Cutting Edge: Hyperinflammatory Monocytes Expressing CD56 Abound in Severe COVID-19 Patients.

Proinflammatory monocytes play a preponderant role in the development of a cytokine storm causing fatal consequences in coronavirus disease 2019 (COVID-19) patients, highlighting the importance of analyzing in more detail monocyte distribution in these patients. In this study, we identified an atypical monocyte subpopulation expressing CD56 molecules that showed a low level of HLA-DR and high level of l-selectin. They released higher amounts of TNF-α and IL-6 and expressed genes associated with an excessive inflammatory process. Remarkably, the frequency of CD56+ monocytes inversely correlated with that of CD16+ monocytes and a high CD56+/CD16+monocyte ratio was associated with both disease severity and mortality, as well as with serum concentration of type I IFN, a factor able to induce the appearance of CD56+ monocytes. In conclusion, severe COVID-19 is characterized by the abundance of hyperinflammatory CD56+ monocytes, which could represent a novel marker with prognostic significance and, possibly, a therapeutic target for controlling the inflammatory process occurring during COVID-19.

Safety profile of immune checkpoint inhibitors: An analysis of the Italian spontaneous reporting system database.

AIMS: To provide an overview of immune checkpoint inhibitors (ICIs) safety profile using the Italian spontaneous adverse drug reaction (ADR) reporting system. METHODS: We selected all ADR reports attributed to ipilimumab (CTLA-4 inhibitor), nivolumab, pembrolizumab, atezolizumab (PD-1/PD-L1 inhibitors) from the Italian spontaneous reporting system (2011-2018). Descriptive analyses of reports for ICIs have been conducted. Time to onset of adverse effects was stratified by system organ class. Reporting odds ratio was used as measure of ADR reporting disproportionality. ICI-related ADR reports were compared with 2 reference groups, i.e. all other suspected drugs or all other antineoplastic agents. RESULTS: Overall, 2217 (0.7%) reports were related to ICIs (nivolumab: 72.2% of those reports; ipilimumab: 14.3%; pembrolizumab: 10.3%; and atezolizumab: 3.5%). ICI-related ADR reports mostly involved males (65%) and median age was 67 (interquartile range 59-73) years. Serious reports accounted for 48.8%. Frequencies of endocrine, general, hepatobiliary, metabolism, musculoskeletal, respiratory disorders, infections and neoplasms were significantly higher for ICIs than for all other drugs (P < .001). Except for infections, similar results emerged through comparison with other anticancer drugs. Colitis, hypophysitis and skin disorders were more frequently reported for anti-CTLA-4 drugs than PD-1/PD-L1 ICIs, and the opposite for musculoskeletal effects, pneumonia, and thyroid dysfunctions. ICIs were disproportionally associated also with less known risks, e.g. ischaemic heart disease, cardiac failure and optic nerve disorders. CONCLUSION: The most frequently reported safety issues were probably immune-related adverse events including general, gastrointestinal and respiratory disorders. Potentially emerging safety signals, such as ischaemic heart disease and cardiac failure, requiring further investigation were detected.

CD56(bright)perforin(low) noncytotoxic human NK cells are abundant in both healthy and neoplastic solid tissues and recirculate to secondary lymphoid organs via afferent lymph.

As limited information is available regarding the distribution and trafficking of NK cells among solid organs, we have analyzed a wide array of tissues derived from different human compartments. NK cells were widely distributed in most solid tissues, although their amount varied significantly depending on the tissue/organ analyzed. Interestingly, the distribution appeared to be subset specific, as some tissues were preferentially populated by CD56(bright)perforin(low) NK cells, with others by the CD56(dim)perforin(high) cytotoxic counterpart. Nevertheless, most tissues were highly enriched in CD56(bright)perforin(low) cells, and the distribution of NK subsets appeared in accordance with tissue gene expression of chemotactic factors, for which receptors are differently represented in the two subsets. Remarkably, chemokine expression pattern of tissues was modified after neoplastic transformation. As a result, although the total amount of NK cells infiltrating the tissues did not significantly change upon malignant transformation, the relative proportion of NK subsets infiltrating the tissues was different, with a trend toward a tumor-infiltrating NK population enriched in noncytotoxic cells. Besides solid tissues, CD56(bright)perforin(low) NK cells were also detected in seroma fluids, which represents an accrual of human afferent lymph, indicating that they may leave peripheral solid tissues and recirculate to secondary lymphoid organs via lymphatic vessels. Our results provide a comprehensive mapping of NK cells in human tissues, demonstrating that discrete NK subsets populate and recirculate through most human tissues and that organ-specific chemokine expression patterns might affect their distribution. In this context, chemokine switch upon neoplastic transformation might represent a novel mechanism of tumor immune escape.

Characterization of human afferent lymph dendritic cells from seroma fluids.

Dendritic cells (DCs) migrate from peripheral tissues to secondary lymphoid organs (SLOs) through the afferent lymph. Owing to limitations in investigating human lymph, DCs flowing in afferent lymph have not been properly characterized in humans until now. In this study, DCs present in seroma, an accrual of human afferent lymph occurring after lymph node surgical dissection, were isolated and analyzed in detail. Two main DC subsets were identified in seroma that corresponded to the migratory DC subsets present in lymph nodes, that is, CD14(+) and CD1a(+). The latter also included CD1a(bright) Langerhans cells. The two DC subsets appeared to share the same monocytic precursor and to be developmentally related; both of them spontaneously released high levels of TGF-ß and displayed similar T cell-activating and -polarizing properties. In contrast, they differed in the expression of surface molecules, including TLRs; in their phagocytic activity; and in the expression of proteins involved in Ag processing and presentation. It is worth noting that although both subsets were detected in seroma in the postsurgical inflammatory phase, only CD1a(+) DCs migrated via afferent lymph under steady-state conditions. In conclusion, the high numbers of DCs contained in seroma fluids allowed a proper characterization of human DCs migrating via afferent lymph, revealing a continuous stream of DCs from peripheral regions toward SLOs under normal conditions. Moreover, we showed that, in inflammatory conditions, distinct subsets of DCs can migrate to SLOs via afferent lymph.

The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production.

BACKGROUND: CTLA-4 (Cytotoxic T lymphocyte antigen-4) is traditionally known as a negative regulator of T cell activation. The blocking of CTLA-4 using human monoclonal antibodies, such as Ipilimumab, is currently used to relieve CTLA-4-mediated inhibition of anti-tumor immune response in metastatic melanoma. Herein, we have analyzed CTLA-4 expression and Ipilimumab reactivity on melanoma cell lines and tumor tissues from cutaneous melanoma patients. Then, we investigated whether Ipilimumab can trigger innate immunity in terms of antibody dependent cellular cytotoxicity (ADCC) or Tumor Necrosis Factor (TNF)-α release. Finally, a xenograft murine model was set up to determine in vivo the effects of Ipilimumab and NK cells on melanoma. METHODS: CTLA-4 expression and Ipilimumab reactivity were analyzed on 17 melanoma cell lines (14 primary and 3 long-term cell lines) by cytofluorimetry and on 33 melanoma tissues by immunohistochemistry. CTLA-4 transcripts were analyzed by quantitative RT-PCR. Soluble CTLA-4 and TNF-α were tested by ELISA. Peripheral blood mononuclear cells (PBMC), NK and γδT cells were tested in ADCC assay with Ipilimumab and melanoma cell lines. TNF-α release was analyzed in NK-melanoma cell co-cultures in the presence of ipilimumab. In vivo experiments of xenotransplantation were carried out in NOD/SCID mice. Results were analyzed using unpaired Student's t-test. RESULTS: All melanoma cell lines expressed mRNA and cytoplasmic CTLA-4 but surface reactivity with Ipilimumab was quite heterogeneous. Accordingly, about 2/3 of melanoma specimens expressed CTLA-4 at different level of intensity.Ipilimumab triggered, via FcγReceptorIIIA (CD16), ex vivo NK cells as well as PBMC, IL-2 activated NK and γδT cells to ADCC of CTLA-4+ melanoma cells. No ADCC was detected upon interaction with CTLA-4- FO-1 melanoma cell line. TNF-α was released upon interaction of NK cells with CTLA-4+ melanoma cell lines. Remarkably, Ipilimumab neither affected proliferation and viability nor triggered ADCC of CTLA-4+ T lymphocytes. In a chimeric murine xenograft model, the co-engraftment of Ipilimumab-treated melanoma cells with human allogeneic NK cells delayed and significantly reduced tumor growth, as compared to mice receiving control xenografts. CONCLUSIONS: Our studies demonstrate that Ipilimumab triggers effector lymphocytes to cytotoxicity and TNF-α release. These findings suggest that Ipilimumab, besides blocking CTLA-4, can directly activate the elimination of CTLA-4+ melanomas.

Mechanical bacterial lysate enhances antimicrobial barrier mechanisms in human airway epithelial cells.

Polyvalent mechanical bacterial lysate is effective in the prevention of respiratory tract infections, although its mechanism of action is not entirely elucidated. Because epithelial cells constitute the frontline defense against infections, we investigated the molecular mechanisms of innate response exerted by bronchial epithelial cells in the presence of polyvalent mechanical bacterial lysate. By using primary human bronchial epithelial cells, we observed that polyvalent mechanical bacterial lysate was able to increase the expression of cellular adhesion molecules such as ICAM-1 and E-cadherin, as well as the expression of amphiregulin, a growth factor able to support human bronchial epithelial cell proliferation. Remarkably, polyvalent mechanical bacterial lysate promoted in human bronchial epithelial cells the de novo expression of human ß-defensin-2, a major antimicrobial peptide, conferring them a direct antimicrobial activity. Moreover, polyvalent mechanical bacterial lysate-stimulated human bronchial epithelial cells provided signals for increased IL-22 production by innate lymphoid cells via IL-23, which could further contribute to the release of antimicrobial peptides by epithelial cells. In agreement with these in vitro data, the concentration of both IL-23 and antimicrobial peptides (human ß-defensin-2 and LL-37) increased in the saliva of healthy volunteers after sublingual administration of polyvalent mechanical bacterial lysate. Altogether, these results indicate that polyvalent mechanical bacterial lysate administration might support mucosal barrier integrity and promote mechanisms of antimicrobial activity in airway epithelial cells.

Murine models to study human NK cells in human solid tumors.

Since the first studies, the mouse models have provided crucial support for the most important discoveries on NK cells, on their development, function, and circulation within normal and tumor tissues. Murine tumor models were initially set to study murine NK cells, then, ever more sophisticated human-in-mice models have been developed to investigate the behavior of human NK cells and minimize the interferences from the murine environment. This review presents an overview of the models that have been used along time to study NK cells, focusing on the most popular NOG and NSG models, which work as recipients for the preparation of human-in-mice tumor models, the study of transferred human NK cells, and the evaluation of various enhancers of human NK cell function, including cytokines and chimeric molecules. Finally, an overview of the next generation humanized mice is also provided along with a discussion on how traditional and innovative in-vivo and in-vitro approaches could be integrated to optimize effective pre-clinical studies.

Human NK cells of mice with reconstituted human immune system components require preactivation to acquire functional competence.

To investigate human natural killer (NK)-cell reactivity in vivo we have reconstituted human immune system components by transplantation of human hematopoietic progenitor cells into NOD-scid IL2Rγ(null) mice. We demonstrate here that this model allows the development of all NK-cell subsets that are also found in human adult peripheral and cord blood, including NKp46(+)CD56(-) NK cells. Similar to human cord blood, NK cells from these reconstituted mice require preactivation by interleukin-15 to reach the functional competence of human adult NK cells. Mainly the terminally differentiated CD16(+) NK cells demonstrate lower reactivity without this stimulation. After preactivation, both CD16(+) and CD16(-) NK cells efficiently produce interferon-γ and degranulate in response to stimulation with NK cell-susceptible targets, including K562 erythroleukemia cells. NK-cell lines, established from reconstituted mice, demonstrate cytotoxicity against this tumor cell line. Importantly, preactivation can as well be achieved by bystander cell maturation via poly I:C stimulation in vitro and injection of this maturation stimulus in vivo. Preactivation in vivo enhances killing of human leukocyte antigen class I negative tumor cells after their adoptive transfer. These data suggest that a functional, but resting, NK-cell compartment can be established in immune-compromised mice after human hematopoietic progenitor cell transfer.

Immune Checkpoint Blockade: A Strategy to Unleash the Potential of Natural Killer Cells in the Anti-Cancer Therapy.

Immune checkpoint inhibitors (ICIs) immunotherapy has represented a breakthrough in cancer treatment. Clinical use of ICIs has shown an acceptable safety profile and promising antitumor activity. Nevertheless, some patients do not obtain clinical benefits after ICIs therapy. In order to improve and cure an increasing number of patients, the field has moved toward the discovery of new ICIs expressed by cells of innate immunity with an elevated inherent antitumor activity, such as natural killer cells. This review will focus on the recent findings concerning the role of classical and non-classical immune checkpoint molecules and receptors that regulate natural killer cell function, as potential targets, and their future clinical application.

ILC in chronic inflammation, cancer and targeting with biologicals.

Since their discovery, Innate Lymphoid Cells (ILC) have emerged as important effector cells, serving multiple roles in maintaining tissue homeostasis and responding to tissue insults. As such, dysregulations of their function and distribution have been observed in a variety of immune-mediated diseases, suggesting a specific role for ILC in the pathophysiology of several disorders including chronic inflammation and cancer. Here, we provide an updated view on ILC biology dissecting their pathological or protective contribution in chronic inflammatory diseases such as multiple sclerosis, inflammatory bowel diseases, psoriasis, rheumatoid arthritis, asthma and COPD, atherosclerosis, also exploring ILC role in tumor surveillance and progression. Throughout the review, we will also highlight how the potential dual role of these cells for protective or pathogenic immunity in many inflammatory diseases makes them interesting targets for the development of novel therapeutic strategies, particularly promising.

Therapeutic Implications of Tumor Microenvironment in Lung Cancer: Focus on Immune Checkpoint Blockade.

In the last decade, the treatment of non-small cell lung cancer (NSCLC) has been revolutionized by the introduction of immune checkpoint inhibitors (ICI) directed against programmed death protein 1 (PD-1) and its ligand (PD-L1), or cytotoxic T lymphocyte antigen 4 (CTLA-4). In spite of these improvements, some patients do not achieve any benefit from ICI, and inevitably develop resistance to therapy over time. Tumor microenvironment (TME) might influence response to immunotherapy due to its prominent role in the multiple interactions between neoplastic cells and the immune system. Studies investigating lung cancer from the perspective of TME pointed out a complex scenario where tumor angiogenesis, soluble factors, immune suppressive/regulatory elements and cells composing TME itself participate to tumor growth. In this review, we point out the current state of knowledge involving the relationship between tumor cells and the components of TME in NSCLC as well as their interactions with immunotherapy providing an update on novel predictors of benefit from currently employed ICI or new therapeutic targets of investigational agents. In first place, increasing evidence suggests that TME might represent a promising biomarker of sensitivity to ICI, based on the presence of immune-modulating cells, such as Treg, myeloid derived suppressor cells, and tumor associated macrophages, which are known to induce an immunosuppressive environment, poorly responsive to ICI. Consequently, multiple clinical studies have been designed to influence TME towards a pro-immunogenic state and subsequently improve the activity of ICI. Currently, the mostly employed approach relies on the association of "classic" ICI targeting PD-1/PD-L1 and novel agents directed on molecules, such as LAG-3 and TIM-3. To date, some trials have already shown promising results, while a multitude of prospective studies are ongoing, and their results might significantly influence the future approach to cancer immunotherapy.

NK cells provide helper signal for CD8+ T cells by inducing the expression of membrane-bound IL-15 on DCs.

NK cell recognition of cells that do not express or express low amounts of MHC class I molecules results not only in direct killing of target cells but also in the generation of specific T cell responses consequent to the induction of dendritic cell (DC) activation. While IL-12 production by NK cell-activated DCs is generally thought to play a critical role, a similar DC-mediated NK cell help has been reported also in IL-12-knockout mice. Here, we show that human NK cells can induce on DC surface membrane, via IFN-gamma secretion, the expression of high levels of IL-15. Remarkably, we show that DC expression of this membrane-bound form of IL-15, which is only partially associated with IL-15R molecules, is essential to promote specific CD8(+) T lymphocyte response in the absence of DC-derived IL-12.

Myeloma cells induce the accumulation of activated CD94low NK cells by cell-to-cell contacts involving CD56 molecules.

Natural killer (NK) cells represent innate effector cells potentially able to play a role during the immune response against multiple myeloma (MM). To better define the distribution and the specific properties of NK cell subsets during MM disease, we analyzed their features in the bone marrow and peripheral blood of newly diagnosed MM patients. Our findings revealed that, in both compartments, NK cells were more abundant than in healthy donors. Among total MM-NK cells, a significant increase of CD94lowCD56dim NK cell subset was observed, which already appears in clinical precursor conditions leading to MM, namely monoclonal gammopathy of undetermined significance and smoldering MM, and eventually accumulates with disease progression. Moreover, a consistent fraction of CD94lowCD56dim NK cells was in a proliferation phase. When analyzed for their killing abilities, they represented the main cytotoxic NK cell subset against autologous MM cells. In vitro, MM cells could rapidly induce the expansion of the CD94lowCD56dim NK cell subset, thus reminiscent of that observed in MM patients. Mechanistically, this accumulation relied on cell to cell contacts between MM and NK cells and required both activation via DNAM-1 and homophilic interaction with CD56 expressed on MM cells. Considering the growing variety of combination treatments aimed at enhancing NK cell-mediated cytotoxicity against MM, these results may also be informative for optimizing current immunotherapeutic approaches.

Association Between Response to Nivolumab Treatment and Peripheral Blood Lymphocyte Subsets in Patients With Non-small Cell Lung Cancer.

Immune checkpoint blockade represents a major breakthrough in advanced non-small cell lung cancer (NSCLC) therapy. However, success is limited to a subset of patients and there is a critical need to identify robust biomarkers associated with clinical response. In this study, we assessed whether pre-existing immunological characteristics, as well as immune parameters measured during treatment, might provide such clinical guidance. We studied blood samples collected at baseline and during treatment in a cohort of advanced NSCLC patients (n = 74) treated with nivolumab. Several lymphocyte subsets and biomarkers were then correlated with overall survival (OS) as well as clinical response, assessed using RECIST criteria. We found that patients characterized by longer OS had higher levels of CD3+, CD4+, and CD8+ T cells but lower levels of NK cells at baseline. Moreover, that they displayed a statistically significant lower expression of PD-1 on both CD3+ and CD8+ T cells (p = 0.013 and p = 0.033, respectively). The pre-treatment level of exhausted T cells (CD8+PD1+Eomes+) was significantly lower in patients with controlled disease (CD), defined as partial response (PR), and stable disease (SD), compared to those with progressive disease (PD) (p = 0.046). In CD patients, the frequency of exhausted CD8+ T cells further decreased during treatment cycles (p = <0.0001, p = 0.0032, and p = 0.0239, respectively). In conclusion, our results suggest that the distribution of lymphocyte subsets and expression of PD-1 on T cells before treatment may help predict the outcome of anti-PD-1 treatment in NSCLC patients. In addition, assessing the initial levels of exhausted T cells as well as their decrease upon treatment may also predict response and clinical outcome.

Isolation, Expansion, and Characterization of Natural Killer Cells and Their Precursors as a Tool to Study Cancer Immunosurveillance.

This chapter will describe the current methodologies to isolate and expand NK cells from Peripheral Blood (PB) or tissues for "in vitro" studies, including NK cell antitumor immune function. In addition, methods to induce NK cell maturation, differentiation, and expansion from CD34+ precursors will also be described. Finally, it will also be treated the topical issue of the characterization of new functionally and phenotypically defined NK cell subsets.

Symptomatic Carotid Atherosclerotic Plaques Are Associated With Increased Infiltration of Natural Killer (NK) Cells and Higher Serum Levels of NK Activating Receptor Ligands.

A wide array of immune cells, including lymphocytes, is known to be present and to play a pathogenetic role in atherosclerotic lesions. However, limited information is currently available regarding the presence of Natural Killer (NK) cell subsets within vessel plaque, and more in general, regarding their role in human atherosclerosis. We evaluated the distribution of NK cells in human carotid atherosclerotic plaques, dissecting asymptomatic and symptomatic patients (identified as affected by stroke, transient ischemic attack, or amaurosis fugax within 6 months) with the aim of shedding light on the putative contribution of NK cells to the pathogenic process that leads to plaque instability and subsequent clinical complications. We observed that carotid plaques were consistently infiltrated by NK cells and, among them, CD56brightperforinlow NK cells were abundantly present and displayed different markers of tissue residency (i.e., CD103 CD69 and CD49a). Interestingly, carotid atherosclerotic plaques of symptomatic patients showed a higher content of NK cells and an increased ratio between CD56brightperforinlow NK cells and their CD56dimperforinhigh counterpart. NK cells isolated from plaques of symptomatic patients were also stronger producers of IFN-γ. Analysis of the expression of NK activating receptor ligands (including MICA/B, ULBP-3, and B7-H6) in atherosclerotic carotid plaques revealed that they were abundantly expressed by a HLA-DR+CD11c+ myeloid cell population resident in the plaques. Remarkably, sera of symptomatic patients contained significant higher levels of soluble ligands for NK activating receptors. Our observations indicate that CD56bright NK cells accumulate within human atherosclerotic lesions and suggest a possible contribution of NK cells to the process determining plaque instability.

Influence of Vitamin D in Advanced Non-Small Cell Lung Cancer Patients Treated with Nivolumab.

Nivolumab is one of the most commonly used monoclonal antibodies for advanced non-small cell lung cancer treatment, to the extent that the presence of its anti-antibody is considered a negative prognostic factor. Vitamin D (VD) modulates expression of the genes involved in drug metabolism and elimination. Immune system regulation and immunodeficiency is frequent in non-small cell lung cancer patients. To date, no data have been reported about the relationship between nivolumab and VD. The aim of this study was to quantify plasma 25-hydroxyVD (25-VD) and 1,25-VD, nivolumab, and its anti-antibody before starting treatment (baseline) and at 15, 45 and 60 days of therapy. VD-pathway-associated gene single nucleotide polymorphisms (SNPs) were also evaluated. Molecules were quantified through enzyme-linked immunosorbent assay, and SNPs through real-time PCR. Forty-five patients were enrolled. Median nivolumab concentrations were 12.5 ug/mL, 22.3 ug/mL and 27.1 ug/mL at 15, 45 and 60 days respectively. No anti-nivolumab antibodies were found. Correlations were observed between nivolumab concentrations and 25-VD levels. Nivolumab concentrations were affected by VD-pathway-related gene SNPs. VDBP AC/CC genotype and baseline 25-VD < 10 ng/mL predicted a nivolumab concentration cut-off value of <18.7ug/mL at 15 days, which was associated with tumor progression. This is the first study showing VD marker predictors of nivolumab concentrations in a real-life context of non-small cell lung cancer treatment.

Molecular Mechanisms Directing Migration and Retention of Natural Killer Cells in Human Tissues.

A large body of data shows that Natural Killer (NK) cells are immune effectors exerting a potent cytolytic activity against tumors and virus infected cells. The discovery and characterization of several inhibitory and activating receptors unveiled most of the mechanisms allowing NK cells to spare healthy cells while selectively attacking abnormal tissues. Nevertheless, the mechanisms ruling NK cell subset recirculation among the different compartments of human body have only lately started to be investigated. This is particularly true for pathological settings such as tumors or infected tissues but also for para-physiological condition like pregnant human uterine mucosa. It is becoming evident that the microenvironment associated to a particular clinical condition can deeply influence the migratory capabilities of NK cells. In this review we describe the main mechanisms and stimuli known to regulate the expression of chemokine receptors and other molecules involved in NK cell homing to either normal or pathological/inflamed tissues, including tumors or organs such as lung and liver. We will also discuss the role played by the chemokine/chemokine receptor axes in the orchestration of physiological events such as NK cell differentiation, lymphoid organ retention/egress and recruitment to decidua during pregnancy.