Neutrophils are an essential part of the innate immune system. To study their importance, experimental studies often aim to deplete these cells, generally by injecting anti-Ly6G or anti-Gr1 antibodies. However, these approaches are only partially effective, transient or lack specificity. Here we report that neutrophils remaining after anti-Ly6G treatment are newly derived from the bone marrow, instead of depletion escapees. Mechanistically, newly generated, circulating neutrophils have lower Ly6G membrane expression, and consequently reduced targets for anti-Ly6G-mediated depletion. To overcome this limitation, we develop a double antibody-based depletion strategy that enhances neutrophil elimination by anti-Ly6G treatment. This approach achieves specific, durable and controlled reduction of neutrophils in vivo, and may be suitable for studying neutrophil function in experimental models.

Dendritic cells play a key role in the orchestration of antitumor immune responses. The cDC1 (conventional dendritic cell 1) subset has been shown to be essential for antitumor responses and response to immunotherapy, but its precise role in humans is largely unexplored. Using a multidisciplinary approach, we demonstrate that human cDC1 play an important role in the antitumor immune response through their capacity to produce type III interferon (IFN-λ). By analyzing a large cohort of breast primary tumors and public transcriptomic datasets, we observed specific production of IFN-λ1 by cDC1. In addition, both IFN-λ1 and its receptor were associated with favorable patient outcomes. We show that IFN-III promotes a T_H1 microenvironment through increased production of IL-12p70, IFN-γ, and cytotoxic lymphocyte–recruiting chemokines. Last, we showed that engagement of TLR3 is a therapeutic strategy to induce IFN-III production by tumor-associated cDC1. These data provide insight into potential IFN- or cDC1-targeting antitumor therapies.

Background: Never-smokers and never-drinkers patients (NSND) suffering from oral squamous cell carcinoma (OSCC) are epidemiologically different from smokers drinkers (SD). We therefore hypothesized that they harbored distinct targetable molecular alterations. Patients and methods: Data from The Cancer Genome Atlas (TCGA) (discovery set), Gene Expression Omnibus and Centre Léon Bérard (CLB) (3 validation sets) with available gene expression profiles of HPV-negative OSCC from NSND and SD were mined. Protein expression profiles and genomic alterations were also analyzed from TCGA, and a functional pathway enrichment analysis was performed. Formalin-fixed paraffin-embedded (FFPE) samples from 44 OSCC including 20 NSND and 24 SD treated at CLB were retrospectively collected to perform targeted-sequencing of 2,559 transcripts (HTG EdgeSeq system), and CD3, CD4, CD8, IDO1 and PD-L1 expression analyses by immunohistochemistry (IHC). Enrichment of a six-gene interferon-γ signature of clinical response to pembrozulimab (PD-1 inhibitor) was evaluated in each sample from all cohorts, using the single sample gene set enrichment analysis method. Results: A total of 854 genes and 29 proteins were found to be differentially expressed between NSND and SD in TCGA. Functional pathway analysis highlighted an overall enrichment for immune-related pathways in OSCC from NSND, especially involving T-cell activation. Interferon-γ response and PD1 signaling were strongly enriched in NSND. IDO1 and PD-L1 were overexpressed and the score of response to pembrolizumab was higher in NSND compared to SD, although the mutational load was lower in NSND. IHC analyses in the CLB cohort evidenced IDO1 and PD-L1 overexpression in tumor cells that was associated with a higher rate of tumor-infiltrating T-cells in NSND compared to SD. Conclusion: The main biological and actionable difference between OSCC from NSND and SD lies in the immune microenvironment, suggesting a higher clinical benefit of PD-L1 and IDO1 inhibition in OSCC from NSND.

Objectives: The accumulation of tumor‐associated macrophages (TAMs) is correlated with poor clinical outcome, but the mechanisms governing their differentiation from circulating monocytes remain unclear in humans.Methods: Using multicolor flow cytometry, we evaluated TAMs phenotype in 93 breast cancer (BC) patients. Furthermore, monocytes from healthy donors were cultured in the presence of supernatants from dilacerated primary tumors to investigate their differentiation into macrophages (MΦ) in vitro. Additionally, we used transcriptomic analysis to evaluate BC patients’ blood monocytes profiles.Results: We observed that high intra‐tumor CD163‐expressing TAM density is predictive of reduced survival in BC patients. In vitro, M‐CSF, TGF‐β and VEGF from primary tumor supernatants skewed the differentiation of healthy donor blood monocytes towards CD163^highCD86^lowIL‐10^high M2‐like MΦ that strongly suppressed CD4⁺ T‐cell expansion via PD‐L1 and IL‐10. In addition, blood monocytes from about 40% of BC patients displayed an altered response to in vitro stimulation, being refractory to type‐1 MΦ (M1‐MΦ) differentiation and secreting higher amounts of immunosuppressive, metastatic‐related and angiogenic cytokines. Aside from showing that monocyte transcriptome is significantly altered by the presence of BC, we also demonstrated an overall metabolic de‐activation in refractory monocytes of BC patients. In contrast, monocytes from sensitive BC patients undergoing normal M1‐MΦ differentiation showed up‐regulation of IFN‐response genes and had no signs of metabolic alteration.Conclusion: Altogether, our results suggest that systemic factors skew BC patient blood monocytes towards a pro‐metastatic profile, resulting in the accumulation of further polarised CD163^high TAMs resembling type‐2 MΦ (M2‐MΦ) in the local BC microenvironment. These data indicate that monitoring circulating monocytes in BC patients may provide an indication of early systemic alterations induced by cancer and, thus, be instrumental in the development of improved personalised immunotherapeutic interventions.

Tumor-associated macrophages (TAMs) are a critical determinant of resistance to PD-1/PD-L1 blockade. This phase 1 study (MEDIPLEX, NCT02777710) investigated the safety and efficacy of pexidartinib, a CSF-1R–directed tyrosine kinase inhibitor (TKI), and durvalumab (anti–PD-L1) in patients with advanced colorectal and pancreatic carcinoma with the aim to enhance responses to PD-L1 blockade by eliminating CSF-1–dependent suppressive TAM. Forty-seven patients were enrolled. No unexpected toxicities were observed, one (2%) high microsatellite instability CRC patient had a partial response, and seven (15%) patients experienced stable disease as their best response. Increase of CSF-1 concentrations and decrease of CD14 ^low CD16 ^high monocytes in peripheral blood mononuclear cells (PBMCs) confirmed CSF-1R engagement. Treatment decreased blood dendritic cell (DC) subsets and impaired IFN-λ/IL-29 production by type 1 conventional DCs in ex vivo TLR3-stimulated PBMCs. Pexidartinib also targets c-KIT and FLT3, both key growth factor receptors of DC development and maturation. In patients, FLT3-L concentrations increased with pexidartinib treatment, and AKT phosphorylation induced by FLT3-L ex vivo stimulation was abrogated by pexidartinib in human blood DC subsets. In addition, pexidartinib impaired the FLT3-L– but not GM-CSF–dependent generation of DC subsets from murine bone marrow (BM) progenitors in vitro and decreased DC frequency in BM and tumor-draining lymph node in vivo. Our results demonstrate that pexidartinib, through the inhibition of FLT3 signaling, has a deleterious effect on DC differentiation, which may explain the limited antitumor clinical activity observed in this study. This work suggests that inhibition of FLT3 should be considered when combining TKIs with immune checkpoint inhibitors.