Background Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients. Methods To test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages. Results We demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk. Conclusions Our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies.

CD49a ⁺ CD16 ⁻ NK cells from the liver and contact dermatitis lesions specifically recognize antigens and contribute to adaptive immunity.

Although γδ T cells are known to participate in immune dysregulation in solid tumors, their relevance to human microsatellite-stable (MSS) colorectal cancer (CRC) is still undefined. Here, using integrated gene expression analysis and T cell receptor sequencing, we characterized γδ T cells in MSS CRC, with a focus on Vδ1 + T cells. We identified Vδ1⁺ T cells with shared motifs in the third complementarity-determining region of the δ-chain, reflective of antigen recognition. Changes in gene and protein expression levels suggested a dysfunctional effector state of Vδ1⁺ T cells in MSS CRC, distinct from Vδ1⁺ T cells in microsatellite-instable (MSI). Interaction analysis highlighted an immunosuppressive role of fibroblasts in the dysregulation of Vδ1⁺ T cells in MSS CRC via the TIGIT-NECTIN2 axis. Blocking this pathway with a TIGIT antibody partially restored cytotoxicity of the dysfunctional Vδ1 phenotype. These results define an operative pathway in γδ T cells in MSS CRC.

Mounting experimental evidence hints to an import role for natural killer (NK) cells in adaptive immune responses to pathogens. NK cells with adaptive features are heterogeneous and belong to different subsets according to their phenotype as well as the nature of their adaptive recall reactions. Three types of adaptive NK cell responses have been described: (i) NK cells with long-lived memory of multiple different haptens and viral antigens were described in murine liver tissue with a possible human counterpart; (ii) infection of human and mouse cytomegalovirus is associated with an expansion of NKG2C⁺ and Ly49H⁺ NK cells, respectively, that selectively recognize CMV-encoded peptides thereby facilitating recall responses; (iii) cytokine-stimulated NK cells respond to different stimuli with enhanced production of IFN-γ after re-stimulation. These exciting findings not only support the idea of NK cells with adaptive features, but define a novel field of harnessing memory NK cell subsets for therapeutic strategies.