Macrophages Impair TLR9 Agonist Antitumor Activity through Interacting with the Anti-PD-1 Antibody Fc Domain

Abstract

Background. A combination of TLR9 agonists and an anti-PD-1 antibody has been reported to be effective in immunocompetent mice but the role of innate immunity has not yet been completely elucidated. Therefore, we investigated the contribution of the innate immune system to this combinatorial immunotherapeutic regimens using an immunodeficient mouse model in which the effector functions of innate immunity can clearly emerge without any interference from T lymphocytes. Methods. Athymic mice xenografted with IGROV-1 human ovarian cells, reported to be sensitive to TLR9 agonist therapy, were treated with cytosine–guanine (CpG)-oligodeoxynucleotides (ODNs), an anti-PD-1 antibody or their combination. Results. We found that PD-1 blockade dampened CpG-ODN antitumor activity. In vitro studies indicated that the interaction between the anti-PD-1 antibody fragment crystallizable (Fc) domain and macrophage Fc receptors caused these immune cells to acquire an immunoregulatory phenotype, contributing to a decrease in the efficacy of CpG-ODNs. Accordingly, in vivo macrophage depletion abrogated the detrimental effect exerted by the anti-PD-1 antibody. Conclusion. Our data suggest that if TLR signaling is active in macrophages, coadministration of an anti-PD-1 antibody can reprogram these immune cells towards a polarization state able to negatively affect the immune response and eventually promote tumor growth.

Toll-like receptor 9 agonists and combination therapies: strategies to modulate the tumour immune microenvironment for systemic anti-tumour immunity

“…In other studies, Zhou et al found that TLR9 activation in HCC cells affected PARP1 and STAT3 pathways, resulting in PD-L1 expression and ultimately inducing immune escape of cancer cells [75]. The latest results suggest that if TLR9 signaling activation in macrophages of breast cancer, the co-administration of anti-PD-1 antibodies with TLR9 agonists may induce macrophages to reprogram and polarise into an immunosuppressive phenotype [76]. This interesting observation is contrary to the prevailing results of the current two-drug combination trials, most of which have shown that TLR9 agonists are effective in combination with PD-1.…”

The Educational Program of Macrophages toward a Hyperprogressive Disease-Related Phenotype Is Orchestrated by Tumor-Derived Extracellular Vesicles

“…We found an enrichment of the TLR-related signaling pathway in Group 1 macrophages and TLR can represent a double-edged sword in the context of cancer [ 55 ]. We previously reported that macrophages treated with a TLR9 agonist in combination with an anti-PD1 antibody acquired a phenotype characterized by immunosuppressive and pro-tumor activity, as demonstrated in vivo and in vitro [ 56 ]. We also demonstrated that this effect is mediated by the anti-PD1 antibody Fc domain [ 56 ].…”

“…We previously reported that macrophages treated with a TLR9 agonist in combination with an anti-PD1 antibody acquired a phenotype characterized by immunosuppressive and pro-tumor activity, as demonstrated in vivo and in vitro [ 56 ]. We also demonstrated that this effect is mediated by the anti-PD1 antibody Fc domain [ 56 ]. We speculated that the binding of an anti-PD1 Fc portion to macrophage Fc receptors in combination with TLR stimulation determined the generation of M2b macrophages, a subtype of regulatory/immunosuppressive macrophages secreting both pro- and anti-inflammatory cytokines and associated with cancer progression [ 57 ].…”

“…BMDMs, generated as previously described [ 56 ], were seeded in 12-well plates (Thermo Fisher Scientific, Waltham, MA, USA) at a density of 8 × 10 5 cells/well in Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin solution (Thermo Fisher Scientific, Waltham, MA, USA). The following day, culture medium was removed and BMDMs were exposed to 2 mL of the different NSCLC CMs for 24 h. For cytokine-induced macrophage polarization, BMDMs were incubated for 24 h with 2 mL of serum-free medium containing 10 ng/mL lipopolysaccharide (LPS, Merck KGaA, Darmstadt, Germany) plus 20 ng/mL of IFN-γ (Peprotech, Cranbury, NJ, USA) or with 20 ng/mL IL-4 (Peprotech) to obtain classically activated (M1) or alternatively activated (M2) macrophages, respectively [ 4 ].…”

Understanding the role of Toll-like receptors in lung cancer immunity and immunotherapy

“…TLR7 agonists in combination with checkpoint inhibitors targeting PD-1 and CTLA-4 have been shown to be safe and effective in immunotherapy-resistant tumor models to promote more long-term immune responses ( 104 ). However, combination therapy has opposingly been reported to inhibit antitumor activity upon PD-1 blockade diminishing CpG-ODNs antitumor activity on macrophages to promote tumor growth ( 105 ). Additional mechanistic insights will lead to the elucidation of more effective regimens to target both innate and adaptive immunity against cancer and unravel the molecular determinants of the TLR response in tumor cells.…”