Glioblastomas (GBMs) are lethal brain cancers that are resistant to current therapies. We investigated the cytotoxicity of human allogeneic NK cells against patient-derived GBM in vitro and in vivo, as well as mechanisms mediating their efficacy. We demonstrate that KIR2DS2 immunogenotype NK cells were more potent killers, notwithstanding the absence of inhibitory killer Ig–like receptor (KIR)-HLA ligand mismatch. FACS-sorted and enriched KIR2DS2+ NK cell subpopulations retained significantly high levels of CD69 and CD16 when in contact with GBM cells at a 1:1 ratio and highly expressed CD107a and secreted more soluble CD137 and granzyme A. In contrast, KIR2DS2− immunogenotype donor NK cells were less cytotoxic against GBM and K562, and, similar to FACS-sorted or gated KIR2DS2− NK cells, significantly diminished CD16, CD107a, granzyme A, and CD69 when in contact with GBM cells. Furthermore, NK cell–mediated GBM killing in vitro depended upon the expression of ligands for the activating receptor NKG2D and was partially abrogated by Ab blockade. Treatment of GBM xenografts in NOD/SCID mice with NK cells from a KIR2DS2+ donor lacking inhibitory KIR-HLA ligand mismatch significantly prolonged the median survival to 163 d compared with vehicle controls (log-rank test, p = 0.0001), in contrast to 117.5 d (log-rank test, p = 0.0005) for NK cells with several inhibitory KIR-HLA ligand mismatches but lacking KIR2DS2 genotype. Significantly more CD56+CD16+ NK cells from a KIR2DS2+ donor survived in nontumor-bearing brains 3 wk after infusion compared with KIR2DS2− NK cells, independent of their proliferative capacity. In conclusion, KIR2DS2 identifies potent alloreactive NK cells against GBM that are mediated by commensurate, but dominant, activating signals.
There are currently no established radiological parameters that predict response to immunotherapy. We hypothesised that multiparametric, longitudinal magnetic resonance imaging (MRI) of physiological parameters and pharmacokinetic models might detect early biological responses to immunotherapy for glioblastoma targeting NG2/CSPG4 with mAb9.2.27 combined with natural killer (NK) cells. Contrast enhanced conventional T1-weighted MRI at 7±1 and 17±2 days post-treatment failed to detect differences in tumour size between the treatment groups, whereas, follow-up scans at 3 months demonstrated diminished signal intensity and tumour volume in the surviving NK+mAb9.2.27 treated animals. Notably, interstitial volume fraction (ve), was significantly increased in the NK+mAb9.2.27 combination therapy group compared mAb9.2.27 and NK cell monotherapy groups (p = 0.002 and p = 0.017 respectively) in cohort 1 animals treated with 1 million NK cells. ve was reproducibly increased in the combination NK+mAb9.2.27 compared to NK cell monotherapy in cohort 2 treated with increased dose of 2 million NK cells (p<0.0001), indicating greater cell death induced by NK+mAb9.2.27 treatment. The interstitial volume fraction in the NK monotherapy group was significantly reduced compared to mAb9.2.27 monotherapy (p<0.0001) and untreated controls (p = 0.014) in the cohort 2 animals. NK cells in monotherapy were unable to kill the U87MG cells that highly expressed class I human leucocyte antigens, and diminished stress ligands for activating receptors. A significant association between apparent diffusion coefficient (ADC) of water and ve in combination NK+mAb9.2.27 and NK monotherapy treated tumours was evident, where increased ADC corresponded to reduced ve in both cases. Collectively, these data support histological measures at end-stage demonstrating diminished tumour cell proliferation and pronounced apoptosis in the NK+mAb9.2.27 treated tumours compared to the other groups. In conclusion, ve was the most reliable radiological parameter for detecting response to intralesional NK cellular therapy.
Glioblastoma is a deadly brain cancer with limited treatment options. Targeting chondroitin sulfate proteoglycan 4 (CSPG4, best known as NG2) with the monoclonal antibody mAb9.2.27 and activated natural killer (NK) cells abrogated the tumor growth and prolonged the survival of glioblastoma-bearing animals by favoring the establishment of a pro-inflammatory microenvironment. The combination of NK cells and mAb9.2.27 recruited ED1+CCR2low macrophages that stimulated ED1+ED2lowMHCIIhigh microglial cells to exert robust cytotoxicity. Our findings demonstrate the therapeutic potential of targeting salient tumor associated-antigens.
CNS immunosurveillance; CSPG4; glioblastoma; NK cells; passive immunotherapy
Natural killer (NK) cells are lymphocytes that play an important role in anti-tumour immunity. Their potential against brain cancer has been demonstrated in vitro and in vivo, both as a direct anti-tumour agent and in experimental therapies stimulating endogenous NK cell cytotoxicity. However, the clinical translation of these promising results requires detailed knowledge about the immune status of brain tumour patients, with focus on the NK cell population. In this report, we provide an overview of the studies investigating NK cell infiltration into the tumour, emphasizing the need of revision of the methodologies and further research in this field. We also discuss the potential of using autologous or allogeneic NK cells as effector cells in cellular therapy against brain cancer and developing immunotherapies stimulating endogenous NK cell-mediated anti-tumour response, such as blocking inhibitory killer immunoglobulin-like receptors. Combination of NK cell adoptive transfer with targeted therapies, such as anti-EGFR therapeutic antibody (CetuximAb) could also be a potent strategy.
Tumour infiltrating lymphocytes; NK cells; Brain tumour; Prognosis; Immunotherapy
Glioblastoma (GBM) is the most malignant brain tumor where patients' survival is only 14.6 months, despite multimodal therapy with debulking surgery, concurrent chemotherapy and radiotherapy. There is an urgent, unmet need for novel, effective therapeutic strategies for this devastating disease. Although several immunotherapies are under development for the treatment of GBM patients, the use of natural killer (NK) cells is still marginal despite this being a promising approach to treat cancer. In regard of our knowledge on the role of NG2/CSPG4 in promoting GBM aggressiveness we investigated the potential of an innovative immunotherapeutic strategy combining mAb9.2.27 against NG2/CSPG4 and NK cells in preclinical animal models of GBM. Multiple immune escape mechanisms maintain the tumor microenvironment in an anti-inflammatory state to promote tumor growth, however, the distinct roles of resident microglia versus recruited macrophages is not elucidated. We hypothesized that exploiting the cytokine release capabilities of activated NK cells to reverse the anti-inflammatory axis combined with mAb9.2.27 targeting the NG2/CSPG4 may favor tumor destruction by editing pro-GBM immune responses. Combination treatment with NK+mAb9.2.27 diminished tumor growth that was associated with reduced tumor proliferation, increased cellular apoptosis and prolonged survival compared to vehicle and monotherapy controls. The therapeutic efficacy was mediated by recruitment of CCR2low macrophages into the tumor microenvironment, increased ED1 and MHC class II expression on microglia that might render them competent for GBM antigen presentation, as well as elevated IFN-γ and TNF-α levels in the cerebrospinal fluid compared to controls. Depletion of systemic macrophages by liposome-encapsulated clodronate decreased the CCR2low macrophages recruited to the brain and abolished the beneficial outcomes. Moreover, mAb9.2.27 reversed tumor-promoting effects of patient-derived tumor-associated macrophage/ microglia (TAM) ex vivo. Taken together, these findings indicate that NK+mAb9.2.27 treatment may be an amenable therapeutic strategy to treat NG2/CSPG4 expressing GBMs. We provide a novel conceptual approach of combination immunotherapy for glioblastoma. The results traverse beyond the elucidation of NG2/CSPG4 as a therapeutic target, but demonstrate a proof of concept that this antibody may hold potential for the treatment of GBM by activation of tumor infiltrated microglia/macrophages.
Microglia; NK cells; glioblastoma; immunotherapy; NG2/CSPG4
NK cells are lymphocytes of the innate immune system which are a first line of defense against infections and tumor cells, in bone marrow and peripheral organs like lung and spleen. The lung is an organ in contact with respiratory pathogens and the site of inflammatory disorders triggered by the respiratory environment. In contrast, spleen is a lymphatic organ connected to the blood system which regulates the systemic immune response. Here we compare NK cell maturation and expansion as well as expression of NK cell receptors in spleen and lung compartments. We show that spleen and lung NK cells differ in phenotypic and functional characteristics due to a difference of maturity and cellular microenvironment. Indeed we observe that spleen and lung macrophages have the capacity to influence the cytotoxicity of NK cells by cell-to-cell contact. This suggests that the differences of NK cell subsets are in part due to a modulation by the organ environment.
The interaction between natural killer (NK) cells and different other immune cells like T cells and dendritic cells is well-described, but the crosstalk with monocytes or macrophages and the nature of ligands/receptors implicated are just emerging. The macrophage-NK interaction is a major first-line defense against pathogens (bacteria, viruses, fungi, and parasites). The recruitment and the activation of NK cells to perform cytotoxicity or produce cytokines at the sites of inflammation are important to fight infections. The two main mechanisms by which macrophages can prime NK cells are (1) activation through soluble mediators such as IL-12, IL-18, and (2) stimulation through direct cell-to-cell contact. We will discuss the progress in matters of modulation of NK cell functions by monocytes and macrophages, in the steady state and during diseases.
NK cells; macrophages; monocytes; receptors; cytokines; activation; infection
Nerve growth factor (NGF) is a neurotrophin crucial for the development and survival of neurons. It also acts on cells of the immune system which express the NGF receptors TrkA and p75NTR and can be produced by them. However, mouse NK cells have not yet been studied in this context.
We used cell culture, flow cytometry, confocal microscopy and ELISA assays to investigate the expression of NGF receptors by NK cells and their secretion of NGF. We show that resting NK cells express TrkA and that the expression is different on NK cell subpopulations defined by the relative presence of CD27 and CD11b. Expression of TrkA is dramatically increased in IL-2-activated NK cells. The p75NTR is expressed only on a very low percentage of NK cells. Functionally, NGF moderately inhibits NK cell degranulation, but does not influence proliferation or cytokine production. NK cells do not produce NGF.
We demonstrate for the first time that mouse NK cells express the NGF receptor TrkA and that this expression is dynamically regulated.
Cobalamin (vitamin B12) deficiency is particularly common in the elderly (>65 years of age) but is often unrecognized because its clinical manifestations are subtle; however, they are also potentially serious, particularly from a neuropsychiatric and hematological perspective. In the elderly, the main causes of cobalamin deficiency are pernicious anemia and food-cobalamin malabsorption. Food-cobalamin malabsorption syndrome is a disorder characterized by the inability to release cobalamin from food or its binding proteins. This syndrome is usually caused by atrophic gastritis, related or unrelated to Helicobacter pylori infection, and long-term ingestion of antacids and biguanides. Management of cobalamin deficiency with cobalamin injections is currently well documented but new routes of cobalamin administration (oral and nasal) are being studied, especially oral cobalamin therapy for food-cobalamin malabsorption.
Peripheral blood natural killer (NK) cells from patients with transporter associated with antigen processing (TAP) deficiency are hyporesponsive. The mechanism of this defect is unknown, but the phenotype of TAP-deficient NK cells is almost normal. However, we noticed a high percentage of CD56bright cells among total NK cells from two patients. We further investigated TAP-deficient NK cells in these patients and compared them to NK cells from two other TAP-deficient patients with no clinical symptoms and to individuals with chronic inflammatory diseases other than TAP deficiency (chronic lung diseases or vasculitis). Peripheral blood mononuclear cells isolated from venous blood were stained with fluorochrome-conjugated antibodies and the phenotype of NK cells was analyzed by flow cytometry. In addition, 51Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56bright NK cells represented 28% and 45%, respectively, of all NK cells (higher than in healthy donors). The patients also displayed a higher percentage of CD56dimCD16− NK cells than controls. Interestingly, this unusual NK cell subtype distribution was not found in the two asymptomatic TAP-deficient cases, but was instead present in several of the other patients. Over-expression of the inhibitory receptor CD94/NKG2A by TAP-deficient NK cells was confirmed and extended to the inhibitory receptor ILT2 (CD85j). These inhibitory receptors were not involved in regulating the cytotoxicity of TAP-deficient NK cells. We conclude that expansion of the CD56bright NK cell subtype in peripheral blood is not a hallmark of TAP deficiency, but can be found in other diseases as well. This might reflect a reaction of the immune system to pathologic conditions. It could be interesting to investigate the relative distribution of NK cell subsets in various respiratory and autoimmune diseases.
In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM–ITAM signaling is likely to play an important role in the developmental program of NKT cells.
NKT cell development; ITAM-containing receptors; ITIM-containing receptors; TCR repertoire; NK receptor repertoire
To study the adaptation of natural killer (NK) cells to their major histocompatibility complex (MHC) class I environment we have established a novel mouse model with mosaic expression of H-2Dd using a Cre/loxP system. In these mice, we noticed that NK cells expressing the inhibitory receptor for Dd, Ly49A, were specifically underrepresented among cells with low Dd levels. That was due to the acquisition of Dd molecules by the Ly49A+ NK cells that have lost their Dd transgene. The uptake of H-2D molecules via the Ly49A receptor was restricted to strong ligands of Ly49A. Surprisingly, when Ly49A+ NK cells were Dd+, uptake of the alternative ligand Dk was not detectable. Similarly, one anti-Ly49A mAb (A1) bound inefficiently when Ly49A was expressed on Dd+ NK cells. Concomitantly, functional assays demonstrated a reduced capacity of Ly49A to inhibit H-2bDd as compared with H-2b NK cells, rendering Ly49A+ NK cells in Dd+ mice particularly reactive. Minor reductions of Dd levels and/or increases of activating ligands on environmental cells may thus suffice to abrogate Ly49A-mediated NK cell inhibition. The mechanistic explanation for all these phenomena is likely the partial masking of Ly49A by Dd on the same cell via a lateral binding site in the H-2Dd molecule.
ligand uptake; NK cells; Ly49A; H-2D; Cre/loxP
The transporter associated with antigen processing (TAP), which is composed of two subunits (TAP1 and TAP2) that have different biochemical and functional properties, plays a key role in peptide loading and the cell surface expression of HLA class I molecules. Three cases of HLA class I deficiency have previously been shown to result from the absence of a functional TAP2 subunit. In the present study, we analyzed two cases displaying not only the typical lung syndrome of HLA class I deficiency but also skin lesions, and found these patients to be TAP1-deficient. This defect leads to unstable HLA class I molecules and their retention in the endoplasmic reticulum. However, the absence of TAP1 is compatible with life and does not seem to result in higher susceptibility to viral infections than TAP2 deficiency. This work also reveals that vasculitis is often observed in HLA class I–deficient patients.
In this paper we describe the function and phenotype of natural killer (NK) lymphocytes from HLA class I–deficient patients. These cells are, as has been previously reported, unable to lyse HLA class I− K562 cells, but are able to perform antibody-dependent cellular cytotoxicity (ADCC), although with lower efficiency as compared to NK cells from normal individuals. Transporter associated to antigen processing (TAP)− NK cells proliferate when cultured in the presence of lymphoblastoid B cells (B-LCs) and interleukin 2 and develop a spectrum of cytotoxicity similar to that of activated normal NK cells. Importantly, activation of the TAP− NK cells induces strong cytotoxicity to autologous B-LCs. Analysis of the phenotype of circulating TAP− NK lymphocytes showed them to display a normal diverse repertoire of HLA class I–specific NK receptors. These receptors were expressed at normal levels, apart from the CD94–NKG2A complex, which appeared to be overexpressed. This latter finding could reflect an adaptation to the low expression of HLA class I molecules. Finally, functional analyses indicated that the inhibitory receptors in TAP− individuals can transduce inhibitory signals. Our results suggest that in vivo, the NK cells of TAP− patients could participate in immune defense, at least through ADCC, but upon activation, may be involved in autoimmune processes.