The effect of physical exercise on anticancer immunity.

Regular physical activity is associated with lower cancer incidence and mortality, as well as with a lower rate of tumour recurrence. The epidemiological evidence is supported by preclinical studies in animal models showing that regular exercise delays the progression of cancer, including highly aggressive malignancies. Although the mechanisms underlying the antitumorigenic effects of exercise remain to be defined, an improvement in cancer immunosurveillance is likely important, with different immune cell subtypes stimulated by exercise to infiltrate tumours. There is also evidence that immune cells from blood collected after an exercise bout could be used as adoptive cell therapy for cancer. In this Perspective, we address the importance of muscular activity for maintaining a healthy immune system and discuss the effects of a single bout of exercise (that is, 'acute' exercise) and those of 'regular' exercise (that is, repeated bouts) on anticancer immunity, including tumour infiltrates. We also address the postulated mechanisms and the clinical implications of this emerging area of research.

The secretome atlas of two mouse models of progeria.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease caused by nuclear envelope alterations that lead to accelerated aging and premature death. Several studies have linked health and longevity to cell-extrinsic mechanisms, highlighting the relevance of circulating factors in the aging process as well as in age-related diseases. We performed a global plasma proteomic analysis in two preclinical progeroid models (LmnaG609G/G609G and Zmpste24-/- mice) using aptamer-based proteomic technology. Pathways related to the extracellular matrix, growth factor response and calcium ion binding were among the most enriched in the proteomic signature of progeroid samples compared to controls. Despite the global downregulation trend found in the plasma proteome of progeroid mice, several proteins associated with cardiovascular disease, the main cause of death in HGPS, were upregulated. We also developed a chronological age predictor using plasma proteome data from a cohort of healthy mice (aged 1-30 months), that reported an age acceleration when applied to progeroid mice, indicating that these mice exhibit an "old" plasma proteomic signature. Furthermore, when compared to naturally-aged mice, a great proportion of differentially expressed circulating proteins in progeroid mice were specific to premature aging, highlighting secretome-associated differences between physiological and accelerated aging. This is the first large-scale profiling of the plasma proteome in progeroid mice, which provides an extensive list of candidate circulating plasma proteins as potential biomarkers and/or therapeutic targets for further exploration and hypothesis generation in the context of both physiological and premature aging.

BTLA dysregulation correlates with poor outcome and diminished T cell-mediated antitumor responses in chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) progressively develop marked immunosuppression, dampening innate and adaptive-driven antitumor responses. However, the underlying mechanisms promoting immune exhaustion are largely unknown. Herein, we provide new insights into the role of BTLA/HVEM axis promoting defects in T cell-mediated responses against leukemic cells. Increased expression of BTLA, an inhibitory immune checkpoint, was detected on the surface of CD4 + and CD8 + T lymphocytes in patients with CLL. Moreover, high levels of BTLA on CD4 + T cells correlated with diminished time to treatment. Signaling through BTLA activation led to decreased IL-2 and IFN-γ production ex vivo, whereas BTLA/HVEM binding disruption enhanced IFN-γ + CD8 + T lymphocytes. Accordingly, BTLA blockade in combination with bispecific anti-CD3/anti-CD19 antibody promoted CD8 + T cell-mediated anti-leukemic responses. Finally, treatment with an anti-BLTA blocking monoclonal antibody alone or in combination with ibrutinib-induced leukemic cell depletion in vitro. Altogether, our data reveal that BTLA dysregulation has a prognostic role and is limiting T cell-driven antitumor responses, thus providing new insights about immune exhaustion in patients with CLL.

Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells.

Better understanding on interactions between SARS-CoV-2 and host cells should help to identify host factors that may be targetable to combat infection and COVID-19 pathology. To this end, we have conducted a genome-wide CRISPR/Cas9-based loss-of-function screen in human lung cancer cells infected with SARS-CoV-2-pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS-CoV-2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS-CoV-2 viruses confirm that loss-of-function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single-cell RNA-Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS-CoV-2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.

Exercise training effects on natural killer cells: a preliminary proteomics and systems biology approach.

BACKGROUND: Regular exercise, particularly moderate-intensity continuous training (MICT), can improve immune function. Natural killer (NK) cells, a subset of lymphocytes that react to infections, are the most responsive innate immune cells to exercise, but the mechanisms underlying this are poorly understood. A type of exercise training that is gaining popularity in recent years is high-intensity interval training (HIIT), but how it affects NK cells is largely unknown. In fact, intense exercise has been traditionally viewed as a potential stressor to immune homeostasis. The purpose of this study was to determine in healthy, previously untrained adults (N=8 [3 male; 40±6 years]) the effects of an intervention consisting of 4-week MICT followed by 4-week HIIT on NK cells as compared with a pre-training (baseline) state. METHODS: Participants were studied at three time points: baseline, mid-intervention (after MICT), and post-intervention (after HIIT). Main assessments included cytotoxicity assays, flow-cytometry analysis of NK cell surface markers, and interrogation of the cellular proteome using a systems biology approach. RESULTS: A significant time effect was found for NK cell cytotoxicity (p<0.001), which was increased ~10-fold at both midand post-intervention versus baseline. No significant intervention effect was found for NK surface receptor expression, except for CXCR3 determined as mean fluorescence intensity (p=0.044, although with no significant differences in post hoc pairwise comparisons). The proteins showing a higher differential expression (Log2 fold-change > 10 and false discovery rate [FDR] q-value < 0.001) were COP9 signalosome subunit 3 (COPS3), DnaJ heat shock protein family member B11 (DNAJB11), histidyl-TRNA synthetase 1 (HARS), NIMA related kinase 9 (NEK9), nucleoporin 88 (NUP88), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), regulator of chromosome condensation 2 (RCC2), TAO kinase 3 (TAOK3), transducin beta like 2 (TBL2), and ring finger protein 40 (RNF40). All were upregulated at mid-intervention compared with baseline, with the exception of HARS, which was downregulated. Four enriched pathways (FDR p<25%) were found: two related to transmembrane transport and cellular composition (downregulated at mid-intervention vs baseline), and two related to oxidation- reduction reactions (regulated at post-intervention versus baseline). CONCLUSION: A progressive exercise intervention of MICT followed by HIIT induces a remarkable improvement in NK function compared with the untrained state, although at the mechanistic level the pathways involved seem to differ over time during the intervention.

LAG-3 Blockade with Relatlimab (BMS-986016) Restores Anti-Leukemic Responses in Chronic Lymphocytic Leukemia.

The inclusion of monoclonal antibodies targeting immune checkpoints such PD-1/PD-L1 or CTLA-4 has revolutionized the landscape of anti-cancer therapy. However, PD-1 and CTLA-4 blockade failed to achieve clinical benefit in CLL, thus attention has been focused on emerging checkpoints in this malignancy. LAG-3 is an immune checkpoint receptor that negatively regulates T cell-mediated responses by inducing an hyporesponsive state, thus promoting tumor escape. Patients with chronic lymphocytic leukemia (CLL) develop a profound immune suppression that leads to lessened immunosurveillance and increased risk of developing a secondary neoplasia. In the study herein, we report the profound dysregulation of LAG-3 on leukemic cells in CLL. Likewise, natural killer (NK) and T cells showed increased LAG-3 expression, hence suggesting a role for this checkpoint in CLL-associated immunosuppression. High LAG-3 expression, as well as high levels of soluble LAG-3 (sLAG-3), correlated with adverse cytogenetics and poor outcome in patients with CLL, highlighting the clinical relevance of this immune checkpoint. Treatment of peripheral blood mononuclear cells (PBMCs) from patients with CLL with relatlimab, a new anti-LAG-3 blocking antibody currently evaluated in numerous clinical trials, depleted leukemic cells and restored NK cell- and T cell-mediated responses. Moreover, combination of LAG-3 with the immunomodulatory drug (IMiD) lenalidomide significantly increased IL-2 production by T cells and antibody-dependent cytotoxicity (ADCC) mediated by NK cells. Altogether, these data provide new insights into the potential anti-leukemic effects of relatlimab, currently in clinical trials in CLL, and provides the rationale to further investigate its combination with IMiDs for the management of hematological malignancies.

BTLA/HVEM Axis Induces NK Cell Immunosuppression and Poor Outcome in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by progressive immunosuppression and diminished cancer immunosurveillance. Immune checkpoint blockade (ICB)-based therapies, a major breakthrough against cancer, have emerged as a powerful tool to reinvigorate antitumor responses. Herein, we analyzed the role of the novel inhibitory checkpoint BTLA and its ligand, HVEM, in the regulation of leukemic and natural killer (NK) cells in CLL. Flow cytometry analyses showed that BTLA expression is upregulated on leukemic cells and NK cells from patients with CLL, whereas HVEM is downregulated only in leukemic cells, especially in patients with advanced Rai-Binet stage. In silico analysis revealed that increased HVEM, but not BTLA, mRNA expression in leukemic cells correlated with diminished overall survival. Further, soluble BTLA (sBTLA) was found to be increased in the sera of patients with CLL and highly correlated with poor prognostic markers and shorter time to treatment. BTLA blockade with an anti-BTLA monoclonal antibody depleted leukemic cells and boosted NK cell-mediated responses ex vivo by increasing their IFN-γ production, cytotoxic capability, and antibody-dependent cytotoxicity (ADCC). In agreement with an inhibitory role of BTLA in NK cells, surface BTLA expression on NK cells was associated with poor outcome in patients with CLL. Overall, this study is the first to bring to light a role of BTLA/HVEM in the suppression of NK cell-mediated immune responses in CLL and its impact on patient's prognosis, suggesting that BTLA/HVEM axis may be a potential therapeutic target in this disease.

Mechanisms of Apoptosis Resistance to NK Cell-Mediated Cytotoxicity in Cancer.

Natural killer (NK) cells are major contributors to immunosurveillance and control of tumor development by inducing apoptosis of malignant cells. Among the main mechanisms involved in NK cell-mediated cytotoxicity, the death receptor pathway and the release of granules containing perforin/granzymes stand out due to their efficacy in eliminating tumor cells. However, accumulated evidence suggest a profound immune suppression in the context of tumor progression affecting effector cells, such as NK cells, leading to decreased cytotoxicity. This diminished capability, together with the development of resistance to apoptosis by cancer cells, favor the loss of immunogenicity and promote immunosuppression, thus partially inducing NK cell-mediated killing resistance. Altered expression patterns of pro- and anti-apoptotic proteins along with genetic background comprise the main mechanisms of resistance to NK cell-related apoptosis. Herein, we summarize the main effector cytotoxic mechanisms against tumor cells, as well as the major resistance strategies acquired by tumor cells that hamper the extrinsic and intrinsic apoptotic pathways related to NK cell-mediated killing.

Mechanisms of Resistance to NK Cell Immunotherapy.

Immunotherapy has recently been a major breakthrough in cancer treatment. Natural killer (NK) cells are suitable targets for immunotherapy owing to their potent cytotoxic activity that may target cancer cells in a major histocompatibility complex (MHC) and antigen-unrestricted manner. Current therapies targeting NK cells include monoclonal antibodies that promote NK cell antibody-dependent cell-mediated cytotoxicity (ADCC), hematopoietic stem cell transplantation (HSCT), the adoptive transfer of NK cells, the redirection of NK cells using chimeric antigen receptor (CAR)-NK cells and the use of cytokines and immunostimulatory drugs to boost the anti-tumor activity of NK cells. Despite some encouraging clinical results, patients receiving these therapies frequently develop resistance, and a myriad of mechanisms of resistance affecting both the immune system and cancer cells have been reported. A first contributing factor that modulates the efficacy of the NK cell therapy is the genetic profile of the individual, which regulates all aspects of NK cell biology. Additionally, the resistance of cancer cells to apoptosis and the immunoediting of cancer cells, a process that decreases their immunogenicity and promotes immunosuppression, are major determinants of the resistance to NK cell therapy. Consequently, the efficacy of NK cell anti-tumor therapy is specific to each patient and disease. The elucidation of such immunosubversive mechanisms is crucial to developing new procedures and therapeutic strategies to fully harness the anti-tumor potential of NK cells.

A cytofluorimetric assay to evaluate intracellular cytokine production by NK cells.

Natural killer (NK) cells are innate lymphoid cells that, together with CD8+ T lymphocytes, are tightly correlated with immunesurveillance and the elimination of transforming and malignant cells both during early steps of tumorigenesis and metastasis. This capacity is due, but not limited to, their great cytotoxic capacity upon recognition of stress-related activating ligands of NK cells on the surface of tumor cells. Due to the emerging role of NK cells in the response to treatment with immune checkpoint blockers (ICBs) and other immunotherapies, it has become essential to deeply study this immune subset. As mentioned above, NK cell antitumor responses not only rely on their high cytotoxic capacity, cytokine production by this immune subset, such as interferon gamma (IFN-γ) or tumor necrosis factor (TNF-α), also plays a key role on NK cell function. In this chapter, we provide a detailed protocol to measure the intracellular expression of cytokines produced by NK cells upon stimulation. A step-by-step guide to isolate peripheral blood mononuclear cells, purify NK cells from whole blood, co-culture with tumor cell lines and evaluate their cytokine production capacity by flow cytometry is here provided.

Evaluation of NK cell cytotoxic activity against malignant cells by the calcein assay.

The immune system exerts a tight cancer surveillance to eradicate nascent and developing tumors. Natural killer (NK) cells rely on their cytotoxic activity to eliminate abnormal or stressed cells, including those undergoing malignant transformation. Thereupon, NK cells, along with CD8+ T cells, are effector immune cells with a pivotal role in cancer immunosurveillance. Due to the outstanding effectivity of immunotherapy in cancer management, strategies aimed at boosting NK cell-mediated tumor recognition and elimination are being thoroughly studied, such as approaches to induce tumor surface expression of NK cell activating ligands. Additionally, the development of methods to evaluate NK cell cytotoxic responses towards malignant cells with distinct immunogenic profiles is of paramount relevance. In this chapter, we detail a simple but highly sensitive and reproducible methodological approach to quantify in vitro NK cell-mediated lysis of a given target cell, e.g. a tumor cell, based on calcein-AM staining and fluorescence detection. Further, this method can be employed to determine the impact of chemical modulators or antibodies blocking cell surface proteins on the antitumor capacity of NK cells, with the aim to improve anticancer immune responses and elucidate the underlying mechanisms regulating NK cell activity.

The Mithralog EC-7072 Induces Chronic Lymphocytic Leukemia Cell Death by Targeting Tonic B-Cell Receptor Signaling.

B-cell receptor (BCR)-dependent signaling is central for leukemia B-cell homeostasis, as underscored by the promising clinical results obtained in patients with chronic lymphocytic leukemia (CLL) treated with novel agents targeting components of this pathway. Herein, we demonstrate that the mithralog EC-7072 displays high ex vivo cytotoxic activity against leukemia cells from CLL patients independently from high-risk prognostic markers and IGHV mutational status. EC-7072 was significantly less toxic against T cells and NK cells and did not alter the production of the immune effector molecules IFN-γ and perforin. EC-7072 directly triggered caspase-3-dependent CLL cell apoptosis, which was not abrogated by microenvironment-derived factors that sustain leukemia cell survival. RNA-sequencing analyses revealed a dramatic EC-7072-driven reprograming of the transcriptome of CLL cells, including a wide downregulation of multiple components and targets of the BCR signaling pathway. Accordingly, we found decreased levels of phosphorylated signaling nodes downstream of the BCR. Crosslinking-mediated BCR activation antagonized CLL cell death triggered by EC-7072, increased the phosphorylation levels of the abovementioned signaling nodes and upregulated BCL2 expression, suggesting that the mithralog disrupts CLL cell viability by targeting the BCR signaling axis at multiple levels. EC-7072 exerted similar or higher antileukemic activity than that of several available CLL therapies and displayed additive or synergistic interaction with these drugs in killing CLL cells. Overall, our findings provide rationale for future investigation to test whether EC-7072 may be a potential therapeutic option for patients with CLL and other B-cell malignancies.

Immunosurveillance of cancer cell stress.

Cancer development is tightly controlled by effector immune responses that recognize and eliminate malignantly transformed cells. Nonetheless, certain immune subsets, such as tumor-associated macrophages, have been described to promote tumor growth, unraveling a double-edge role of the immune system in cancer. Cell stress can modulate the crosstalk between immune cells and tumor cells, reshaping tumor immunogenicity and/or immune function and phenotype. Infiltrating immune cells are exposed to the challenging conditions typically present in the tumor microenvironment. In return, the myriad of signaling pathways activated in response to stress conditions may tip the balance toward stimulation of antitumor responses or immune-mediated tumor progression. Here, we explore how distinct situations of cellular stress influence innate and adaptive immunity and the consequent impact on cancer establishment and progression.

CD107a Degranulation Assay to Evaluate Immune Cell Antitumor Activity.

Cancer development is under surveillance by the immune system of the host. Tumor cells can be recognized and killed by cytotoxic lymphocytes- such as CD8+ T lymphocytes and natural killer (NK) cells-mainly through the immune secretion of lytic granules that kill target cells. This process involves the fusion of the granule membrane with the cytoplasmic membrane of the immune effector cell, resulting in surface exposure of lysosomal-associated proteins that are typically present on the lipid bilayer surrounding lytic granules, such as CD107a. Therefore, membrane expression of CD107a constitutes a marker of immune cell activation and cytotoxic degranulation. In this chapter, we detail the steps required to isolate peripheral blood mononuclear cells (PBMCs), coculture them with target tumor cell lines, and evaluate the cytotoxic immune function by means of flow cytometry evaluation of CD107a expression on the surface of NK cells.

A Flow Cytometric NK Cell-Mediated Cytotoxicity Assay to Evaluate Anticancer Immune Responses In Vitro.

The immune system is able to detect and eliminate nascent and developing tumors. Thus, T lymphocytes of the adaptive immunity recognize cancer cells by detecting tumor-associated antigens, whereas certain innate immune cells scan for molecules that are mainly overexpressed on malignant and infected cells. The best example of the latter is natural killer (NK) cells. The activity of these immune cells is strictly governed by a balance between positive and negative signals provided by stress-regulated molecules that bind NK cell membrane receptors, such as the activatory receptor NKG2D. Given the key role of NK cells in eradicating cancer cells, the development of assays to study the cellular and molecular determinants of NK cell antitumor activity are of great relevance. In this chapter, we describe a flow cytometric in vitro assay to evaluate the cytotoxic activity of NK cells against a given target cell, including tumor cells. Moreover, this method is highly versatile, as it can be complemented with the use of antibodies blocking cell surface proteins (receptors or ligands) or a variety of chemical modulators, which allows the elucidation of molecules and signaling pathways that regulate NK cell anticancer function.

WNT Signaling in Cancer Immunosurveillance.

Deregulated WNT signaling has been shown to favor malignant transformation, tumor progression, and resistance to conventional cancer therapy in a variety of preclinical and clinical settings. Accumulating evidence suggests that aberrant WNT signaling may also subvert cancer immunosurveillance, hence promoting immunoevasion and resistance to multiple immunotherapeutics, including immune checkpoint blockers. Here, we discuss the molecular and cellular mechanisms through which WNT signaling influences cancer immunosurveillance and present potential therapeutic avenues to harness currently available WNT modulators for cancer immunotherapy.

Cancer-Induced Endoplasmic Reticulum Stress in T Cells Subverts Immunosurveillance.

Compelling evidence indicates that tumors may evade immunosurveillance by subverting the metabolism of immune cells. In a recent paper in Nature, Song et al. (2018) demonstrate that ovarian cancers activate IRE1α-XBP1 signaling within CD4+ T lymphocytes, thereby disrupting their bioenergetic function and contribution to anticancer immune responses.