VEGFR-1 blockade with the monoclonal antibody D16F7 counteracts VEGF-A-induced tolerogenic and immune regulatory phenotypes without impairing T-cell activation

Objective: VEGF-A exerts complex immunomodulatory effects that foster an immunosuppressive tumour microenvironment, including impaired dendritic cell (DC) maturation, expansion of regulatory T cells (Tregs), and induction of T-cell exhaustion, while transiently enhancing effector T-cell responses. This dual activity underscores the need to clarify the immunosuppressive versus immunostimulatory consequences of VEGF-A receptor activation to optimise selective inhibition. This proof-of-concept study evaluated whether selective VEGFR-1 blockade can counteract VEGF-A-driven immunosuppression in human immune cells using the monoclonal antibody (mAb) D16F7, which inhibits membrane-bound VEGFR-1 while preserving the decoy and anti-angiogenic activity of its soluble form. Methods: The impact of VEGFR-1 blockade by D16F7 mAb on VEGF-A-mediated immune modulation was assessed by multiparametric flow cytometry using primary immune cells from healthy donors. Human DCs were generated from CD14 + monocytes and cultured with VEGF-A; tolerogenic and pro-exhaustion activity was evaluated via maturation markers (CD80, CD83, HLA-DR) and PD-1 expression on DC-stimulated T cells after 14 days. Short-term effects on effector T cells, stratified by PD-1 and CD28 expression, were analysed by intracellular cytokine staining at 5–6 and 18 h. Purified Tregs, isolated via a two-step magnetic separation, were cultured for 1 week to assess the acquisition of an immunosuppressive phenotype by measuring the expression of checkpoint markers (PD-1, ICOS, TIM-3). Results: D16F7 reversed VEGF-A-induced DC maturation defects, reduced DC-mediated T-cell tolerance, and attenuated the emergence of a highly immunosuppressive Treg phenotype, while largely preserving short-term effector T-cell function. Conclusions: Despite the limitations of an in vitro PBMC-based system, selective VEGFR-1 blockade by D16F7 mAb alleviates VEGF-A-driven immune tolerance while preserving T-cell activation, supporting its translational potential as a complementary immunomodulatory approach. • VEGF-A exerts immunosuppressive and immunostimulating effects. • VEGFR-1 activation is mostly involved in VEGF-A immunosuppressive effects. • The anti-VEGFR-1 monoclonal antibody D16F7 counteracts VEGF-A-induced tolerogenic dendritic cells. • VEGFR-1 blockade reduces suppressive Tregs and preserves effector T-cell function. • Anti-VEGFR-1 D16F7-based biologics might be useful to improve antitumor immunity.