NK Cell-mediated Cytotoxic Activity Research Articles

Abstract 3218: Disruption of the CD39 immune checkpoint pathway increases the efficacy of various anticancer therapies in syngeneic mouse models

Abstract The CD39-CD73-adenosine pathway is an emerging regulator of the immune antitumor response. CD39 is expressed within tumors and the tumor microenvironment by several cell population including immune and cancer cells. In tumor tissues, the pathway leads to the accumulation of immunosuppressive adenosine together with decreased levels of immunoactivating peritumoral ATP. We reported previously that CD39 blockade increased T cell and NK cell-mediated cytotoxic activity in vitro and disclose, during this meeting, the development of the first human-CD39-blocking humanized antibody (S. Augier et al., Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy). Here we demonstrated that this pathway is involved in tumor-induced resistance to various cancer therapies in syngeneic mouse melanoma, colon cancer and fibrosarcoma models. We used therapy-resistant mouse models or inefficacious treatment regimens in the CD39 knockout mice to assess the capacity of CD39 to affect the response to chemotherapies, tumor associated antigen (TAA)-targeting antibodies and immunomodulators such as anti-PD1 antibodies. We achieved increased response rates, increased response duration and some complete and long lasting tumor regressions in the CD39 deficient context. These preclinical proof-of-concept studies highlight the role of the CD39 immune checkpoint pathway in limiting the efficacy of various anticancer therapies in syngeneic mouse models and thereby support the potential clinical value of the humanized CD39-neutralizing antibody under development. Citation Format: Marion Lapierre, Cécile Dejou, Carine Paturel, Henri-Alexandre Michaud, Laurent Gros, Armand Bensussan, Jean-François Eliaou, Jeremy Bastid, Nathalie Bonnefoy. Disruption of the CD39 immune checkpoint pathway increases the efficacy of various anticancer therapies in syngeneic mouse models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3218.

NKT cells act through third party bone marrow-derived cells to suppress NK cell activity in the liver and exacerbate hepatic melanoma metastases.

Uveal melanoma (UM) is the most common intraocular tumor in adults and liver metastasis is the leading cause of death in UM patients. We have previously shown that NKT cell-deficient mice develop significantly fewer liver metastases from intraocular melanomas than do wild-type (WT) mice. Here, we examine the interplay between liver NKT cells and NK cells in resistance to liver metastases from intraocular melanomas. NKT cell-deficient CD1d(-/-) mice and WT C57BL/6 mice treated with anti-CD1d antibody developed significantly fewer liver metastases than WT mice following either intraocular or intrasplenic injection of B16LS9 melanoma cells. The increased number of metastases in WT mice was associated with reduced liver NK cytotoxicity and decreased production of IFN-γ. However, liver NK cell-mediated cytotoxic activity was identical in non-tumor bearing NKT cell-deficient mice and WT mice, indicating that liver metastases were crucial for the suppression of liver NK cells. Depressed liver NK cytotoxicity in WT mice was associated with production of IL-10 by bone marrow-derived liver cells that were neither Kupffer cells nor myeloid-derived suppressor cells and by increased IL-10 receptor expression on liver NK cells. IL-10(-/-) mice had significantly fewer liver metastases than WT mice, but were not significantly different from NKT cell-deficient mice. Thus, development of melanoma liver metastases is associated with upregulation of IL-10 in the liver and an elevated expression of IL-10 receptor on liver NK cells. This impairment of liver NK activity is NKT cell-dependent and only occurs in hosts with melanoma liver metastases.

Abstract 4572: The CD30/CD16A RECRUIT TandAb AFM13 activates patient NK cells resulting in specific tumor cell killing.

Abstract AFM13 represents a new class of bispecific antibodies, based on the RECRUIT-TandAb platform, which enables the generation of therapeutics with improved efficacy, convenient administration regimens, and excellent safety profiles. AFM13 was developed for the treatment of Hodgkin Lymphoma (HL) and other CD30+ malignancies. AFM13 targets CD30 on HL tumor cells and recruits NK cells via CD16A. Preclinical data demonstrate specific and highly efficient anti-tumor activity by the selectively recruited NK cell immune response, which addresses key deficiencies of monoclonal antibodies: (i) reduced binding to the 158F allotype of CD16A and (ii) non-selective binding to immune effector cells via both activating CD16A and non-signalling CD16B receptors. In vitro assays demonstrate that AFM13 binds with high affinity to both CD30 and CD16A antigens and rapidly induces lysis of CD30+ cells at picomolar concentrations. Further, AFM13 activates CD16A only in the presence of tumor cells: there is no systemic activation of NK-cells in the absence of target cells. A robust GMP process in mammalian cells and a lyophilized formulation with excellent stability are established. Finally, no toxicity is observed in Cynomolgus monkeys. AFM13 was investigated in an open-label single-arm phase I dose escalation trial in heavily pre-treated patients with relapsed/refractory HL. Each patient received 4 weekly doses of AFM13. Seven dose levels from 0.01 to 7.0 mg/kg were escalated in cohorts of 3 patients. In addition, one cohort of 4 patients received AFM13 twice a week at 4.5 mg/kg for 4 weeks. All dose levels of AFM13 proved to be well tolerated and safe and clear signs of anti-tumor activity were demonstrated. We have performed an analysis of the activation of NK cells and clearance of soluble CD30 in humans since these are key parameters for the mode of action of AFM13. We confirmed a correlation between anti-tumor activity parameters and biomarkers based on a statistically significant dose-dependent increase in the activation of NK cells and reduction in soluble CD30 levels. Since the activation of NK cells has been reported to facilitate low-level expression of CD30 on NK cells, which we also observed in several patients in the highest AFM13 dose groups, we investigated whether this may affect NK cell cytotoxicity. Our ex vivo evaluation of samples from patients that received AFM13 and in vitro evaluation of activated NK cells demonstrated no inhibition of NK cell-mediated cytotoxic activity. Furthermore, AFM13 exhibits a remarkable half-life of 1 day which represents a substantial increase relative to that of diabody-like formats currently being evaluated in the clinic for haematological malignancies. Thus, AFM13 has demonstrated encouraging biologic activity and represents a new targeted therapy for heavily pre-treated patients with HL. Citation Format: Achim Rothe, Max S. Topp, Anas Younes, Uwe Reusch, Stefan Knackmuss, Erich Rajkovic, Dennis A. Eichenauer, Horst Hummel, Katrin S. Reiners, Markus Dietlein, Joerg Kessler, Miroslav Ravic, Christian Hucke, Eugene Zhukovsky, Elke Pogge von Strandmann, Andreas Engert. The CD30/CD16A RECRUIT TandAb AFM13 activates patient NK cells resulting in specific tumor cell killing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4572. doi:10.1158/1538-7445.AM2013-4572

Comparison of Atlantic salmon individuals with different outcomes of cardiomyopathy syndrome (CMS)

BackgroundCardiomyopathy syndrome (CMS) is a severe disease of Atlantic salmon (Salmo salar L.) associated with significant economic losses in the aquaculture industry. CMS is diagnosed with a severe inflammation and degradation of myocardial tissue caused by a double-stranded RNA virus named piscine myocarditis virus (PMCV), with structural similarities to the Totiviridae family. In the present study we characterized individual host responses and genomic determinants of different disease outcomes.ResultsFrom time course studies of experimentally infected Atlantic salmon post-smolts, fish exhibited different outcomes of infection and disease. High responder (HR) fish were characterized with sustained and increased viral load and pathology in heart tissue. Low responder (LR) fish showed declining viral load from 6–10 weeks post infection (wpi) and absence of pathology. Global gene expression (SIQ2.0 oligonucleotide microarray) in HR and LR hearts during infection was compared, in order to characterize differences in the host response and to identify genes with expression patterns that could explain or predict the different outcomes of disease. Virus-responsive genes involved in early antiviral and innate immune responses were upregulated equally in LR and HR at the first stage (2–4 wpi), reflecting the initial increase in virus replication. Repression of heart muscle development was identified by gene ontology enrichment analyses, indicating the early onset of pathology. By six weeks both responder groups had comparable viral load, while increased pathology was observed in HR fish. This was reflected by induced expression of genes implicated in apoptosis and cell death mechanisms, presumably related to lymphocyte regulation and survival. In contrast, LR fish showed earlier activation of NK cell-mediated cytotoxicity and NOD-like receptor signaling pathways. At the late stage of infection, increased pathology and viral load in HR was accompanied by a broad activation of genes involved in adaptive immunity and particularly T cell responses, probably reflecting the increased infiltration and homing of virus-specific T cells to the infected heart. This was in sharp contrast to LR fish, where recovery and reduced viral load was associated with a significantly reduced transcription of adaptive immunity genes and activation of genes involved in energy metabolism.ConclusionsIn contrast to LR, a stronger and sustained expression of genes involved in adaptive immune responses in heart tissue of HR at the late stage of disease probably reflected the increased lymphocyte infiltration and pathological outcome. In addition to controlled adaptive immunity and activation of genes involved in cardiac energy metabolism in LR at the late stage, recovery of this group could also be related to an earlier activation of NOD-like receptor signaling and NK cell-mediated cytotoxicity pathways.

CS1 (CRACC, CD319) Induces Proliferation and Autocrine Cytokine Expression on Human B Lymphocytes

CS1 (CRACC, CD319), a member of the CD2 family of cell surface receptors, is implicated in the activation of NK cell-mediated cytotoxicity. Previous studies showed that CS1 is also expressed on activated B cells. However, the functional role of CS1 in human B-lymphocytes is not known. Two isoforms of CS1, CS1-L and CS1-S, are expressed in human NK cells that differentially regulate NK cell function. CS1-L contains immunoreceptor tyrosine-based switch motifs in its cytoplasmic domain whereas CS1-S lacks immunoreceptor tyrosine-based switch motifs. In this study, we show that human B lymphocytes express only the CS1-L isoform, and its expression is up-regulated upon B cell activation with various stimulators. Moreover, anti-CS1 mAb strongly enhanced proliferation of both freshly isolated as well as activated B cells. The enhanced proliferation effects of CS1 were most prominent on B cells activated by anti-CD40 mAbs and/or hrIL-4. The effects of CS1 on B cell proliferation were shown on both naive and memory B cells. Human cytokine microarray and quantitative real-time PCR results indicated that CS1 activation enhanced mRNA transcripts of flt3 ligand, lymphotoxin A, TNF, and IL-14. Neutralizing Abs against lymphotoxin A, TNF-alpha, and/or flt3 ligand abolished the ability of CS1 on the B cell proliferation. These results suggest that activation of B lymphocytes, through surface CS1, may be mediated through secretion of autocrine cytokines and CS1 may play a role in the regulation of B lymphocyte proliferation during immune responses.

Tumoricidal Potential of Native Blood Dendritic Cells: Direct Tumor Cell Killing and Activation of NK Cell-Mediated Cytotoxicity

Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity for DC-based cancer vaccination protocols. Novel findings reveal that besides their role as potent inducers of tumor-specific T cells, human DCs display additional antitumor effects. Most of these data were obtained with monocyte-derived DCs, whereas studies investigating native blood DCs are limited. In the present study, we analyze the tumoricidal capacity of M-DC8(+) DCs, which represent a major subpopulation of human blood DCs. We demonstrate that IFN-gamma-stimulated M-DC8(+) DCs lyse different tumor cell lines but not normal cells. In addition, we show that tumor cells markedly enhance the production of TNF-alpha by M-DC8(+) DCs via cell-to-cell contact and that this molecule essentially contributes to the killing activity of M-DC8(+) DCs. Furthermore, we illustrate the ability of M-DC8(+) DCs to promote proliferation, IFN-gamma production, and tumor-directed cytotoxicity of NK cells. The M-DC8(+) DC-mediated enhancement of the tumoricidal potential of NK cells is mainly dependent on cell-to-cell contact. These results reveal that, in addition to their crucial role in activating tumor-specific T cells, blood DCs exhibit direct tumor cell killing and enhance the tumoricidal activity of NK cells. These findings point to the pivotal role of DCs in triggering innate and adaptive immune responses against tumors.

Tumoricidal Potential of Native Human Blood Dendritic Cells: Direct Tumor Cell Killing and Activation of NK Cell-Mediated Cytotoxicity.

Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity of DC-based cancer vaccination protocols. Recently, we defined a novel major subset of human blood DCs by using the monoclonal antibody M-DC8 which recognizes a carbohydrate modification of P selectin glycoprotein ligand-1 (PSGL-1) selectively expressed on these cells (Immunity 2002; 17:289-301). In addition to a marked capacity to activate tumor-reactive cytotoxic T cells M-DC8+ DCs efficiently mediate antibody-dependent cytotoxicity (Blood ; 2002; 100:1502-1504). In the present study, we analyzed the capacity of M-DC8+ DCs to kill tumor cells in the absence of antibodies and to enhance the tumor-directed cytotoxicity of NK cells. To determine whether M-DC8+ DCs exhibit tumoricidal activity, DCs were isolated at high purity (>93%) from the blood of healthy donors by immunomagnetic separation. These cells were cultured for 6 h in the presence or absence of 200 U/ml interferon (IFN)-gamma. Subsequently, DCs were coincubated with four chromium-labeled tumor cell lines and two normal cell lines for 18 h. Whereas unstimulated DCs demonstrated only moderate tumor-directed cytotoxicity (specific lysis: 7–13%), IFN-gamma-stimulated M-DC8+ DCs displayed potent killing of each of these tumor cell lines (specific lysis: 27–35%). Only a marginal cytotoxic effect was seen when normal human cells such as lung fibroblasts or endothelial cells were used as targets. When evaluating the cytotoxic effector mechanisms FACS analysis and ELISA assays revealed that IFN-gamma-stimulated M-DC8+ DCs secreted a high amount of tumor necrosis factor (TNF)-alpha induced by direct cell-to-cell contact with the different tumor cell lines. This effect was already observed after 3 h of cocultivation. Interestingly, no significant induction of TNF-alpha was detected during contact of M-DC8+ DCs with the normal human cell lines. These results suggest that tumor-associated surface molecules are important for the observed increase of TNF-alpha production in M-DC8+ DCs. Inhibition experiments with neutralizing antibodies clearly demonstrated that tumor cell-induced TNF-alpha play an important role in tumor-directed cytotoxicity mediated by M-DC8+ DCs. To investigate whether M-DC8+ DCs enhance the tumoricidal activity of NK cells freshly isolated DCs were cultured for 6 h in the presence or absence of IFN-gamma. Thereafter, DCs were coincubated with highly enriched (>90%) NK cells. The cytotoxic potential of the stimulated NK cells was tested towards various tumor cell lines in a 4 h chromium release assay. We observed a two- to threefold increase of NK cell-mediated cytotoxicity towards all analyzed tumor cell lines by IFN-gamma-stimulated M-DC8+ DCs. In addition, transwell experiments demonstrated that this triggering effect was mainly dependent on cell-to-cell contact. In conclusion, our data provide evidence that a major subpopulation of circulating human blood DCs exhibits efficient tumoricidal activity and clearly enhances NK cell-mediated tumor-directed cytotoxicity. The capacity of DCs to induce tumor-specific T cell responses and to kill tumor cells either directly or by activating NK cells points to the pivotal role of DCs in triggering the innate and adaptive immune response against tumors.

NKT cells act as regulatory cells rather than killer cells during activation of NK cell-mediated cytotoxicity by α-galactosylceramide in vivo

Administration of NKT cell ligands, α-galactosylceramide (α-GalCer) resulted in the activation of both cytokine production and natural killing. These responses were abolished in both CD1d-deficient mice and Vα14NKT-deficient mice. Therefore, NKT cells have been considered to be responsible cells for both cytokine production and natural killing. Here, we reevaluated a critical role of NKT and NK cells at early time after α-GalCer administration. Intracellular staining experiments demonstrated that NKT cells were the earliest source of both IL-4 and IFN-γ production after α-GalCer administration in vivo. However, these α-GalCer-activated NKT cells exhibited no significant natural killing activity. In contrast, isolated NK1.1 +CD3 − classical NK cells exhibited greatly enhanced natural killing activity 6 h after α-GalCer administration. NKT cells, however, exhibited a strong cytotoxicity when they were activated and expanded with α-GalCer plus IL-2 in vitro. These results indicated that NKT cells act as regulatory cells via production of cytokines for activation of NK cell-mediated cytotoxicity in vivo at early phase after α-GalCer administration. Thus, NK cells rather than NKT cells may be a crucial early activated killer induced by α-GalCer in vivo.

NKT cells act as regulatory cells rather than killer cells during activation of NK cell-mediated cytotoxicity by $alpha;-galactosylceramide in vivo

NKT cells act as regulatory cells rather than killer cells during activation of NK cell-mediated cytotoxicity by $alpha;-galactosylceramide in vivo

NK cells and HIV infection: lessons from other viruses.

Although the means by which NK cells may contribute to anti viral defense are still incompletely understood, various studies merge to a better comprehension of pathways that mediate NK cell activation (NK cell mediated cytotoxic activity and cytokine production) and their implications during the immune response towards a variety of viruses. Characterization of a specific expression pattern of ligands for NK receptors on virally infected cells and consequent modulation of NK cell activity have provided new insights in the field. A major break through to a direct evidence of a role for NK cells and NK cell receptors in immune protection against viral infection, was the recent implication of the murine activating Ly49H receptors in immune protection against MCMV infection. Although much remains to be learned concerning implication of NK cells in HIV infection, various reports have documented alteration in NK cell function and numbers during the course of HIV infection or treatment of AIDS. This review will focus on the current knowledge about the factors which might influence NK cell activation during various viral challenge and an emerging view of their alteration during HIV infection.

Activation of NK cell-mediated cytotoxicity by a SAP-independent receptor of the CD2 family.

Some CD2 family receptors stimulate NK cell-mediated cytotoxicity through a signaling pathway, which is dependent on the recruitment of an adapter protein called SLAM-associated protein (SAP). In this work we identify a novel leukocyte cell surface receptor of the CD2 family called CD2-like receptor activating cytotoxic cells (CRACC). CRACC is expressed on cytotoxic lymphocytes, activated B cells, and mature dendritic cells, and activates NK cell-mediated cytotoxicity. Remarkably, although CRACC displays cytoplasmic motifs similar to those recruiting SAP, CRACC-mediated cytotoxicity occurs in the absence of SAP and requires activation of extracellular signal-regulated kinases-1/2. Thus, CRACC is a unique CD2-like receptor which mediates NK cell activation through a SAP-independent extracellular signal-regulated kinase-mediated pathway.

Decreased expression of signaling lymphocytic-activation molecule-associated protein (SAP) transcripts in T cells from patients with rheumatoid arthritis.

The function of Epstein-Barr virus (EBV)-specific cytotoxic T cells is disturbed in rheumatoid arthritis (RA) patients but the mechanism for this disturbance has remained unknown. In a recent study searching for the causative gene of X-linked lymphoproliferative syndrome, the gene possibly linked to EBV-specific cytotoxic T cells or NK cell-mediated cytotoxic activity to EBV-infected cells was discovered, and its product is now referred to as signaling lymphocytic-activation molecule-associated protein (SAP) or Src homology 2 domain-containing protein (SH2D1A). In the present study, we attempted to investigate the involvement of the SAP gene in RA using a quantitative real-time PCR; the expression level of SAP transcripts in peripheral leukocytes or T cells was examined for patients with RA. The expression level of SAP transcripts in peripheral leukocytes of 21 RA patients was significantly lower than that of 13 normal individuals (P = 0.0007), four patients with palindromic RA, 11 with inactive systemic lupus erythematosus (SLE) or 17 with chronic renal diseases. The decreased expression of SAP transcripts in RA patients was also observed in peripheral CD2(+) T cells compared with normal individuals. There was no mutation in the coding region of SAP cDNAs derived from peripheral leukocytes of five RA patients. The decreased expression of SAP transcripts in peripheral leukocytes or T cells of RA patients might lead to the failure of the immune system to eliminate the EBV-infected synovial lining cells in joints of RA patients. Our findings have suggested that decreased expression of the SAP gene might be involved in the onset or progress of RA.

Lack of immunotoxicity of saquinavir (Ro 31-8959) used alone or in double or triple combination with AZT and ddC.

Saquinavir (Ro 31-8959; SQV) has been demonstrated to be a potent inhibitor of human immunodeficiency virus (HIV) proteinases and acts synergistically with dideoxynucleoside analogues. The aim of this study was to investigate the in vitro immunomodulatory effects of SQV on normal human peripheral blood mononuclear cells (PBMC) and on lamina propria mononuclear cells (LPMC). We used the drug either alone or in double and triple combination with AZT and ddC to assess whether SQV enhances the immunomodulatory effects induced by AZT and ddC that we previously observed. We demonstrated that SQV did not induce any modulation of the proliferative response either in PBMC or in LPMC. Similarly, NK cell-mediated cytotoxic activity and cytokine production were not modified by SQV. More importantly, SQV/AZT, SQV/ddC, and SQV/AZT/ddC combinations did not strengthen neither the inhibition of PBMC and LPMC proliferative response or the modulation of cytokine production induced by AZT, ddC, and AZT/ddC. On the other hand, the increased IL-2 production induced by AZT and ddC was not observed adding SQV to the dideoxynucleoside analogues. In conclusion, we demonstrated that SQV used in combination with AZT and ddC did not add any further immunotoxicity.

In vitro and in vivo effect of growth hormone on cytotoxic activity

The presence of specific growth hormone receptors on peripheral blood mononuclear cells 1-3 raises the possibility that GH may affect the immune system directly. Data obtained in a mouse model suggest that GH may infleunce cytotoxic activity mediated by natural killer cells, 4 which are lymphocytes involved in immune surveillance for viral infections and neoplastic diseases. 58 The effects of GH on cell-mediated cytotoxic activity in human subjects are still controversial. Impaired NK cell activity has been found in patients with GH deficiency. 91~ Although it has been reported that short-term administration of GH and GH-releasing hormone fails to normalize NK cell function in GH-deficient children, 9 a previous work demonstrated that daily exogenous GH treat~nent increases this activity in GH-deficient women. 1~ The aim of our work was to study NK cell-mediated cytotoxic activity in GH-deficient children before and during long-term therapy with biosynthetic GH.