Modulation of T-cell Proliferation by Tumor-Conditioned Neutrophils

In a 5-day incubation, bead-purified peripheral CD3⁺ T cells (1×10⁵ cells/well in 96-well plates) were labelled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with autologous neutrophils isolated from tumour or non-tumour tissues at a 2:1 ratio in 200 μL RPMI-1640 medium containing rhIL-2 (20 IU/mL), anti-CD3 (2 μg/mL), and anti-CD28 (1 μg/mL) antibodies, with or without a human PD-L1 neutralising antibody (20 μg/mL). In another co-culture system, TTCS-conditioned or NTCS-conditioned blood neutrophils were co-cultured with autologous or allogeneic CFSE-labelled bead-purified blood CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells or CFSE-labelled FACS-sorted blood PD-1⁺ or PD-1⁻ CD3⁺ T cells at a 1:1 ratio in a similar condition as above, in the presence or absence of a neutralising antibody against human PD-L1 (20 μg/mL), an arginase-1 inhibitor nor-NOHA (250 μM), or an induced nitric oxide synthase (iNOS) inhibitor 1400 W (10 μM). After 5-day incubation, the cells were harvested for intracellular cytokine staining.