Search

Immunotherapy utilizing checkpoint inhibitors has shown remarkable success in the treatment of cancers. In addition to immune checkpoint inhibitors, immune co-stimulation has the potential to enhance immune activation and destabilize the immunosuppressive tumor microenvironment. CD137, also known as 4-1BB, is one of the potent immune costimulatory receptors that could be targeted for effective immune co-stimulation. The interaction of the 4-1BB receptor with its natural ligand (4-1BBL) generates a strong costimulatory signal for T cell proliferation and survival. 4-1BBL lacks costimulatory activity in soluble form. To obtain co-stimulatory activity in soluble form, a recombinant 4-1BBL protein was generated by fusing the extracellular domains of murine 4-1BBL to a modified version of streptavidin (SA-4-1BBL). Treatment with SA-4-1BBL inhibited the development of lung tumors in A/J mice induced by weekly injections of the tobacco carcinogen NNK for eight weeks. The inhibition was dependent on the presence of T cells and NK cells; depletion of these cells diminished the SA-4-1BBL antitumor protective effect. The number of lung tumor nodules was significantly reduced by the administration of SA-4-1BBL to mice during ongoing exposure to NNK. The data presented in this paper suggest that utilizing an immune checkpoint stimulator as a single agent generate a protective immune response against lung cancer in the presence of a carcinogen. More broadly, this study suggests that immune checkpoint stimulation can be extended to a number of other cancer types, including breast and prostate cancers, for which improved diagnostics can detect disease at the preneoplastic stage. [ABSTRACT FROM AUTHOR]

This paper addresses the controversy of Granzyme B (GzmB) expression by murine Myeloid Derived Suppressor Cells (MDSCs). MDSCs are a heterogenous immature myeloid population that are generated in chronic inflammatory pathologies for the purpose to suppress inflammatory responses. MDSCs express a multitude of factors to induce suppressive function such as PD-L1, reactive oxygen species (ROS), nitric oxide synthase (iNOS), and Arginase-1. Recently, Dufait et al. sought to demonstrate GzmB as an additional mechanism for suppression by MDSCs. They reported that murine MDSCs not only significantly express GzmB as well as Perforin (Prf1), but this expression is functionally important for tumor growth in vivo as well as tumor migration in vitro. We conducted experiments to address the same question but made confounding observations: MDSCs under stringent developmental process do not express GzmB. Our results show that not only GzmB protein is not produced at functional level, but the mRNA transcript is not detectable either. In fact, the GzmB protein found in the media of MDSC culture was due to T cells or natural killer cells contained in bone marrow and cultured alongside MDSCs. We strengthen this finding by genetically deleting GzmB from the myeloid lineage and measuring tumor burden compared to WT counterpart. Our results show no significant difference in tumor burden, suggesting that even if there is minor expression of GzmB, it is not produced at a functional amount to affect tumor growth. Therefore, this paper proposes alternative theories that align with the known understanding of GzmB expression and secretion. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are key components of innate immunity and substantially contribute to anti-tumor immune responses. The role of NK cells in immune surveillance is linked to many aspects of NK cell biology, but the age of the animal being studied or the human under treatment is rarely taken into account. The solicited reviews constituting a collection of papers presented here as a 'Symposium-in-Writing' on the topic of NK cells, ageing and cancer were inspired by the increasing knowledge of NK cell biology and genetics, and emerging data on their impact in the clinic (disease associations and therapies), together with the realization that older individuals also differ from younger ones regarding innate as well as adaptive immunity. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are functionally suppressed in the glioblastoma multiforme (GBM) tumor microenvironment. We have recently shown that survival and differentiation of cancer stem-like cells (CSCs)/poorly differentiated tumors are controlled through two distinct phenotypes of cytotoxic and non-cytotoxic/split anergized NK cells, respectively. In this paper, we studied the function of NK cells against brain CSCs/poorly differentiated GBM and their NK cell-differentiated counterparts. Brain CSCs/poorly differentiated GBM, differentiated by split anergized NK supernatants (supernatants from NK cells treated with IL-2 + anti-CD16mAb) expressed higher levels of CD54, B7H1 and MHC-I and were killed less by the NK cells, whereas their CSCs/poorly differentiated counterparts were highly susceptible to NK cell lysis. Resistance to NK cells and differentiation of brain CSCs/poorly differentiated GBM by split anergized NK cells were mediated by interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Brain CSCs/poorly differentiated GBM expressed low levels of TNFRs and IFN-γRs, and when differentiated and cultured with IL-2-treated NK cells, they induced increased secretion of pro-inflammatory cytokine interleukin (IL)-6 and chemokine IL-8 in the presence of decreased IFN-γ secretion. NK-induced differentiation of brain CSCs/poorly differentiated GBM cells was independent of the function of IL-6 and/or IL-8. The inability of NK cells to lyse GBM tumors and the presence of a sustained release of pro-inflammatory cytokines IL-6 and chemokine IL-8 in the presence of a decreased IFN-γ secretion may lead to the inadequacy of NK cells to differentiate GBM CSCs/poorly differentiated tumors, thus failing to control tumor growth. [ABSTRACT FROM AUTHOR]

The human cutaneous metastatic melanoma is the deadliest skin cancer. Partial, or less frequently complete spontaneous regressions could be observed, mainly mediated by T cells. Nevertheless, the underlying mechanisms are not fully unraveled. We investigated the first events of the immune response related to cancer regression in Melanoma-bearing Libechov Minipigs (MeLiM), a unique swine model of cutaneous melanoma that regresses spontaneously. Using a multiparameter flow cytometry strategy and integrating new clinical and histological criteria of the regression, we show that T cells and B cells are present only in the late stages, arguing against their role in the initial destruction of malignant cells. NK cells infiltrate the tumors before T cells and therefore might be involved in the induction of the regression process. Myeloid cells represent the main immune population within the tumor microenvironment regardless of the regression stage. Among those, MHCII+ CD163− macrophages that differ phenotypically and functionally compared to other tumor-associated macrophages, increase in number together with the first signs of regression suggesting their crucial contribution to initiating the regression process. Our study supports the importance of macrophage reprogramming in humans to improve current immunotherapy for metastatic melanoma. [ABSTRACT FROM AUTHOR]

HLA-B alleles are associated with outcomes in various pathologies, including autoimmune diseases and malignancies. The encoded HLA-B proteins are pivotal in antigen presentation to cytotoxic T cells, and some variants containing a Bw4 motif also serve as ligands to the killer immunoglobulin-like receptors (KIR) 3DL1/S1 of NK cells. We investigated the potential impact of HLA-B genotypes on the efficacy of immunotherapy for relapse prevention in acute myeloid leukemia (AML). Seventy-eight non-transplanted AML patients receiving HDC/IL-2 in the post-consolidation phase were genotyped for HLA-B and KIR genes. HLA-B*44 heralded impaired LFS (leukemia-free survival) and overall survival (OS), but the negative association with outcome was not shared across alleles of the HLA-B44 supertype. Notably, HLA-B*44 is one of few HLA-B44 supertype alleles containing a Bw4 motif with a threonine at position 80, which typically results in weak binding to the inhibitory NK receptor, KIR3DL1. Accordingly, a strong interaction between KIR3DL1 and Bw4 was associated with superior LFS and OS (p = 0.014 and p = 0.027, respectively). KIR3DL1+ NK cells from 80 T-Bw4 donors showed significantly lower degranulation responses and cytokine responses than NK cells from 80I-Bw4 donors, suggesting impaired KIR3DL1-mediated education in 80 T-Bw4 subjects. We propose that presence of a strong KIR3DL1+–Bw4 interaction improves NK cell education and thus is advantageous in AML patients receiving HDC/IL-2 immunotherapy for relapse prevention. [ABSTRACT FROM AUTHOR]

Our knowledge of the mechanisms underlying tumor-specific immune response and tumor escape has considerably increased. HLA class I antigen defects remain an important tumor escape mechanism since they influence the interactions between tumor cells and specific T and NK cells in the course of malignant disease. We have studied here HLA class I expression in six subcutaneous metastases obtained from a melanoma patient immunized with an autologous melanoma cell vaccine (M-VAX). We report in this paper that HLA class I antigen expression on these metastatic lesions strongly correlated with the course of the disease. The three metastases that were partially regressing at the time of their excision showed a strong HLA class I expression, whereas the progressing ones showed a very weak or negative staining with most of the anti-HLA class I mAbs used. Real-time quantitative PCR of the samples obtained from microdissected tumor tissue revealed a significant difference in the mRNA levels of HLA-ABC heavy chain and β2m between the two types of metastases, i.e., lower levels in progressing metastases and high levels in regressing ones, confirming the immunohistological findings. This is, to our knowledge, the first report where the clinical outcome of different HLA class I positive and negative melanoma metastases can be clearly correlated with the regression and progression of the disease, respectively. [ABSTRACT FROM AUTHOR]

The aim of this study is to identify candidate factors which may be responsible for the functional inactivation and depletion of NK cells by tumor cells. Inhibition of NFκB activity by an IκB super-repressor in HEp2 cells, a cell line commonly used as an oral tumor model, blocked tumor-induced NK cell death, and increased the function of NK cells significantly. Increased expression of CD69 early activation antigen on NK cells as well as augmented proliferation and secretion of IFN-γ by NK cells were observed when these cells were co-incubated with IκB super-repressor transfected HEp2 cells (HEp2-IκB(S32AS36A)). More importantly, the secretion of IL-6 was significantly inhibited when NK cells were co-cultured with HEp2-IκB(S32AS36A) cells. In addition, the survival and function of cytotoxic effector cells remained significantly elevated in the presence of IFN-γ-treated HEp2-IκB(S32AS36A) cells when compared to either untreated or IFN-γ-treated vector-alone transfected HEp2 cells. Similar findings to those obtained using purified peripheral blood NK cells were also observed when non-fractionated peripheral blood mononuclear cells were used in the co-cultures of immune effectors with HEp2 cell transfectants. Addition of recombinant human IL-6 to the co-cultures of immune effectors with the NFκB knockdown HEp2 tumor cells substantially decreased the levels of secreted IFN-γ. Thus, the results presented in this paper suggest that the inhibition of NFκB function in oral tumors may serve to activate and expand the function and numbers of NK cells. Moreover, NFκB-mediated increase in IL-6 secretion by oral tumors may in part be responsible for the observed inactivation and death of the immune effectors. [ABSTRACT FROM AUTHOR]

Although the activation of innate immunity to treat a wide variety of cancers is gaining increasing attention, it has been poorly investigated in human papillomavirus (HPV)-associated malignancies. Because these tumors harbor a severely impaired cGAS-STING axis, but they still retain a largely functional RIG-I pathway, another critical mediator of adaptive and innate immune responses, we asked whether RIG-I activation by the 5'ppp-RNA RIG-I agonist M8 would represent a therapeutically viable option to treat HPV+ cancers. Here, we show that M8 transfection of two cervical carcinoma-derived cell lines, CaSki and HeLa, both expressing a functional RIG-I, triggers intrinsic apoptotic cell death, which is significantly reduced in RIG-I KO cells. We also demonstrate that M8 stimulation potentiates cisplatin-mediated cell killing of HPV+ cells in a RIG-I dependent manner. This combination treatment is equally effective in reducing tumor growth in a syngeneic pre-clinical mouse model of HPV16-driven cancer, where enhanced expression of lymphocyte-recruiting chemokines and cytokines correlated with an increased number of activated natural killer (NK) cells in the tumor microenvironment. Consistent with a role of RIG-I signaling in immunogenic cell killing, stimulation of NK cells with conditioned medium from M8-transfected CaSki boosted NK cell proliferation, activation, and migration in a RIG-I-dependent tumor cell-intrinsic manner. Given the highly conserved molecular mechanisms of carcinogenesis and genomic features of HPV-driven cancers and the remarkably improved prognosis for HPV+ oropharyngeal cancer, targeting RIG-I may represent an effective immunotherapeutic strategy in this setting, favoring the development of de-escalating strategies. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is an incurable hematological cancer, in which immune checkpoint inhibition (ICI) with monoclonal antibodies (mAbs) has failed due to uncontrollable immune responses in combination therapies and lack of efficacy in monotherapies. Although NK cell-specific checkpoint targets such as NKG2A and KIRs are currently being evaluated in clinical trials, the clinical impact of NK cells on the PD1 cascade is less well understood compared to T cells. Furthermore, while NK cells have effector activity within the TME, under continuous ligand exposure, NK cell dysfunctionality may occur due to interaction of PD1 and its ligand PD-L1. Due to above-mentioned factors, we designed novel NK cell specific PD1-based chimeric switch receptors (PD1-CSR) by employing signaling domains of DAP10, DAP12 and CD3ζ to revert NK cell inhibition and retarget ICI. PD1-CSR modified NK cells showed increased degranulation, cytokine secretion and cytotoxicity upon recognition of PD-L1+ target cells. Additionally, PD1-CSR+ NK cells infiltrated and killed tumor spheroids. While primary NK cells (pNK), expressing native PD1, showed decreased degranulation and cytokine production against PD-L1+ target cells by twofold, PD1-CSR+ pNK cells demonstrated increased activity upon PD-L1+ target cell recognition and enhanced antibody-dependent cellular cytotoxicity. PD1-CSR+ pNK cells from patients with MM increased degranulation and cytokine expression against autologous CD138+PD-L1+ malignant plasma cells. Taken together, the present results demonstrate that PD1-CSR+ NK cells enhance and sustain potent anti-tumor activity in a PD-L1+ microenvironment and thus represent a promising strategy to advance adoptive NK cell-based immunotherapies toward PD-L1+ cancers. [ABSTRACT FROM AUTHOR]

While natural killer (NK) cells are essential players in detection and elimination of malignant cells, these surveillance properties can be compromised by cancer cells. Since NK cell education primarily occurs in the bone marrow and lymphoid tissue, this process might be particularly affected by their infiltration with lymphoma cells. This study aimed to explore functional properties of diffuse large B-cell lymphoma (DLBCL) patient NK cells, which could potentially promote tumour immune evasion and disease propagation. NK cells isolated from the peripheral blood (PB) of 26 DLBCL patients and 13 age-matched healthy controls (HC) were analysed. The cytotoxic CD56dim subtype was the only one identified in patients. Compared to HC, patient cells demonstrated low levels of inhibitory CD158a/b along with decreased expression of activating NKG2D and CD161 and increased inhibitory NKG2A levels. Patient NK cell cytotoxic activity was impaired, as were their degranulation and inflammatory cytokine production, which partially recovered following non-receptor-dependant stimulation. The phenotypically skewed and restricted population of patient NK cells, along with their blunted cytotoxic and immune-regulatory activity, appear to be driven by exposure to lymphoma environment. These NK cell functional aberrations could support lymphoma immune evasion and should be considered in the era of cellular therapy. [ABSTRACT FROM AUTHOR]

NK cells, especially FDA-approved NK-92 cells, could be used for TCR engineering owing to their specialized cytotoxicity against tumors, safety profile and potential use as an off-the-shelf cellular therapy. The TCR complex requires assembly of TCR- α/ β chains with CD3 molecules (CD3δ, CD3γ, CD3ε, CD3ζ) to be correctly expressed at the cell membrane, and yet NK cells lack expression of these CD3 subunits besides CD3ζ. Since transmembrane regions of TCR α and β chains are involved in TCR complex assembly, transmembrane regions of TCR replaced by CD28 transmembrane domain could result in the expression of TCR independent of its companion CD3 subunits. However, since the absence of CD3 signaling components can influence the transmission of TCR signals to NK cells, it is necessary to add the signaling molecules of NK cells followed by CD28 transmembrane domain. Both CD3ζ and DAP10 play an important role in the activation and cytotoxicity of NK cells; moreover, 2B4 and 4-1BB are the main costimulatory molecules in NK cells. Therefore, we designed a chimeric TCR that consisted of the extracellular domains of the TCR α and β chains specific for NYESO-1 fused to the CD28 transmembrane domain followed by the 41BB and CD3ζ signaling domains as well as the 2B4 and DAP10 signaling domain, respectively. The chimeric TCR genetically engineered NK-92 cells exhibit antigen-specific recognition and lysis of tumor cells both in vitro and in vivo. In addition, TCR-28-2B10/BBζ can be feasibly expressed in primary NK cells and exhibit antigen-reactive recognition and effect function. The overall encouraging data highlight the value of NK-92 cells and primary NK cells engineered to express therapeutic chimeric TCR for adoptive immunotherapies. [ABSTRACT FROM AUTHOR]

We extended our previous observations with other tumor models to study seven ovarian tumor cell lines—OVCAR3, OVCAR4, OVCAR8, SKOV3, Kuramochi, OAW28, and CaOV3. We found that NK cells targeted and killed poorly differentiated OVCAR8 and CAOV3; these two tumor lines express lower MHC-class I and higher CD44 surface receptors. OVCAR3 and OVCAR4 were more resistant to NK cell-mediated cytotoxicity, and SKOV3, Kuramochi and OAW28 had intermediate sensitivity to NK cell-mediated cytotoxicity, likely representing well-differentiated and moderately differentiated ovarian tumor cell lines, respectively. Similar trends were observed for secretion of IFN-γ by the NK cells when co-cultured with different ovarian tumor cell lines. Treatment with both IFN-γ and TNF-α upregulated MHC-class I in all ovarian tumor cell lines and resulted in tumor resistance to NK cell-mediated cytotoxicity and decreased secretion of IFN-γ in co-cultures of NK cells with tumors cells with the exception of OVCAR8 and CAOV3 which did not upregulate MHC-class I and remained sensitive to NK cell-mediated cytotoxicity and increased secretion of IFN-γ when co-cultured with NK cells. Similarly, treatment with NK cell supernatants induced resistance to NK cell-mediated cytotoxicity in OVCAR4 but not in OVCAR8, and the resistance to killing was correlated with the increased surface expression of MHC-class I in OVCAR4 but not in OVCAR8. In addition, OVCAR4 was found to be carboplatin sensitive before and after treatment with IFN-γ and NK cell supernatants, whereas OVCAR8 remained carboplatin resistant with and without treatment with IFN-γ and NK cell supernatants. Overall, sensitivity to NK cell-mediated killing correlated with the levels of tumor differentiation and aggressiveness, and more importantly, poorly differentiated ovarian tumors were unable to upregulate MHC-class I under the activating conditions for MHC-class I, a feature that was not seen in other tumor models and may likely be specific to ovarian tumors. Such tumors may also pose a significant challenge in elimination by the T cells; however, NK cells are capable of targeting such tumors and can be exploited to eliminate these tumors in immunotherapeutic strategies. [ABSTRACT FROM AUTHOR]

The CD57 antigen (alternatively HNK-1, LEU-7, or L2) is routinely used to identify terminally differentiated 'senescent' cells with reduced proliferative capacity and altered functional properties. In this article, we review current understanding of the attributes of CD57-expressing T-cells and NK cells in both health and disease and discuss how this marker can inform researchers about their likely functions in human blood and tissues in vivo. While CD57 expression on human lymphocytes indicates an inability to proliferate, these cells also display high cytotoxic potential, and CD57 NK cells exhibit both memory-like features and potent effector functions. Accordingly, frequencies of CD57-expressing cells in blood and tissues have been correlated with clinical prognosis in chronic infections or various cancers and with human aging. Functional modulation of senescent CD57 T-cells and mature CD57 NK cells may therefore represent innovative strategies for protection against human immunological aging and/or various chronic diseases. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are considered crucial for the elimination of emerging tumor cells. Effector NK-cell functions are controlled by interactions of inhibitory and activating killer-cell immunoglobulin-like receptors (KIRs) on NK cells with human leukocyte antigen (HLA) class I ligands on target cells. KIR and HLA are highly polymorphic genetic systems segregating independently, creating a great diversity in KIR/HLA gene profiles in different individuals. There is an increasing evidence supporting the relevance of KIR and HLA ligand gene background for the occurrence and outcome of certain cancers. However, the data are still controversial and the mechanisms of receptor-ligand mediated NK-cell action remain unclear. Here, the main characteristics and functions of KIRs and their HLA class I ligands are reviewed. In addition, we review the HLA and KIR correlations with different hematological malignancies and discuss our current understanding of the biological significance and mechanisms underlying these associations. [ABSTRACT FROM AUTHOR]

Chordoma is a rare and aggressive bone tumor. An accurate investigation of tumor heterogeneity is necessary for the development of effective therapeutic strategies. This study aims to assess the poorly understood tumor heterogeneity of chordomas and identify potential therapeutic targets. Single-cell RNA sequencing was performed to delineate the transcriptomic landscape of chordomas. Six tumor samples of chordomas were obtained, and 33,737 cells passed the quality control test and were analyzed. The main cellular populations identified with specific markers were as follows: chordoma cells (16,052 [47.6%]), fibroblasts (6945 [20.6%]), mononuclear phagocytes (4734 [14.0%]), and T/natural killer (NK) cells (3944 [11.7%]). Downstream analysis of each cell type was performed. Six subclusters of chordomas exhibited properties of an epithelial-like extracellular matrix, stem cells, and immunosuppressive activity. Although few immune checkpoints were detected on cytotoxic immune cells such as T and NK cells, a strong immunosuppressive effect was exerted on the Tregs and M2 macrophages. In addition, the cellular interactions were indicative of enhancement of the TGF-β signaling pathway being the main mechanism for tumor progression, invasion, and immunosuppression. These findings, especially from the analysis of molecular targeted therapy and tumor immune microenvironment, may help in the identification of therapeutic targets in chordomas. [ABSTRACT FROM AUTHOR]

Despite recent advance in immunotherapy agents, safe new therapies that enhance the effects of immune checkpoint inhibitors are still required to develop. We previously demonstrated that hemagglutinating virus of Japan-envelope (HVJ-E) induced not only direct tumor cell death but also antitumor immunity through the activation of T and natural killer (NK) cells, thereafter, developed a manufacturing process of HVJ-E (GEN0101) for clinical use. We here performed a phase Ia clinical trial of intratumoral GEN0101 administration in six patients with stage IIIC or IV malignant melanoma. The primary aim was to evaluate the safety and tolerability of GEN0101, and the secondary aim was to examine the objective tumor response. Patients were separated into two groups (n = 3 each) and received a low dose of 30,000 and high dose of 60,000 mNAU of GEN0101. All patients completed a two-week follow-up evaluation without severe adverse events. The overall response rate was 33% (2 of 6), with 2 partial responses in the high-dose group and 2 with stable disease, and 2 with progressive disease in the low-dose group. Local complete or partial responses were observed in 11 of 18 (61%) target lesions. One patient demonstrated shrinkage of lung metastases after the treatment. The activity of NK cells and interferon-γ levels were increased in the circulation, indicating augmentation of antitumor immunity by GEN0101. This trial showed not only the safety and tolerability but also the significant antitumor effect of GEN0101, suggesting that GEN0101 might be a promising new drug for patients with advanced melanoma. [ABSTRACT FROM AUTHOR]

The tumor microenvironment (TME) has a central role in restraining tumor growth, and a deep understanding of the interplay between cancer cells and their local environment is relevant to designing immunotherapies that fit each tumor best. Today, treatment allocation of PDAC patient is currently based on clinical, biochemical, and radiologic features but, to reach a personalized treatment, it is mandatory to identify new tools to predict tumor response and develop a more effective patient treatment. Activation of tumor-specific CD8 T cells that mediate regression of the irradiated tumor as well as a non-irradiated synchronous tumor (abscopal effect) is seen only when radiation is combined with CTLA-4 blockade, whereas each treatment alone was ineffective, demonstrating a therapeutic synergy of radiation and CTLA4 blockade. Keywords: Immunotherapy; Cancer vaccines; Checkpoint blockade agents; Adoptive immunotherapy; Targeted therapies; NIBIT EN Immunotherapy Cancer vaccines Checkpoint blockade agents Adoptive immunotherapy Targeted therapies NIBIT 1777 1786 10 06/15/22 20220701 NES 220701 Introduction Oncology embrace Immunology: the way forward captioned the XVII international meeting of the Italian Network for Tumor Biotherapy (NIBIT) held in Verona on October 11-13, 2019. [Extracted from the article]

An important mechanism of oncolytic virotherapy in ameliorating cancer immunotherapy is by inducing significant changes in the immune landscape in the tumor microenvironment (TME). Despite this notion and the potential therapeutic implications, a comprehensive analysis of the immune changes in carcinomas induced by virotherapy has not yet been elucidated. We conducted single-cell RNA sequencing analysis on carcinomas treated with an HSV-2-based oncolytic virus to characterize the immunogenic changes in the TME. We specifically analyzed and compared the immune cell composition between viral treated and untreated tumors. We also applied CellChat to analyze the complex interactions among the infiltrated immune cells. Our data revealed significant infiltration of B cells in addition to other important immune cells, including CD4+, CD8+, and NK cells following virotherapy. Further analysis identified distinct subset compositions of the infiltrated immune cells and their activation status upon virotherapy. The intensive interactions among the infiltrated immune cells as revealed by CellChat analysis may further shape the immune landscape in favor of generating antitumor immunity. Our findings will facilitate the design of new strategies in incorporating immunotherapy into virotherapy for clinical translation. Moreover, the significant infiltration of B cells makes it suitable for combining virotherapy with immune checkpoint inhibitors. [ABSTRACT FROM AUTHOR]

The majority of the previous reports on NK cells use cross-sectional studies to establish the status of patient NK cell function, however such studies fail to evaluate the immune status of the patients on a continuous basis from the disease-free stage to progression of cancer. In this study, we performed a prospective study of the immune function by continuously monitoring the NK numbers, expansion and function of a pancreatic cancer patient from 1/6/2016 to 2/14/2019. The results indicated that at initial stages of the disease where no overt disease was identified, the patient had consistently higher percentages of NK and B cells and lower percentages of CD3 + T cells in the peripheral blood. The percentages of CD14 + monocytes were similar at the initial stages of the disease, and at the later stages of the disease, it increased and remained higher in the patient when compared to those from healthy donors. The numbers of expanded NK cells and the cytotoxic function, as well as secretion of IFN-γ from primary and osteoclast expanded patient NK cells remained consistently low throughout the years of follow up. Similarly, the majority of cytokines in patient's serum remained lower with the exception of IL-6 which was higher. The IFN-γ secreted from the patients' NK cells had much lower ability to differentiate the poorly differentiated oral tumors as assessed by their lack of ability to upregulate differentiation antigens. Overall, before any evidence of overt disease, patient NK cells exhibited significant dysfunction. Intervention at the stage of no disease or minimal disease may be important for the prevention of pancreatic cancer progression. [ABSTRACT FROM AUTHOR]

Background: Natural killer (NK) cell-based immunotherapy is a promising treatment approach for multiple myeloma (MM), but obtaining a sufficient number of activated NK cells remains challenging. Here, we report an improved method to generate ex vivo expanded NK (eNK) cells from MM patients based on genetic engineering of K562 cells to express OX40 ligand and membrane-bound (mb) IL-18 and IL-21. Methods: K562-OX40L-mbIL-18/-21 cells were generated by transducing K562-OX40L cells with a lentiviral vector encoding mbIL-18 and mbIL-21, and these were used as feeder cells to expand NK cells from peripheral blood mononuclear cells of healthy donors (HDs) and MM patients in the presence of IL-2/IL-15. Purity, expansion rate, receptor expression, and functions of eNK cells were determined over four weeks of culture. Results: NK cell expansion was enhanced by short exposure of soluble IL-18 and IL-21 with K562-OX40L cells. Co-culture of NK cells with K562-OX40L-mbIL-18/-21 cells resulted in remarkable expansion of NK cells from HDs (9,860-fold) and MM patients (4,929-fold) over the 28-day culture period. Moreover, eNK cells showed increased expression of major activation markers and enhanced cytotoxicity towards target K562, U266, and RPMI8226 cells. Conclusions: Our data suggest that genetically engineered K562 cells expressing OX40L, mbIL-18, and mbIL-21 improve the expansion of NK cells, increase activation signals, and enhance their cytolytic activity towards MM cells. [ABSTRACT FROM AUTHOR]

AML is the most common blood cancer in adults with a high relapse and an overall poor survival rate. NK cells have been demonstrated to have the capacity to eradicate AML blast, and an impaired NK cell function is involved in AML development and progression. Immune checkpoints are involved in immune escape in various cancers. Immune checkpoints blockade therapy mainly aimed to unleash CD8+T cells function, but NK cells have emerged as new target. However, immune checkpoints profile on NK cells has not been observed in AML patients. Here, we studied the immune checkpoints expression of NK cells from AML patients at initial diagnosis and found increased PD-1, TIGIT and TIM-3 expression compared to NK cells from healthy donors. Further analysis showed that TIGIT expressing NK cells from AML patients had a dysfunctional phenotype, as TIGIT+NK cells exhibit lower antileukemia effect, cytokine production and degranulation compared to TIGIT−NK cells. TIGIT blockade could significantly enhance the function of NK cells. Moreover, AML patients with high frequency of TIGIT+NK cells had higher frequency of poor prognosis risk. Further analysis found that IL-10 upregulated TIGIT expression on NK cells. Thus, TIGIT blockade alone or in combination with other therapy might be potential strategy to treat AML. [ABSTRACT FROM AUTHOR]

Similar to pediatric acute myeloid leukemia (AML) the subgroup of biphenotypic acute lymphoblastic leukemia (ALL) is a rare complex entity with adverse outcome, characterized by the surface expression of CD33. Despite novel and promising anti-CD19 targeted immunotherapies such as chimeric antigen receptor T cells and bispecific anti-CD19/CD3 antibodies, relapse and resistance remain a major challenge in about 30% to 60% of patients. To investigate the potential role of the fully humanized bispecific antibody CD16 × CD33 (BiKE) in children with CD33+ acute leukemia, we tested whether the reagent was able to boost NK cell effector functions against CD33+ AML and biphenotypic ALL blasts. Stimulation of primary NK cells from healthy volunteers with 16 × 33 BiKE led to increased cytotoxicity, degranulation and cytokine production against CD33+ cell lines. Moreover, BiKE treatment significantly increased degranulation, IFN-γ and TNF-α production against primary ALL and AML targets. Importantly, also NK cells from leukemic patients profited from restoration of effector functions by BiKE treatment, albeit to a lesser extent than NK cells from healthy donors. In particular, those patients with low perforin and granzyme expression showed compromised cytotoxic function even in the presence of BiKE. In patients with intrinsic NK cell deficiency, combination therapy of CD16xCD33 BiKE and allogeneic NK cells might thus be a promising therapeutic approach. Taken together, CD16xCD33 BiKE successfully increased NK cell effector functions against pediatric AML and biphenotypic ALL blasts and constitutes a promising new option for supporting maintenance therapy or "bridging" consolidation chemotherapy before hematopoietic stem cell transplantation. [ABSTRACT FROM AUTHOR]

Objective: Chemoradiotherapy (CRT) is the standard treatment for limited-stage small cell lung cancer (LS-SCLC), which can exert anti-tumor effects by regulating immune cells. Different immune cell subsets are associated with a specific sensitivity to CRT. The purpose of this study was to characterize the proportion or composition of peripheral lymphocytes in patients with LS-SCLC before and after CRT, and evaluate their prognostic value. Methods: A total of 98 patients with LS-SCLC were enrolled. The expression of CD3, CD4, CD8, CD45RA, CD45RO, CD38, CD56, and CD19 on the surface of peripheral blood cells was detected by flow cytometry and retrospectively analyzed. The relationship between the proportion of lymphocyte subsets, progression-free survival (PFS), and overall survival (OS) was evaluated using a log-rank test and Cox regression model. Results: The median PFS was 12.3 months and the median OS was 21.7 months. Compared with the pre-treatment specimens, post-treatment lymphocytes had increased proportions of CD3+, CD3+CD8+, CD8+CD38+ T cells, and NKT cells, and a decreased proportion of CD3+CD4+ T cells, CD4+CD45RA+ T cells, B cells, NK cells, and CD4/CD8 ratio. Univariate and multivariate analyses showed that prophylactic cranial irradiation, high percentages of CD4+CD45RA+, CD8+CD38+ T cells after CRT independently predicted superior PFS. Male patients with a high baseline CD4+CD45RO+ T cell ratio predicted a poor OS. Conclusions: CRT induced changes in the proportion of circulating lymphocyte subsets in LS-SCLC, which is helpful for designing a regimen of immune drugs to be combined with CRT. The prognostic value of the proportion of lymphocytes aids in understanding the role of peripheral immune profiles in LS-SCLC. [ABSTRACT FROM AUTHOR]

Competent type I IFN signaling is the lynchpin of most immune surveillance mechanisms and has recently proven critical to the efficacy of several anticancer agents. Expression of the type I IFN receptor, IFNAR, underpins type I IFN responsiveness in all cells and facilitates the activation and cytotoxic potential of lymphocytes, while loss of IFNAR on lymphocytes has previously been associated with tumor progression and poor patient survival. This study underscores the importance of intact type I IFN signaling to NK cells in the regulation of tumorigenesis and metastasis, whereby ablation of NK cell IFNAR1 impairs antitumor activity and tumor clearance. Using a preclinical model of triple negative breast cancer, we identified that intact IFNAR on NK cells is required for an effective response to type I IFN-inducing immunotherapeutics that may be mediated by pathways associated with NK cell degranulation. Taken together, these data provide a rationale for considering the IFNAR status on NK cells when devising therapeutic strategies aimed at inducing systemic type I IFN signaling in breast cancer. [ABSTRACT FROM AUTHOR]

Introduction: Endometriosis is a risk factor for low-grade serous, clear cell, and endometroid ovarian carcinoma. In both endometriosis and ovarian carcinoma, immunological factors are associated with clinical outcome. Chronic inflammation in endometriosis may be linked to tumorigenesis, but exact processes contributing to endometriosis-associated ovarian carcinoma remain unknown. This review aims to describe potential immunological factors involved in the malignant transformation of endometriosis into ovarian carcinoma. Methods: PubMed and Embase were searched from inception up to October 2020 for studies comparing immunological processes in endometriosis and endometriosis-associated ovarian carcinoma. Results: Detailed analysis of immune components in the malignant transformation of endometriosis into endometriosis-associated ovarian carcinoma is lacking. Altered levels of chemokines and cytokines as IL-6, IL-8, IL-10, and TNF-α are reported and the function, number and polarization of NK cells, dendritic cells, and monocytes differ between endometriosis and associated ovarian carcinoma compared to healthy tissue. In addition, altered inflammasome and complement systems, indicate a role for the immune system in the carcinogenesis of endometriosis. Conclusion: Chronic inflammation in endometriosis may potentially drive inflammation-induced carcinogenesis in endometriosis-associated ovarian carcinoma. Exact immunological pathways and cellular processes remain unknown and require more thorough investigation. [ABSTRACT FROM AUTHOR]

Purpose: Natural Killer (NK) cells are a vital part of immune surveillance and have been implicated in colorectal cancer development and prognosis. This systematic review aims to distil the literature on NK cells as it relates to colorectal cancer. Methods: All published studies over 10 years relating to NK cells and colorectal cancer were reviewed. All studies publishing in English, searchable via pubmed or through reference review and reporting directly on the nature or function of NK cells in colorectal cancer patients were included. Outcomes were determined as alterations or new information regarding NK cells in colorectal cancer patients. Results: Natural killer cells may be implicated in the development of colorectal cancer and may play a role in prognostication of the disease. NK cells are altered by the treatment (both surgical and medical) of colorectal cancer and it seems likely that they will also be a target for manipulation to improve colorectal cancer survival. Conclusions: NK cell morphology and function are significantly affected by the development of colorectal cancer. Observation of NK cell changes may lead to earlier detection and better prognostication in colorectal cancer. Further study is needed into immunological manipulation of NK cells which may lead to improved colorectal cancer survival. [ABSTRACT FROM AUTHOR]

Objective: The subset distribution and immunophenotype of circulating immune cells ("peripheral blood immune cell profile") may reflect tumor development and response to cancer treatment. In order to use the peripheral blood immune cell profile as biomarker to monitor patients over time, it is crucial to know how immune cell subsets respond to therapeutic interventions. In this study, we investigated the effects of tumor resection and adjuvant therapy on the peripheral blood immune cell profile in patients with colon carcinoma (CC). Methods: The subset distribution and immunophenotype of T cells (CD3+CD56−), CD56dim NK cells (CD3−CD56dim), CD56bright NK cells (CD3−CD56bright) and NKT-like cells (CD3+CD56+) were studied in preoperative and postoperative peripheral blood mononuclear cell (PBMC) samples of 24 patients with CC by multiparameter flow cytometry. Changes in immunophenotype of circulating immune cells after tumor resection were studied in patients treated with and without (capecitabine-based) adjuvant therapy. Results: The NKT-like cell (% of total PBMCs) and CD8+ T cell (% of total T cells) populations expanded in the peripheral blood of non-adjuvant-treated CC patients after surgery. NK- and NKT-like cells showed upregulation of activating receptors and downregulation of inhibitory receptors in non-adjuvant-treated CC patients after surgery. These changes were not observed in the peripheral blood of adjuvant-treated CC patients. Conclusions: Our results suggest tumor-induced suppression of NK- and NKT-like cells in CC patients, an effect that could not be detected after tumor resection. In contrast, adjuvant therapy maintained tumor-induced immunosuppression of NK- and NKT-like cells in CC patients. [ABSTRACT FROM AUTHOR]

Following excision of colorectal tumors, metastatic disease is prevalent, primarily occurs in the liver, and is highly predictive of poor prognosis. The perioperative period is now recognized as critical in determining the incidence of postoperative metastases and long-term cancer outcomes. Thus, various perioperative prophylactic interventions are currently studied during this time frame. However, immune stimulation during the perioperative period has rarely been attempted due to specific contraindications to surgery and various adverse effects. Here, to prevent liver metastases, we perioperatively employed a TLR-9 agonist, CpG-C, which exhibits minimal pyrogenic and other adverse effects in patients. We found that marginating-hepatic (MH) cells in BALB/c mice contained high percentage of NK cells, but exhibited negligible NK cytotoxicity, as previously reported in humans. However, a single CpG-C administration (25-100 µg/mouse) doubled MH-NK cell numbers, increased NK cell activation and maturation markers (NKp46, CD11b), decreased the inhibitory NKG2A ligand, and dramatically increased MH-NK-cell cytotoxicity against the syngeneic CT26 colon cancer line. Moreover, in operated mice, this innocuous intervention also markedly improved resistance to CT26 and MC38 hepatic metastases in BALB/c and C57BL/6 mice, respectively. Beneficial effects of CpG-C were mediated through activation of MH-NK cells, as indicated by an in vivo NK depletion study. Last, CpG-C protected against surgery-induced suppression of MH-NK cytotoxicity and improved their activation indices. Thus, we suggest that systemic perioperative CpG-C treatment should be considered and studied as a novel therapeutic approach to improve long-term cancer outcomes in colorectal cancer patients. [ABSTRACT FROM AUTHOR]

Hemagglutinating virus of Japan (HVJ; Sendai virus) is an RNA virus that has cell fusion activity. HVJ-envelope (HVJ-E) is a UV-irradiated HVJ particle that loses viral replication and protein synthesis activity but retains cell fusion activity. We recently reported that HVJ-E has antitumor effects on several types of tumors. Here, we describe the results of a first-in-human phase I/IIa study in patients with advanced melanoma, receiving intratumoral administration of HVJ-E. The primary aim was to evaluate the safety and tolerability of HVJ-E, and the secondary aim was to examine the objective tumor response and antitumor immunity. Six patients with stage IIIC or IV progressive malignant melanoma with skin or lymph metastasis were enrolled. Patients were separated into two groups (n = 3 each) and received low and high doses of HVJ-E. Five of the six patients completed 4 weeks of follow-up evaluation; one patient discontinued treatment owing to progressive disease. Complete or partial responses were observed in 3 of 6 (50%) injected target lesions, 7 of 15 (47%) noninjected target lesions, and 10 of 21 (48%) target lesions. Induction of antitumor immunity was observed: activation of natural killer cells, a marked increase in interferon-γ levels in the peripheral blood, and infiltration of cytotoxic T cells into both injected and noninjected tumor lesions. Thus, intratumoral injection of HVJ-E in advanced melanoma patients showed safety and tolerability with local regression of the tumor mediated by antitumor immunity. The results suggest that HVJ-E might be a new treatment approach in patients with advanced melanoma. [ABSTRACT FROM AUTHOR]

Obinutuzumab is a glycoengineered tumor-targeting anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for the FcγRIIIA/CD16 receptor, which was recently approved for clinical use in CLL and follicular lymphoma. Here we extend our previous observation that, in human NK cells, the sustained CD16 ligation by obinutuzumab-opsonized targets leads to a markedly enhanced IFN-γ production upon a subsequent cytokine re-stimulation. The increased IFN-γ competence in response to IL-2 or IL-15 is attributable to post-transcriptional regulation, as it does not correlate with the upregulation of IFN-γ mRNA levels. Different from the reference molecule rituximab, we observe that the stimulation with obinutuzumab promotes the upregulation of microRNA (miR)-155 expression. A similar trend was also observed in NK cells from untreated CLL patients stimulated with obinutuzumab-opsonized autologous leukemia. miR-155 upregulation associates with reduced levels of SHIP-1 inositol phosphatase, which acts in constraining PI3K-dependent signals, by virtue of its ability to mediate phosphatidylinositol 3,4,5-trisphosphate (PIP3) de-phosphorylation. Downstream of PI3K, the phosphorylation status of mammalian target of rapamycin (mTOR) effector molecule, S6, results in amplified response to IL-2 or IL-15 stimulation in obinutuzumab-experienced cells. Importantly, NK cell treatment with the PI3K or mTOR inhibitors, idelalisib and rapamycin, respectively, prevents the enhanced cytokine responsiveness, thus, highlighting the relevance of the PI3K/mTOR axis in CD16-dependent priming. The enhanced IFN-γ competence may be envisaged to potentiate the immunoregulatory role of NK cells in a therapeutic setting. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of the CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (MoAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells that mediate in vivo tumour immunosurveillance. NK cells also play an important role during MoAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their FcγRIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring variant of CD16 harbouring a single-point polymorphism (F158V). However, the contribution of NK cells to the efficacy of daratumumab remains debatable as clinical data clearly indicate the rapid depletion of CD38high peripheral blood NK cells in patients upon daratumumab administration. In contrast, CD38low peripheral blood NK cells have been shown to survive daratumumab mediated fratricide in vivo, while still retaining their potent anti-MM cytolytic effector functions ex vivo. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach on CD38low NK cells in combination with daratumumab could represent a novel therapeutic option for treatment of MM. In the present study, we investigate a NK cell line (KHYG-1), derived from a patient with aggressive NK cell leukemia, as a platform for generating CD38low NK cells expressing CD16F158V which can be administered as an "off-the-shelf" therapy to target both CD38high and CD38low tumour clones in patients receiving daratumumab. [ABSTRACT FROM AUTHOR]

Cathepsins are lysosomal peptidases involved in intracellular protein catabolism as well as in various other physiological and pathological processes. Several members of the family, most notably cathepsins B, S, K and L, are frequently overexpressed in cancer and have been associated with remodeling of the proteins of the extracellular matrix, a process leading to tumor cell migration, invasion and metastasis. In addition, lysosomal cathepsins play a role in innate and adaptive immunity, regulation of antigen presentation, Toll-like receptor signaling, cytokine secretion, apoptosis, autophagy, differentiation, migration and cytotoxicity. In cancer, the cells of innate immunity, such as myeloid cells, are often subverted to the regulatory immunosuppressive phenotype. Most studies indicate that lysosomal cathepsins reinforce the pro-tumoral activity of myeloid-derived suppressor cells and tumor-associated macrophages as well as of neutrophils. On the other hand, in cytotoxic natural killer cells, tumor cells suppress lysosomal peptidases in their activation of perforin and granzymes, thus diminishing their killing ability. With multifaceted actions, lysosomal peptidases constitute an important regulatory mechanism for fine-tuning the anti-tumor immune response. [ABSTRACT FROM AUTHOR]

Chemotherapy is still the backbone of systemic treatment in the majority of cancers. However, immunotherapies, especially those based on checkpoint inhibition, are additional therapy options for many. For this, functional T cells are a mandatory requirement. The aim of this prospective study was to investigate the influence of chemotherapy on the cellular immune status of individual patients. Peripheral blood samples of 26 patients with solid malignancies undergoing chemotherapy were analyzed for lymphocyte populations and their subsets in a longitudinal approach. Chemotherapy decreased total B lymphocyte counts [median value (25–75 percentile): before chemotherapy 76/µl (39–160) vs. after chemotherapy 49/µl (24–106); p = 0.001]. Among B cells, specific subsets decreased particularly [naïve B cells (49/µl (21–111) vs. 25/µl (13–56); p = 0.001], memory B cells [3/µl (2–8) vs. 2/µl (1–4); p = 0.001], and class-switched B cells [11/µl (6–20) vs. 6/µl (3–12); p = 0.011]. In contrast, chemotherapy had no influence on the total numbers of CD4 + and CD8 + T lymphocytes or on their subsets (T helper cells 1, 2, and 17 as well as cytotoxic T cells in early, intermediate, late, terminal effector and exhausted status as well as both T-cell types with naïve, center memory, effector memory, activated, or regulatory phenotype). Furthermore, the count of natural killer (NK) lymphocytes showed no significant change before and after chemotherapy. In summary, this study shows a decrease of B lymphocytes during systemic chemotherapy, but no relevant effect on T lymphocytes, NK lymphocytes and their subsets. This could support the idea of an effective additive T-cell-dependent immunotherapy to chemotherapy. [ABSTRACT FROM AUTHOR]

Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are innate immune lymphocytes which express an array of activating and inhibitory receptors. These receptors bind a large spectrum of ligands, which are expressed on stressed, malignantly transformed or virally infected cells, as well as on bacterial, fungal, and parasitic pathogens. The decision on whether or not to kill the target is based on the integration of activating and inhibitory signals sent downstream from NK cell receptors. One of the most prominent NK cell activating receptor families is the family of natural cytotoxicity receptors (NCRs) which includes NKp30, NKp44, and NKp46. NKp46 is the only NCR to have a fully functional mouse orthologue denoted Ncr1. Despite a large body of evidence highlighting its importance in the clearance of both solid and liquid tumors, the membrane-bound tumor ligand for NKp46 and its mouse orthologue Ncr1 is still unknown. Here we review the discovery of a novel role for NKp46/Ncr1, not only in tumor clearance but also in prevention of metastasis by structural editing of primary tumors. [ABSTRACT FROM AUTHOR]

The incidence of some types of tumours has increased progressively in recent years and is expected to continue growing in the coming years due in part to the aging of the population. The design of new therapies based on natural killer (NK) cells opens new possibilities especially for the treatment of elderly patients who are particularly susceptible to the toxicity of conventional chemotherapy treatments. In recent years, the potential use of NK cells in cancer immunotherapy has been of great interest thanks to advances in the study of NK cell biology. The identification of key points (checkpoints) in the activation of NK cells that can be regulated by monoclonal antibodies has allowed the design of new therapeutic strategies based on NK cells. However, there are still limitations for its use and the first clinical trials blocking KIR inhibitory receptors have shown little efficacy by inhibiting the maturation of NK cells. Blockade of other inhibitory receptors such as TIGIT, TIM3, LAG3 and PD1 may represent novel strategies to increase NK function in cancer patients. Altogether, the identification of NK cell and tumour cell markers of resistance or susceptibility to the action of NK cells will contribute to identifying those patients that will most likely benefit from NK cell-based immunotherapy. [ABSTRACT FROM AUTHOR]

Tumor-infiltrating lymphocytes are an important prognostic factor after neoadjuvant chemotherapy (NAC) in patients with breast cancer. Natural killer (NK) cells play critical roles in antitumor immune surveillance. Here, we assessed the relationship between peripheral natural killer (pNK) cell activity, tumor microenvironmental factors (TMEFs), and the therapeutic efficacy of preoperative chemotherapy in patients with breast cancer. In a cohort of 39 patients diagnosed with stage II–IV breast cancer who received NAC, we measured pNK cell activity by chromium release assay and assessed TMEF levels by next-generation sequencing. Following NAC, pNK cell activity was increased in 24/39 patients but decreased in 15/39 patients. Increased pNK cell activity following preoperative chemotherapy was associated significantly with the disappearance of axillary lymph node metastasis (Ax+; p = 0.0235). Increased pNK cell activity remained significantly associated with the disappearance of Ax+ in multivariate logistic regression analysis (OR 5.41, 95% CI 1.19–24.52, p = 0.0283). A Grade 2 or higher effect of NAC was associated with high pre-NAC cytotoxic T lymphocyte-associated protein 4 (CTLA-4) levels (p = 0.0281) and elevated post-NAC NK (p = 0.0005) cells and transforming growth factor-beta (TGF-β; p = 0.0350) levels. The disappearance of Ax+ was associated with high pre-NAC CTLA-4 levels (p = 0.0278) and elevated CD4 levels after NAC (p = 0.0250). The systemic activation of pNK cells after NAC may improve metastatic tumor elimination in patients with breast cancer owing to a release from local immunosuppression, and immune activation in the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Vitamin D3 (25-OH-D3) deficiency impairs rituximab-dependent cellular cytotoxicity and the outcome of patients with diffuse large B-cell and follicular lymphomas (DLBCL). Since the optimum 25-OH-D3 serum levels for NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) are unknown, we determined the 25-OH-D3 serum levels associated with maximum NK cell-mediated ADCC. CD20 antibody-loaded CD20+ B-cell lymphoma cell lines were cultured with NK cells and ADCC activity was determined by lactate dehydrogenase release assays. Using a newly developed formula, pre-defined 25-OH-D3 serum levels were achieved with high individual precision over a wide range. NK cells from 20 healthy individuals killed antibody-treated CD20+ lymphoma cells in a concentration- and E:T ratio-dependent manner with obinutuzumab displaying a stronger ADCC activity than rituximab. Maximum NK-cell activity and ADCC were observed at 65 ng/ml 25-OH-D3, the middle of the normal range (30-100 ng/ml). 25-OH-D3 serum levels around this range should be the target in interventional trials aiming at improving NK cell-mediated ADCC by 25-OH-D3 substitution. Lower levels do not provide significant ADCC improvements in individuals with 25-OH-D3 deficiency or insufficiency and might result in the failure of interventions with 25-OH-D3. [ABSTRACT FROM AUTHOR]

The monocyte network is important for therapeutic efficacy of antibody therapies against cancer. One mechanism which monocytes/macrophages use to kill cancer cells is phagocytosis. Using trastuzumab and human breast cancer cell lines as a model, we used flow cytometry to evaluate the importance of avidity, antigen density, Fcγ receptor (FcγR) expression, and FcγR polymorphisms in human monocyte phagocytosis. By increasing avidity for the tumor through the addition of pertuzumab to trastuzumab, there was a two-to-threefold increase in phagocytosis potency against the HCC1419 cell line compared to antibodies alone, while NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) failed to increase tumor cell death. Consistent with increasing the avidity through multiple antibodies, antigen density significantly enhanced phagocytosis with breast cancer cell lines that were HER2 gene-amplified compared to non-amplified tumor cells. Confirmation that high antigen density enhanced phagocytosis was obtained when HER2 was overexpressed in HER2 non-amplified cell lines. In contrast, NK cell ADCC failed to distinguish differences in tumor cell death when comparing gene-amplified and non-amplified breast cancer cell lines. The level of phagocytosis was influenced by FcγRIIa and FcγRIIIa expression. Most monocytes are FcγRIIIa−, and the induction of the receptor significantly enhances antibody-dependent phagocytosis. Although both receptors are involved, when blocked FcγRIIIa had a greater influence on phagocytosis. Furthermore, the polymorphism FcγRIIIa 158V significantly enhanced phagocytosis; whereas FcγRIIa 131H polymorphism appeared to improve phagocytosis but was not statistically significant. Targeting of monocytes for enhanced phagocytosis may improve the effectiveness of therapeutic antibodies to improve clinical outcomes. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cell-based immunotherapy is a promising novel approach to treat cancer. However, NK cell function has been shown to be potentially diminished by factors common in the tumor microenvironment (TME). In this study, we assessed the synergistic potential of antibody-dependent cell-mediated cytotoxicity (ADCC) and killer immunoglobin-like receptor (KIR)-ligand mismatched NK cells to potentiate NK cell antitumor reactivity in multiple myeloma (MM). Hypoxia, lactate, prostaglandin E2 (PGE2) or combinations were selected to mimic the TME. To investigate this, NK cells from healthy donors were isolated and NK cell ADCC capacity in response to MM cells was assessed in flow cytometry-based cytotoxicity and degranulation (CD107a) assays in the presence of TME factors. Hypoxia, lactate and PGE2 reduced cytotoxicity of NK cells against myeloma target cells. The addition of daratumumab (anti-CD38 antibody) augmented NK-cell cytotoxicity against target cells expressing high CD38, but not against CD38 low or negative target cells also in the presence of TME. Co-staining for inhibitory KIRs and NKG2A demonstrated that daratumumab enhanced degranulation of all NK cell subsets. Nevertheless, KIR-ligand mismatched NK cells were slightly better effector cells than KIR-ligand matched NK cells. In summary, our study shows that combination therapy using strategies to maximize activating NK cell signaling by triggering ADCC in combination with an approach to minimize inhibitory signaling through a selection of KIR-ligand mismatched donors, can help to overcome the NK-suppressive TME. This can serve as a platform to improve the clinical efficacy of NK cells. [ABSTRACT FROM AUTHOR]

Acute lymphoblastic leukemia (ALL) is believed to be resistant to NK cell-mediated killing. To overcome this resistance, we developed an innovative approach based on NK cell stimulation with Toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDC). The translation of this approach into the clinic requires the production of high numbers of human pDC. Herein, we show that in vitro differentiation of cord blood CD34 progenitors in the presence of aryl hydrocarbon receptor antagonists gives rise to clinically relevant numbers of pDC, as about 10 pDC can be produced from a typical cord blood unit. Blocking the aryl hydrocarbon receptor (AHR) pathway significantly increased the yield of pDC. When compared to pDC isolated from peripheral blood, in vitro differentiated pDC (ivD-pDC) exhibited an increased capacity to induce NK cell-mediated killing of ALL. Although ivD-pDC produced lower amounts of IFN-α than peripheral blood pDC upon TLR activation, they produced more IFN-λ2, known to play a critical role in the induction of anti-tumoral NK cell functions. Both TLR-9 and TLR-7 ligands triggered pDC-induced NK cell activation, offering the possibility to use any clinical-grade TLR-7 or TLR-9 ligands in future clinical trials. Finally, adoptive transfer of ivD-pDC cultured in the presence of an AHR antagonist cured humanized mice with minimal ALL disease. Collectively, our results pave the way to clinical-grade production of sufficient numbers of human pDC for innate immunotherapy against ALL and other refractory malignancies. [ABSTRACT FROM AUTHOR]

Cancer is primarily considered a disease of old age. Immunosenescence refers to the age-associated changes in the immune system, and its contribution to the increased risk of cancer in old individuals has been discussed for many years. Natural killer (NK) cells are cytotoxic innate immune cells specialized in defence against tumour and virus-infected cells. NK cell cytotoxicity is the result of a fine balance between activating and inhibitory receptors. Several activating receptors have been identified that recognize different ligands frequently found over-expressed on tumour cells or virus-infected cells. The most important NK cell inhibitory receptors interact with major histocompatibility complex class I molecules expressed on almost all nucleated cells preventing NK cell-mediated lysis of healthy cells. NK cell immunosenescence is characterized by a redistribution of NK cell subsets, a diminished expression of several activating receptors and lower per-cell cytotoxicity. Altered expression of activating receptors has also been described in young and elderly cancer patients probably due to chronic exposure to ligands on tumour cells. Thus, the effect of both age and cancer may act synergistically to diminish NK cell-mediated tumour immunosurveillance. Different strategies harnessing the power of NK cells to target tumour cells have been designed including adoptive therapy with autologous or allogeneic expanded NK cells. In addition, checkpoint blockade of inhibitory receptors and the use of agonist antibodies to stimulate activating receptors are emerging areas of research. In this context, the effect of immunosenescence should be considered to improve the efficiency of cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Down-regulation of HLA in tumor cells, low numbers and dysfunctionality of NK cells are commonly observed in patients with end-stage cervical cancer. Adoptive transfer of high numbers of cytotoxic NK cells might be a promising treatment approach in this setting. Here, we explored the cytotoxic efficacy on ten cervical cancer cell lines of activated allogeneic NK cells from two sources, i.e., peripheral blood (PBNK) with and without cetuximab (CET), a tumor-specific monoclonal antibody directed against EGFR, or derived from umbilical cord blood (UCB-NK). Whereas CET monotherapy was ineffective against the panel of cervical cancer cell lines, irrespective of their EGFR expression levels and despite their RAS status, it significantly enhanced the in vitro cytotoxic efficacy of activated PBNK ( P = 0.002). Equally superior cytotoxicity over activated PBNK alone was achieved by UCB-NK ( P < 0.001). Both PBNK- and UCB-NK-mediated cytotoxic activity was dependent on the NK-activating receptors natural killer group 2, member D receptor (NKG2D) and DNAX accessory molecule-1 (DNAM-1) ( P < 0.05) and unrelated to expression levels of the inhibitory receptors HLA-E and/or HLA-G. Most strikingly, whereas the PBNK's cytotoxic activity was inversely correlated with HLA-ABC levels ( P = 0.036), PBNK + CET and UCB-NK cytotoxicity were entirely independent of HLA-ABC expression. In conclusion, this study provides a rationale to initiate a clinical trial for cervical cancer with adoptively transferred allogeneic NK cells, employing either UCB-NK or PBNK + CET for EGFR-expressing tumors. Adoptive transfer of UCB-NK might serve as a generally applicable treatment for cervical cancer, enabled by HLA-, histology- and HPV-independent killing mechanisms. [ABSTRACT FROM AUTHOR]

The success of checkpoint inhibitors has validated immunomodulatory agents as a valuable class of anticancer therapeutics. A promising co-stimulatory immunologic target is 4-1BB, or CD137, a member of the tumor necrosis factor receptor superfamily. Ligation of 4-1BB induces an activating signal in CD8 T cells and natural killer cells, resulting in increased pro-inflammatory cytokine secretion, cytolytic function, and antibody-dependent cell-mediated cytotoxicity. Targeting 4-1BB with agonistic monoclonal antibody (mAb) therapy demonstrated potent antitumor effects in murine tumor models. While anti-4-1BB mAbs have entered clinical trials, optimal efficacy of 4-1BB-targeted agents will inevitably come from combination therapeutic strategies. Checkpoint blockade is a compelling combination partner for 4-1BB agonism. This novel immunotherapeutic approach has the potential to active antitumor immune effectors by a complementary mechanism: simultaneously 'removing the brakes' via blocking inhibitory signaling and 'stepping on the accelerator' via co-stimulation. While important considerations should be given to 4-1BB-mediated toxicities, the current understanding of 4-1BB biology suggests it may play a key role in advancing the capabilities of cancer combination therapy. [ABSTRACT FROM AUTHOR]

Based on data obtained from oral, pancreatic and lung cancers, glioblastoma, and melanoma, we have established that natural killer (NK) cells target cancer stem-like cells (CSCs). CSCs displaying low MHC class I, CD54, and PD-L1 are killed by cytotoxic NK cells and are differentiated by split anergized NK cells through both membrane bound and secreted forms of TNF-α and IFN-γ. NK cells select and differentiate both healthy and transformed stem-like cells, resulting in target cell maturation and shaping of their microenvironment. In our recent studies, we have observed that oral, pancreatic, and melanoma CSCs were capable of forming large tumors in humanized bone marrow, liver, thymus (hu-BLT) mice with fully reconstituted human immune system. In addition, major human immune subsets including NK cells, T cells, B cells, and monocytes were present in the spleen, bone marrow, peripheral blood, and tumor microenvironment. Similar to our previously published in vitro data, CSCs differentiated with split anergized NK cells prior to implantation in mice formed smaller tumors. Intravenous injection of functionally potent osteoclast-expanded NK cells inhibited tumor growth through differentiation of CSCs in humanized mice. In this review, we present current approaches, advances, and existing limitations in studying interactions of the immune system with the tumor, in particular NK cells with CSCs, using in vivo preclinical hu-BLT mouse model. In addition, we discuss the use of osteoclast-expanded NK cells in targeting cancer stem-like tumors in humanized mice-a strategy that provides a much-needed platform to develop effective cancer immunotherapies. [ABSTRACT FROM AUTHOR]

The expression of N-glycolyl-monosialodihexosyl-ganglioside (NGcGM3) in humans is restricted to cancer cells; therefore, it is a tumor antigen. There are measurable quantities of circulating anti-NGcGM3 antibodies (aNGcGM3 Abs) in human serum. Interestingly, some people have circulating Ag-specific immunoglobulins G (IgGs) that are capable of complement mediated cytotoxicity against NGcGM3 positive cells, which is relevant for tumor surveillance. In light of the chemical nature of Ag, we postulated it as a candidate ligand for CD1d. Furthermore, we hypothesize that the immune mechanism involved in the generation of these Abs entails cross talk between B lymphocytes (Bc) and invariant natural killer T cells (iNKT). Combining cellular techniques, such as flow cytometry and biochemical assays, we demonstrated that CD1d binds to NGcGM3 and that human Bc present NGcGM3 in a CD1d context according to two alternative strategies. We also showed that paraformaldehyde treatment of cells expressing CD1d affects the presentation. Finally, by co-culturing primary human Bc with iNKT and measuring Ki-67 expression, we detected a reproducible increment in the proliferation of the iNKT population when Ag was on the medium. Our findings identify a novel, endogenous, human CD1d ligand, which is sufficiently competent to stimulate iNKT. We postulate that CD1d-restricted Bc presentation of NGcGM3 drives effective iNKT activation, an immunological mechanism that has not been previously described for humans, which may contribute to understanding aNGcGM3 occurrence. [ABSTRACT FROM AUTHOR]

It is well established that natural killer (NK) cells play an important role in the immunity against cancer, while the involvement of other recently identified, NK-related innate lymphoid cells is still poorly defined. In the haploidentical hematopoietic stem cell transplantation for the therapy of high-risk leukemias, NK cells have been shown to exert a key role in killing leukemic blasts residual after conditioning. While the clinical results in the cure of leukemias are excellent, the exploitation of NK cells in the therapy of solid tumors is still limited and unsatisfactory. In solid tumors, NK cell function may be inhibited via different mechanisms, occurring primarily at the tumor site. The cellular interactions in the tumor microenvironment involve tumor cells, stromal cells and resident or recruited leukocytes and may favor tumor evasion from the host's defenses. In this context, a number of cytokines, growth factors and enzymes synthesized by tumor cells, stromal cells, suppressive/regulatory myeloid and lymphoid cells may substantially impair the function of different tumor-reactive effector cells, including NK cells. The identification and characterization of such mechanisms may offer clues for the development of new immunotherapeutic strategies to restore effective anti-tumor responses. In order to harness NK cell-based immunotherapies, several approaches have been proposed, including reinforcement of NK cell cytotoxicity by means of specific cytokines, antibodies or drugs. These new tools may improve NK cell function and/or increase tumor susceptibility to NK-mediated killing. Hence, the integration of NK-based immunotherapies with conventional anti-tumor therapies may increase chances of successful cancer treatment. [ABSTRACT FROM AUTHOR]

A key feature of human natural killer (NK) cells, which enables efficient recognition of infected and malignant target cells, is the expression of HLA class I-specific receptors of the KIR and NKG2 gene families. Cell-to-cell variability in receptor expression leads to the formation of complex NK cell repertoires. As outlined here, NK cells go through major changes from newborns to adults characterized by downregulation of the inhibitory NKG2A receptor and concomitant upregulation of KIR family members. This process is completed in young adults, and in the majority of individuals, KIR/NKG2A repertoires remain remarkably stable until old age. Nonetheless, age-related factors have the potential to majorly influence the complexity of NK cell repertoires: Firstly infection with HCMV is associated with major clonal expansions of terminally differentiated NKG2C- and KIR-expressing NK cells in certain individuals. Secondly, ineffective hematopoiesis can lead to immature and less diversified NK cell repertoires as observed in myelodysplastic syndrome (MDS), a malignant disease of the elderly. Thus, whereas in the majority of elderly the NK cell compartment appears to be highly stable in terms of function and phenotype, in a minority of subjects a breakdown of NK cell repertoire diversity is observed that might influence immune surveillance and healthy aging. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are increasingly considered as immunotherapeutic agents in particular in the fight against cancers. NK cell therapies are potentially broadly applicable and, different from their T cell counterparts, do not cause graft-versus-host disease. Efficacy and clinical in vitro or in vivo expansion of primary NK cells will however always remain variable due to individual differences of donors or patients. Long-term storage of clinical NK cell lots to allow repeated clinical applications remains an additional challenge. In contrast, the established and well-characterized cell line NK-92 can be easily and reproducibly expanded from a good manufacturing practice (GMP)-compliant cryopreserved master cell bank. Moreover, no cost-intensive cell purification methods are required. To date, NK-92 has been intensively studied. The cells displayed superior cytotoxicity against a number of tumor types tested, which was confirmed in preclinical mouse studies. Subsequent clinical testing demonstrated safety of NK-92 infusions even at high doses. Despite the phase I nature of the trials conducted so far, some efficacy was noted, particularly against lung tumors. Furthermore, to overcome tumor resistance and for specific targeting, NK-92 has been engineered to express a number of different chimeric antigen receptors (CARs), including targeting, for example, CD19 or CD20 (anti-B cell malignancies), CD38 (anti-myeloma) or human epidermal growth factor receptor 2 (HER2; ErbB2; anti-epithelial cancers). The concept of an NK cell line as an allogeneic cell therapeutic produced 'off-the-shelf' on demand holds great promise for the development of effective treatments. [ABSTRACT FROM AUTHOR]