, ATCC) at different concentrations ranging from 50 mg/mL to 400 ng/mL. Immune checkpoints (ICs) expression was determined by labeling with anti-PD-1 (Clone EH12, BD Biosciences), anti-TIGIT (Clone A15153G, BioLegend), anti-LAG-3 (Clone 11C3C65, BD Biosciences), anti-Tim-3 (Clone F38-2E2, Biolegend) and anti-KLRG1 (Clone 14C2A07, Biolegend) antibodies. An anti-CD25 (clone M-A251, BD Biosciences) specific-antibody was also used as a T cell activation marker. The expression of the main costimulation molecules was assessed on resting T lymphocytes with anti-CD45RO (clone UCHL1, BD Biosciences), anti-CD28 (Clone CD28.2, BD Biosciences), anti-CD27 (Clone L128, BD Biosciences), anti-CD2 (Clone RPA-2.10, BD Biosciences) and anti-LFA-1 (Clone HI111, BD Biosciences) monoclonal antibodies. All the cytometric analyses were performed on a Facs Canto II (BD Biosciences).