Investigating the role of macrophages in the suppression of NK cell functions in mouse models of metastatic breast cancer

Breast cancer is the leading cause of cancer-related death in females worldwide. Although 5-year survival of breast cancer patients in early stages is 89-100%, that of patients with metastatic tumours is reduced to just 21%, suggesting the requirement of effective therapies for metastatic breast cancer (MBC). MBC is primarily treated with chemotherapeutics however the efficacy of such treatments is limited due to resistance. As an alternative approach, NK cell-based immunotherapy (i.e., adoptive transfer of NK cells to patients) has been focused on since it shows significant therapeutic effects on haematopoietic tumours. Nevertheless, its efficacy is limited in MBC probably due to an immune suppressive tumour microenvironment (TME). Tumour-associated macrophages (TAMs) are an abundant cell type within the TME of breast cancer and promote the metastatic process such as cancer cell egress from the primary tumour in mouse models. In mouse models of breast cancer, a distinct population of TAMs in the metastatic site called metastasis-associated macrophages (MAMs) can promote tumour cell seeding, survival and growth. Moreover, we have recently shown that MAMs and their progenitor cells can suppress cytotoxicity of CD8+ T cells in vitro. Interestingly, a recent study suggests that TAMs isolated from the ‘primary’ mammary tumour in mice can suppress tumour killing ability of NK cells in vitro, whereas the effects of MAMs on NK functions in the ‘metastatic’ tumour is largely unknown. We hypothesise that MAMs in the metastatic site suppress NK cell function, and that the depletion of these MAMs can improve NK cell immunotherapy efficacy for MBC. To investigate this hypothesis, we first established an in vitro NK cell cytotoxicity assay whereby mouse breast cancer cells were co-cultured with splenic NK cells, and the resultant tumour cell apoptosis was determined by quantitative fluorescence microscopy. Using this assay, we found that the NK cell-induced tumour cell apoptosis was significantly reduced in the presence of MAMs isolated from metastatic tumours in the lung of tumour cell injected mice. We also found that bone marrow-derived macrophages cultured with M-CSF (M- BMMs) that resemble MAMs also reduced NK cell cytotoxicity in a cell-to-cell contact dependent manner. In contrast, BMMs cultured with GM-CSF that represent pro-inflammatory macrophages did not suppress NK cell cytotoxicity. We further identified by flow cytometry that MAMs and M-BMMs expressed high levels of NK cell inhibitory ligands such as H2-Kb and H2-Db, and NK cells in the metastatic lung expressed high levels of their receptors. However, blockade of H2-Kb or H2-Db did not prevent macrophage mediated NK cell suppression in our assay. Alternatively, we found that M-BMMs expressed higher levels of membrane bound TGF-b than GM-BMMs and blocking TGF-b rescued the macrophage-mediated NK cell suppression, although these data must be confirmed. Using a mouse model of breast cancer metastasis, we further demonstrated that depletion of MAMs promoted maturation of NK cells in the metastatic lung as well as recruitment of NK cells towards the metastatic site. Importantly, the MAM depletion in this model significantly increased the efficacy of transferred NK cells in reducing metastatic tumour burden whereas NK cell transfer on its own did not suppress metastatic tumour growth. Collectively, our data suggest that MAMs in metastatic tumours can suppress NK cell cytotoxicity towards breast cancer cells by direct contact with NK cells that transmit suppressive signals via membrane bound TGF-b as well as by suppressing NK cell maturation and recruitment in the metastatic site. Our data also indicate that the depletion of MAMs can alter the immune suppressive TME and thereby improve the efficacy of NK cell infusion therapy efficacy. Further investigation of the mechanisms behind MAM-mediated NK suppression would lead to the increased success of NK cell-based immunotherapy for MBC

The atypical chemokine receptor Ackr2 constrains NK cell migratory activity and promotes metastasis

Chemokines have been shown to be essential players in a range of cancer contexts. In this study, we demonstrate that mice deficient in the atypical chemokine receptor Ackr2 display impaired development of metastasis in vivo in both cell line and spontaneous models. Further analysis reveals that this relates to increased expression of the chemokine receptor CCR2, specifically by KLRG1+ NK cells from the Ackr2−/− mice. This leads to increased recruitment of KLRG1+ NK cells to CCL2-expressing tumors and enhanced tumor killing. Together, these data indicate that Ackr2 limits the expression of CCR2 on NK cells and restricts their tumoricidal activity. Our data have important implications for our understanding of the roles for chemokines in the metastatic process and highlight Ackr2 and CCR2 as potentially manipulable therapeutic targets in metastasis

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19

Metastasis-associated macrophages constrain antitumor capability of natural killer cells in the metastatic site at least partially by membrane bound transforming growth factor β

Background Metastatic breast cancer is a leading cause of cancer-related death in women worldwide. Infusion of natural killer (NK) cells is an emerging immunotherapy for such malignant tumors, although elimination of the immunosuppressive tumor environment is required to improve its efficacy. The effects of this “metastatic” tumor environment on NK cells, however, remain largely unknown. Previous studies, including our own, have demonstrated that metastasis-associated macrophages (MAMs) are one of the most abundant immune cell types in the metastatic tumor niche in mouse models of metastatic breast cancer. We thus investigated the effects of MAMs on antitumor functions of NK cells in the metastatic tumor microenvironment.Methods MAMs were isolated from the tumor-bearing lung of C57BL/6 mice intravenously injected with E0771-LG mouse mammary tumor cells. The effects of MAMs on NK cell cytotoxicity towards E0771-LG cells were evaluated in vitro by real-time fluorescence microscopy. The effects of MAM depletion on NK cell activation, maturation, and accumulation in the metastatic lung were evaluated by flow cytometry (CD69, CD11b, CD27) and in situ hybridization (Ncr1) using colony-stimulating factor 1 (CSF-1) receptor conditional knockout (Csf1r-cKO) mice. Finally, metastatic tumor loads in the chest region of mice were determined by bioluminescence imaging in order to evaluate the effect of MAM depletion on therapeutic efficacy of endogenous and adoptively transferred NK cells in suppressing metastatic tumor growth.Results MAMs isolated from the metastatic lung suppressed NK cell-induced tumor cell apoptosis in vitro via membrane-bound transforming growth factor β (TGF-β) dependent mechanisms. In the tumor-challenged mice, depletion of MAMs increased the percentage of activated (CD69+) and mature (CD11b+CD27–) NK cells and the number of Ncr1+ NK cells as well as NK cell-mediated tumor rejection in the metastatic site. Moreover, MAM depletion or TGF-β receptor antagonist treatment significantly enhanced the therapeutic efficacy of NK cell infusion in suppressing early metastatic tumor outgrowth.Conclusion This study demonstrates that MAMs are a main negative regulator of NK cell function within the metastatic tumor niche, and MAM targeting is an attractive strategy to improve NK cell-based immunotherapy for metastatic breast cancer

Expansions of adaptive-like NK cells with a tissue-resident phenotype in human lung and blood

Human adaptive-like "memory" CD56(dim)CD16(+) natural killer (NK) cells in peripheral blood from cytomegalovirus-seropositive individuals have been extensively investigated in recent years and are currently explored as a treatment strategy for hematological cancers. However, treatment of solid tumors remains limited due to insufficient NK cell tumor infiltration, and it is unknown whether large expansions of adaptive-like NK cells that are equipped for tissue residency and tumor homing exist in peripheral tissues. Here, we show that human lung and blood contains adaptive-like CD56(bright)CD16(-) NK cells with hallmarks of tissue residency, including expression of CD49a. Expansions of adaptive-like lung tissue-resident NK (trNK) cells were found to be present independently of adaptive-like CD56(dim)CD16(+) NK cells and to be hyperresponsive toward target cells. Together, our data demonstrate that phenotypically, functionally, and developmentally distinct subsets of adaptive-like NK cells exist in human lung and blood. Given their tissue-related character and hyperresponsiveness, human lung adaptive-like trNK cells might represent a suitable alternative for therapies targeting solid tumors.Peer reviewe

Accumulation of tissue-resident natural killer cells, innate lymphoid cells, and CD8+ T cells towards the center of human lung tumors

ABSTRACTLung cancer is a leading cause of cancer-related death worldwide. Despite recent advances in tissue immunology, little is known about the spatial distribution of tissue-resident lymphocyte subsets in lung tumors. Using high-parameter flow cytometry, we identified an accumulation of tissue-resident lymphocytes including tissue-resident NK (trNK) cells and CD8+ tissue-resident memory T (TRM) cells toward the center of human non-small cell lung carcinomas (NSCLC). Chemokine receptor expression patterns indicated different modes of tumor-infiltration and/or residency between trNK cells and CD8+ TRM cells. In contrast to CD8+ TRM cells, trNK cells and ILCs generally expressed low levels of immune checkpoint receptors independent of location in the tumor. Additionally, granzyme expression in trNK cells and CD8+ TRM cells was highest in the tumor center, and intratumoral CD49a+CD16− NK cells were functional and responded stronger to target cell stimulation than their CD49a− counterparts, indicating functional relevance of trNK cells in lung tumors.In summary, the present spatial mapping of lymphocyte subsets in human NSCLC provides novel insights into the composition and functionality of tissue-resident immune cells, suggesting a role for trNK cells and CD8+ TRM cells in lung tumors and their potential relevance for future therapeutic approaches

Natural killer cell immunotypes related to COVID-19 disease severity

Understanding innate immune responses in COVID-19 is important to decipher mechanisms of host responses and interpret disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections but might also contribute to immunopathology. Using 28-color flow cytometry, we here reveal strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients. This pattern was mirrored in scRNA-seq signatures of NK cells in bronchoalveolar lavage from COVID-19 patients. Unsupervised high-dimensional analysis of peripheral blood NK cells furthermore identified distinct NK cell immunotypes that were linked to disease severity. Hallmarks of these immunotypes were high expression of perforin, NKG2C, and Ksp37, reflecting increased presence of adaptive NK cells in circulation of patients with severe disease. Finally, arming of CD56 bright NK cells was observed across COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This study provides a detailed map of the NK cell activation landscape in COVID-19 disease

Type I Interferon Autoantibodies Correlate With Cellular Immune Alterations in Severe COVID-19

BackgroundInfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to severe disease with increased morbidity and mortality among certain risk groups. The presence of autoantibodies against type I interferons (aIFN-Abs) is one mechanism that contributes to severe coronavirus disease 2019 (COVID-19).MethodsThis study aimed to investigate the presence of aIFN-Abs in relation to the soluble proteome, circulating immune cell numbers, and cellular phenotypes, as well as development of adaptive immunity.ResultsaIFN-Abs were more prevalent in critical compared to severe COVID-19 but largely absent in the other viral and bacterial infections studied here. The antibody and T-cell response to SARS-CoV-2 remained largely unaffected by the presence aIFN-Abs. Similarly, the inflammatory response in COVID-19 was comparable in individuals with and without aIFN-Abs. Instead, presence of aIFN-Abs had an impact on cellular immune system composition and skewing of cellular immune pathways.ConclusionsOur data suggest that aIFN-Abs do not significantly influence development of adaptive immunity but covary with alterations in immune cell numbers