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Background ACKR2 is an atypical chemokine receptor that plays a significant role in regulating inflammation by binding to inflammatory CC chemokines and facilitating their degradation. Previous findings suggest that the genetic absence of ACKR2 leads to heightened tumor growth in inflammation-driven models. Conversely, mice lacking ACKR2 exhibit protection against lung metastasis in melanoma and breast cancer models. This study aims to explore the specific cell types expressing ACKR2 and their relative contributions to the protection against lung metastasis.Methods ACKR2 expression was studied by the generation of an inducible and conditional knockout (KO) mouse expressing two reporter genes, luciferase and TdTomato visible by In Vivo Imaging System, flow cytometry and immunofluorescence. Gene expression in lung endothelial cells (ECs) was investigated by RNA sequencing analysis. In vivo models of lung metastasis and inflammation were performed in wild-type (WT) and conditional KO mice by intravenous injection of melanoma and colon cancer cell lines; the induction of acute lung injury model was done by intranasal injection of lipopolysaccharide (LPS). Leukocytes infiltrating lung metastasis were studied by fluorescence-activated cell sorting (FACS) analysis. The serum chemokine levels were studied with a multiplex ELISA.Results The analysis of the reporter mouse revealed that ACKR2 is expressed by lymphatic endothelial cells (LECs) in most murine organs. However, uniquely in the lungs, ACKR2 expression is observed in blood endothelial cells (BECs), specifically in capillaries known as aerocytes specialized for regulating leukocyte trafficking. Selective deletion of Ackr2 from ECs (ACKR2ΔCdh5 mice) but not from LECs (ACKR2ΔProx1 mice) resulted in protection in models of melanoma and colorectal cancer lung metastasis. This protection was associated with an increased presence of activated T lymphocytes infiltrating the lungs compared with WT mice. Additionally, in a model of acute lung injury, mice with selective deletion from the endothelial compartment exhibited heightened extravasation of T lymphocytes compared with both ACKR2 KO and WT mice.Conclusions These results indicate that ACKR2 is selectively expressed by lung vascular capillaries (aerocytes) that are devoted to the regulation of leukocyte extravasation. Selective ACKR2 targeting in this compartment, by modulating chemokine availability, promotes T lymphocyte extravasation resulting in reduced lung metastases.

Background More than 50% of all patients with colorectal cancer (CRC) develop liver metastases (CLM), a clinical condition characterized by poor prognosis and lack of reliable prognostic markers. Vδ1 cells are a subset of tissue-resident gamma delta (γδ) T lymphocytes endowed with a broad array of antitumor functions and showing a natural high tropism for the liver. However, little is known about their impact in the clinical outcomes of CLM.Methods We isolated human γδ T cells from peripheral blood (PB) and peritumoral (PT) tissue of 93 patients undergone surgical procedures to remove CLM. The phenotype of freshly purified γδ T cells was assessed by multiparametric flow cytometry, the transcriptional profiles by single cell RNA-sequencing, the functional annotations by Gene Ontology enrichment analyses and the clonotype by γδ T cell receptor (TCR)-sequencing.Results The microenvironment of CLM is characterized by a heterogeneous immune infiltrate comprising different subsets of γδ tumor-infiltrating lymphocytes (TILs) able to egress the liver and re-circulate in PB. Vδ1 T cells represent the largest population of γδ TILs within the PT compartment of CLM that is greatly enriched in Vδ1 T effector (TEF) cells expressing constitutive high levels of CD69. These Vδ1 CD69+ TILs express a distinct phenotype and transcriptional signature, show high antitumor potential and correlate with better patient clinical outcomes in terms of lower numbers of liver metastatic lesions and longer overall survival (OS). Moreover, intrahepatic CD69+ Vδ1 TILs can egress CLM tissue to re-circulate in PB, where they retain a phenotype, transcriptional signature and TCR clonal repertoires resembling their liver origin. Importantly, even the increased frequencies of the CD69+ terminally differentiated (TEMRA) Vδ1 cells in PB of patients with CLM significantly correlate with longer OS. The positive prognostic score of high frequencies of CD69+ TEMRA Vδ1 cells in PB is independent from the neoadjuvant chemotherapy and immunotherapy regimens administered to patients with CLM prior surgery.Conclusions The enrichment of tissue-resident CD69+ Vδ1 TEMRA cells re-circulating at high frequencies in PB of patients with CLM limits tumor progression and represents a new important clinical tool to either predict the natural history of CLM or develop alternative therapeutic protocols of cellular therapies.