Multimeric immunotherapeutic complexes activating natural killer cells towards HIV-1 cure.

BACKGROUND: Combination antiretroviral therapy (cART) has dramatically extended the life expectancy of people living with HIV-1 and improved their quality of life. There is nevertheless no cure for HIV-1 infection since HIV-1 persists in viral reservoirs of latently infected CD4+ T cells. cART does not eradicate HIV-1 reservoirs or restore cytotoxic natural killer (NK) cells which are dramatically reduced by HIV-1 infection, and express the checkpoint inhibitors NKG2A or KIR2DL upregulated after HIV-1 infection. Cytotoxic NK cells expressing the homing receptor CXCR5 were recently described as key subsets controlling viral replication. METHODS: We designed and evaluated the potency of "Natural killer activating Multimeric immunotherapeutic compleXes", called as NaMiX, combining multimers of the IL-15/IL-15Rα complex with an anti-NKG2A or an anti-KIR single-chain fragment variable (scFv) to kill HIV-1 infected CD4+ T cells. The oligomerization domain of the C4 binding protein was used to associate the IL-15/IL-15Rα complex to the scFv of each checkpoint inhibitor as well as to multimerize each entity into a heptamer (α form) or a dimer (ß form). Each α or ß form was compared in different in vitro models using one-way ANOVA and post-hoc Tukey's tests before evaluation in humanized NSG tg-huIL-15 mice having functional NK cells. RESULTS: All NaMiX significantly enhanced the cytolytic activity of NK and CD8+ T cells against Raji tumour cells and HIV-1+ ACH-2 cells by increasing degranulation, release of granzyme B, perforin and IFN-γ. Targeting NKG2A had a stronger effect than targeting KIR2DL due to higher expression of NKG2A on NK cells. In viral inhibition assays, NaMiX initially increased viral replication of CD4+ T cells which was subsequently inhibited by cytotoxic NK cells. Importantly, anti-NKG2A NaMiX enhanced activation, cytotoxicity, IFN-γ production and CXCR5 expression of NK cells from HIV-1 positive individuals. In humanized NSG tg-huIL-15 mice, we confirmed enhanced activation, degranulation, cytotoxicity of NK cells, and killing of HIV-1 infected cells from mice injected with the anti-NKG2A.α NaMiX, as compared to control mice, as well as decreased total HIV-1 DNA in the lung. CONCLUSIONS: NK cell-mediated killing of HIV-1 infected cells by NaMiX represents a promising approach to support HIV-1 cure strategies.

Allergic airway inflammation delays glioblastoma progression and reinvigorates systemic and local immunity in mice.

BACKGROUND: Numerous patient-based studies have highlighted the protective role of immunoglobulin E-mediated allergic diseases on glioblastoma (GBM) susceptibility and prognosis. However, the mechanisms behind this observation remain elusive. Our objective was to establish a preclinical model able to recapitulate this phenomenon and investigate the role of immunity underlying such protection. METHODS: An immunocompetent mouse model of allergic airway inflammation (AAI) was initiated before intracranial implantation of mouse GBM cells (GL261). RAG1-KO mice served to assess tumor growth in a model deficient for adaptive immunity. Tumor development was monitored by MRI. Microglia were isolated for functional analyses and RNA-sequencing. Peripheral as well as tumor-associated immune cells were characterized by flow cytometry. The impact of allergy-related microglial genes on patient survival was analyzed by Cox regression using publicly available datasets. RESULTS: We found that allergy establishment in mice delayed tumor engraftment in the brain and reduced tumor growth resulting in increased mouse survival. AAI induced a transcriptional reprogramming of microglia towards a pro-inflammatory-like state, uncovering a microglia gene signature, which correlated with limited local immunosuppression in glioma patients. AAI increased effector memory T-cells in the circulation as well as tumor-infiltrating CD4+ T-cells. The survival benefit conferred by AAI was lost in mice devoid of adaptive immunity. CONCLUSION: Our results demonstrate that AAI limits both tumor take and progression in mice, providing a preclinical model to study the impact of allergy on GBM susceptibility and prognosis, respectively. We identify a potentiation of local and adaptive systemic immunity, suggesting a reciprocal crosstalk that orchestrates allergy-induced immune protection against GBM.

A Hot Topic: Cancer Immunotherapy and Natural Killer Cells.

Despite significant progress in recent years, the therapeutic approach of the multiple different forms of human cancer often remains a challenge. Besides the well-established cancer surgery, radiotherapy and chemotherapy, immunotherapeutic strategies gain more and more attention, and some of them have already been successfully introduced into the clinic. Among these, immunotherapy based on natural killer (NK) cells is considered as one of the most promising options. In the present review, we will expose the different possibilities NK cells offer in this context, compare data about the theoretical background and mechanism(s) of action, report some results of clinical trials and identify several very recent trends. The pharmaceutical industry is quite interested in NK cell immunotherapy, which will benefit the speed of progress in the field.

Dual cholinergic signals regulate daily migration of hematopoietic stem cells and leukocytes.

Hematopoietic stem and progenitor cells (HSPCs) and leukocytes circulate between the bone marrow (BM) and peripheral blood following circadian oscillations. Autonomic sympathetic noradrenergic signals have been shown to regulate HSPC and leukocyte trafficking, but the role of the cholinergic branch has remained unexplored. We have investigated the role of the cholinergic nervous system in the regulation of day/night traffic of HSPCs and leukocytes in mice. We show here that the autonomic cholinergic nervous system (including parasympathetic and sympathetic) dually regulates daily migration of HSPCs and leukocytes. At night, central parasympathetic cholinergic signals dampen sympathetic noradrenergic tone and decrease BM egress of HSPCs and leukocytes. However, during the daytime, derepressed sympathetic noradrenergic activity causes predominant BM egress of HSPCs and leukocytes via ß3-adrenergic receptor. This egress is locally supported by light-triggered sympathetic cholinergic activity, which inhibits BM vascular cell adhesion and homing. In summary, central (parasympathetic) and local (sympathetic) cholinergic signals regulate day/night oscillations of circulating HSPCs and leukocytes. This study shows how both branches of the autonomic nervous system cooperate to orchestrate daily traffic of HSPCs and leukocytes.

Revisiting the Functional Impact of NK Cells.

Immune responses are critical for the maintenance of homeostasis but can also upset the equilibrium, depending on the context and magnitude of the response. Natural killer (NK) cells are well known for their important roles in antiviral and antitumor immune responses, and they are currently used, mostly under optimized forms, as immunotherapeutic agents against cancer. Nevertheless, with accumulating examples of deleterious effects of NK cells, it is paramount to consider their negative contributions. Here, we critically review and comment on the literature surrounding undesirable aspects of NK cell activity, focusing on situations where they play a harmful rather than a protective role.

Human CD56bright NK Cells: An Update.

Human NK cells can be subdivided into various subsets based on the relative expression of CD16 and CD56. In particular, CD56(bright)CD16(-/dim) NK cells are the focus of interest. They are considered efficient cytokine producers endowed with immunoregulatory properties, but they can also become cytotoxic upon appropriate activation. These cells were shown to play a role in different disease states, such as cancer, autoimmunity, neuroinflammation, and infection. Although their phenotype and functional properties are well known and have been extensively studied, their lineage relationship with other NK cell subsets is not fully defined, nor is their precise hematopoietic origin. In this article, we summarize recent studies about CD56(bright) NK cells in health and disease and briefly discuss the current controversies surrounding them.

Neurturin influences inflammatory responses and airway remodeling in different mouse asthma models.

Neurturin (NTN) was previously described for its neuronal activities, but recently, we have shown that this factor is also involved in asthma physiopathology. However, the underlying mechanisms of NTN are unclear. The aim of this study was to investigate NTN involvement in acute bronchial Th2 responses, to analyze its interaction with airway structural cells, and to study its implication in remodeling during acute and chronic bronchial inflammation in C57BL/6 mice. We analyzed the features of allergic airway inflammation in wild-type and NTN(-/-) mice after sensitization with two different allergens, OVA and house dust mite. We showed that NTN(-/-) dendritic cells and T cells had a stronger tendency to activate the Th2 pathway in vitro than similar wild-type cells. Furthermore, NTN(-/-) mice had significantly increased markers of airway remodeling like collagen deposition. NTN(-/-) lung tissues showed higher levels of neutrophils, cytokine-induced neutrophil chemoattractant, matrix metalloproteinase 9, TNF-α, and IL-6. Finally, NTN had the capacity to decrease IL-6 and TNF-α production by immune and epithelial cells, showing a direct anti-inflammatory activity on these cells. Our findings support the hypothesis that NTN could modulate the allergic inflammation in different mouse asthma models.

NK cells with KIR2DS2 immunogenotype have a functional activation advantage to efficiently kill glioblastoma and prolong animal survival.

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.

NK cell killer Ig-like receptor repertoire acquisition and maturation are strongly modulated by HLA class I molecules.

The interaction between clonally distributed inhibitory receptors and their activating counterparts on NK cells and HLA class I molecules defines NK cell functions, but the role of HLA class I ligands in the acquisition of their receptors during NK development is still unclear. Although some studies demonstrated that HLA-C affects the expression of killer Ig-like receptors (KIR), other studies showed that NK cells acquire their KIR repertoire in a stochastic manner. Only when infected with human CMV is an expansion of self-specific KIR(+) NKG2C(+) NK cells detected. To gain more insight into this question, we compared the coexpression of different KIR molecules, NKG2A, CD8, and CD57, on NK cells in healthy donors and seven patients with deficient HLA class I expression due to mutations in one of the TAP genes. Our results show a correlation between the presence/absence of HLA class I molecules and the coexpression of their receptors. In an HLA class I low-expression context, an increase in KIR molecules' coexpression is detected on the NKG2A(+) CD8(+) subset. In functional assays, hyporesponsiveness was observed for TAP-deficient NK cells derived from four patients. In contrast, NK cells from patient five were functional, whereas CD107a(+) and IFN-γ(+) CD56(dim) NK cells presented a different pattern of HLA class I receptors compared with healthy donors. Taken together, our results provide strong evidence for the role of HLA class I molecules in NK cell maturation and KIR repertoire acquisition.

Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia.

Recent studies demonstrated that autophagy is an important regulator of innate immune response. However, the mechanism by which autophagy regulates natural killer (NK) cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis. The work presented here provides a cutting-edge advance in our understanding of the mechanism by which hypoxia-induced autophagy impairs NK-mediated lysis in vitro and paves the way for the formulation of more effective NK cell-based antitumor therapies.

NK cells in central nervous system disorders.

NK cells are important players in immunity against pathogens and neoplasms. As a component of the innate immune system, they are one of the first effectors on sites of inflammation. Through their cytokine production capacities, NK cells participate in the development of a potent adaptive immune response. Furthermore, NK cells were found to have regulatory functions to limit and prevent autoimmunity via killing of autologous immune cells. These paradoxical functions of NK cells are reflected in CNS disorders. In this review, we discuss the phenotypes and functional features of peripheral and brain NK cells in brain tumors and infections, neurodegenerative diseases, acute vascular and traumatic damage, as well as mental disorders. We also discuss the implication of NK cells in neurotoxicity and neuroprotection following CNS pathology, as well as the crosstalk between NK cells and brain-resident immune cells.

Natural killer cells in intracranial neoplasms: presence and therapeutic efficacy against brain tumours.

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.

Bridging the gap with multispecific immune cell engagers in cancer and infectious diseases.

By binding to multiple antigens simultaneously, multispecific antibodies are expected to substantially improve both the activity and long-term efficacy of antibody-based immunotherapy. Immune cell engagers, a subclass of antibody-based constructs, consist of engineered structures designed to bridge immune effector cells to their target, thereby redirecting the immune response toward the tumor cells or infected cells. The increasing number of recent clinical trials evaluating immune cell engagers reflects the important role of these molecules in new therapeutic approaches for cancer and infections. In this review, we discuss how different immune cell types (T and natural killer lymphocytes, as well as myeloid cells) can be bound by immune cell engagers in immunotherapy for cancer and infectious diseases. Furthermore, we explore the preclinical and clinical advancements of these constructs, and we discuss the challenges in translating the current knowledge from cancer to the virology field. Finally, we speculate on the promising future directions that immune cell engagers may take in cancer treatment and antiviral therapy.

Increased Th2 cytokine secretion, eosinophilic airway inflammation, and airway hyperresponsiveness in neurturin-deficient mice.

Neurotrophins such as nerve growth factor and brain-derived neurotrophic factor have been described to be involved in the pathogenesis of asthma. Neurturin (NTN), another neurotrophin from the glial cell line-derived neurotrophic factor family, was shown to be produced by human immune cells: monocytes, B cells, and T cells. Furthermore, it was previously described that the secretion of inflammatory cytokines was dramatically stimulated in NTN knockout (NTN(-/-)) mice. NTN is structurally similar to TGF-ß, a protective cytokine in airway inflammation. This study investigates the implication of NTN in a model of allergic airway inflammation using NTN(-/-) mice. The bronchial inflammatory response of OVA-sensitized NTN(-/-) mice was compared with wild-type mice. Airway inflammation, Th2 cytokines, and airway hyperresponsiveness (AHR) were examined. NTN(-/-) mice showed an increase of OVA-specific serum IgE and a pronounced worsening of inflammatory features. Eosinophil number and IL-4 and IL-5 concentration in the bronchoalveolar lavage fluid and lung tissue were increased. In parallel, Th2 cytokine secretion of lung draining lymph node cells was also augmented when stimulated by OVA in vitro. Furthermore, AHR was markedly enhanced in NTN(-/-) mice after sensitization and challenge when compared with wild-type mice. Administration of NTN before challenge with OVA partially rescues the phenotype of NTN(-/-) mice. These findings provide evidence for a dampening role of NTN on allergic inflammation and AHR in a murine model of asthma.

Special Issue "New Developments in Natural Killer Cells for Immunotherapy".

Since their formal discovery in 1975, natural killer (NK) cells have always been proposed in the literature as a potential treatment for cancer and viral infections [...].