Human NK Cell Up-regulation of CD69, HLA-DR, Interferon γ Secretion and Cytotoxic Activity by Plasmacytoid Dendritic Cells is Regulated through Overlapping but Different Pathways

Human plasmacytoid dendritic cells secrete high levels of IFNα and are thus implicated in the activation of NK cells. Activated NK cells are characterised by the up-regulation of CD69 and MHC class II DR expression, secretion of IFN γ and enhanced cytotoxicity. We show that pDC mediate these processes by different mechanisms, some of which overlap. Human NK cells were analysed after co-culture with immature or CpG-matured blood pDC or with supernatant from these cells. Maximal CD69 expression by NK cells was mediated by supernatant from mature pDC and did not require pDC contact. Up-regulation was due in part to IFNα but also to factors in IFNα negative supernatant from immature DC. HLA-DR expression was independent of secreted molecules but required contact with immature or mature DC. Enhanced NK cytotoxicity, measured by killing of K562 targets and expression of CD107a, was mediated by multiple factors including type I IFN, supernatant from immature pDC cultures and contact with immature or mature pDC. These factors act cumulatively to enhance cytotoxcity. Thus different parameters of pDC mediated NK cell activation are regulated by distinct pathways

Loss of NK Stimulatory Capacity by Plasmacytoid and Monocyte-Derived DC but Not Myeloid DC in HIV-1 Infected Patients

Dendritic cells (DC) are potent inducers of natural killer (NK) cells. There are two distinct populations in blood, myeloid (mDC) and plasmacytoid (pDC) but they can also be generated In vitro from monocytes (mdDC). Although it is established that blood DC are lost in HIV-1 infection, the full impact of HIV-1 infection on DC-NK cell interactions remains elusive. We thus investigated the ability of pDC, mDC, and mdDC from viremic and anti-retroviral therapy-treated aviremic HIV-1+ patients to stimulate various NK cell functions. Stimulated pDC and mdDC from HIV-1+ patients showed reduced secretion of IFN-α and IL-12p70 respectively and their capacity to stimulate expression of CD25 and CD69, and IFN-γ secretion in NK cells was also reduced. pDC activation of NK cell degranulation in response to a tumour cell line was severely reduced in HIV-1+ patients but the ability of mDC to activate NK cells was not affected by HIV-1 infection, with the exception of HLA-DR induction. No differences were observed between viremic and aviremic patients indicating that anti-retroviral therapy had minimal effect on restoration on pDC and mdDC-mediated activation of NK cells. Results from this study provide further insight into HIV-1 mediated suppression of innate immune functions

The role of oncolytic virus-induced anti-tumour immunity in haematological malignancies

Haematological malignancies (HM) are a diverse group of relatively rare, but often life-threatening cancers. Over 900 000 people worldwide are expected to receive a HM diagnosis in 2018. Most types of HM have an increasing incidence with age, which is often associated with poor survival due to the inadequate treatment options available. Two types of HM with a particularly poor outlook are acute myeloid leukaemia (AML), which is an acute cancer of immature myeloid blood cells, and multiple myeloma (MM) which is an incurable malignancy with primary focus in the bone marrow (BM). Oncolytic viruses (OV) preferentially infect and kill malignantly transformed cells. OV therapy (OVT) is a promising treatment strategy which has recently been approved for clinical use in the treatment of melanoma. In addition to direct lytic killing, the induction of an anti-tumour immune response is thought to be essential for efficient OVT with establishment of long-term protection against tumour recurrence. While research efforts into OVT for solid malignancies have seen a surge in recent years, HM remains under-investigated in this context, in particular with regards to the anti-tumour immune response. Thus, the overall aim of this study was to examine the role of OV-induced anti-tumour immunity in HM. Two OV were evaluated in the context of MM; both reovirus and coxsackievirus A21 (CVA21) were found to induce anti-tumour immune responses in vitro comprising cytokine-mediated killing, natural killer cellmediated cytotoxicity, and the generation of tumour-specific cytotoxic T cells. Moreover, reovirus treatment showed efficacy in an immunocompetent in vivo model, along with evidence of the onset of an immune response primarily in the spleen. Pivotally, this study is the first to evaluate CVA21 as a treatment for AML. CVA21 was able to induce anti-tumour immunity in AML in vitro using both AML cell lines and a cohort of primary AML samples, despite resistance to lytic killing. Furthermore, examination of peripheral blood samples from patients with solid malignancies following intravenous administration of CVA21 provided “proof of principle” evidence that CVA21 was able to activate immune cells in the peripheral circulation in a state of malignancy, which is crucial for the future development of CVA21 OVT. The obtained results also established the cellular mechanisms responsible for CVA21-induced anti-tumour immunity. Plasmacytoid dendritic cells were identified as the main detectors of CVA21 infection and were responsible for orchestrating both innate and adaptive anti-tumour immune responses. These findings provide novel insights into the immunobiology of CVA21 with importance for the continued clinical development of this OV. In conclusion, this study has demonstrated that OVT, as a well-tolerated, specific, and long-lasting therapy, has the potential to improve the treatment of both MM and AML through the induction of a multi-component anti-tumour immune response

Molecular Aspects and Future Perspectives of Cytokine-Based Anti-cancer Immunotherapy

Cytokine-based immunotherapy is a promising field in the cancer treatment, since cytokines, as proteins of the immune system, are able to modulate the host immune response toward cancer cell, as well as directly induce tumor cell death. Since a low dose monotherapy with some cytokines has no significant therapeutic results and a high dose treatment leads to a number of side effects caused by the pleiotropic effect of cytokines, the problem of understanding the influence of cytokines on the immune cells involved in the pro- and anti-tumor immune response remains a pressing one. Immune system cells carry CD makers on their surface which can be used to identify various populations of cells of the immune system that play different roles in pro- and anti-tumor immune responses. This review discusses the functions and specific CD markers of various immune cell populations which are reported to participate in the regulation of the immune response against the tumor. The results of research studies and clinical trials investigating the effect of cytokine therapy on the regulation of immune cell populations and their surface markers are also discussed. Current trends in the development of cancer immunotherapy, as well as the role of cytokines in combination with other therapeutic agents, are also discussed

Nouvelle approche immunothérapeutique afin de traiter le neuroblastome réfractaire chez l’enfant

Malgré plusieurs chimiothérapies suivies d’une transplantation et d’une immunothérapie, 40% des patients avec un neuroblastome (NB) à haut risque subissent une progression de la maladie ou une rechute. L’échec de ces traitements est attribué à la présence de cellules initiatrices de tumeur (TIC) qui expriment le marqueur CD133 et qui sont souvent résistantes aux agents chimiothérapeutiques. Les cellules Natural Killer (NK), qui possèdent un effet anti-tumoral, peuvent être utilisées dans le cadre du développement de nouvelles approches immuno-thérapeutiques. Nous posons l’hypothèse que les cellules NK activées éliminent efficacement les TIC et contribuent à la réduction des risques de rechute. De plus, il est possible d’augmenter l’effet anti-tumoral des cellules NK contre le NB. L’activité cytotoxique des cellules NK est augmentée par des cellules dendritiques plasmacytoïdes (pDC) activées. A la suite de la stimulation de leurs récepteurs Toll-like les pDC produisent de grandes quantités d'interféron-alpha (IFN-α). Nous avons étudié les propriétés lytiques des cellules NK contre des lignées cellulaires de NB à la suite de leur activation par l’IFN-α ou des pDC activées. Nos résultats révèlent une augmentation de l’activité cytolytique des cellules NK contre ces lignées en réponse à une stimulation par les pDC activées. De plus, les cellules de NB CD133+ ou celles résistantes à l’immunothérapie dirigée contre le GD2 sont sensibles à la lyse médiée par les cellules NK stimulées par les pDC. Nous avons examiné les mécanismes cellulaires impliqués dans la lyse des cellules de NB. Nous montrons que cette cytotoxicité est médiée en partie par TRAIL induisant l'apoptose et en partie par la libération des granules cytotoxiques. Ainsi, ces résultats permettent de proposer une nouvelle approche immuno-thérapeutique complémentaire au traitement par l’anticorps anti-GD2 pour les patients atteints de NB à haut risque.Despite aggressive treatment by chemotherapy followed by transplantation and treatment with anti-tumor cell disialoganglioside (GD2) monoclonal antibody, IL-2, GM-CSF and retinoic acid, 40% of patients with high-risk neuroblastoma (NB) still undergo disease progression or relapse. Furthermore, tumor-initiating cells (TIC) expressing the CD133 marker are present in NB tumors and are more resistant to chemotherapy. To evaluate a new immunotherapeutic approach, we took advantage of the anti-tumor effect of Natural Killer (NK) cells. We hypothesized that activated NK cells would be a potent therapeutic strategy to eliminate TIC and reduce relapse of NB. We aimed to establish the best strategy to increase the NK cell mediated cytotoxicity against NB. NK cell cytotoxic activity is increased by cytokines, chemokines and activated plasmacytoid dendritic cells (pDC) which produce high amounts of interferon-alpha (IFN-α) upon Toll-like receptor stimulation. We investigated NK-cell lytic properties against NB cell lines following activation by IFN-α or activated pDC. Our results reveal an increased cytolytic activity of NK cells against NB cell lines after stimulation by activated pDC, CD133+ (TIC) as well as anti-GD2 resistant NB cells are sensitive to NK cell mediated cytotoxicity following stimulation by activated pDC. We also examined the cellular mechanisms involved in NK cell-mediated lysis of NB cell lines. The increased cytotoxicity is partially mediated by TRAIL induced apoptosis and as well as by the release of cytolytic granules. In conclusion, we propose a new immunotherapeutic approach that can be used in combination with the anti-GD2 therapy for the treatment of high-risk NB patients

Unique and Overlapping Actions of Type I and III IFNs in Influenza A Virus Infection and Implications for Therapy

Influenza A virus (IAV) poses a significant public health burden. Severe disease is characterised by infected lung airway epithelial cells (AECs), inflammation and tissue damage. However, disease severity differs between individuals and this cannot be entirely explained by inter-individual differences in pre-existing immunity or comorbidities. Host-specific, genetically determined factors must contribute to susceptibility. Type I interferon (IFNαβ) is known to have antiviral function in vitro, but its role in restricting IAV infection in vivo is controversial. We demonstrate that responsiveness to IFNαβ signalling is a host-specific determinant with protective or pathogenic potential which determines the severity of IAV-induced disease. IAV infected 129, CBA/J and DBA mouse strains showed dramatically increased mortality and lung damage, yet higher levels of pulmonary IFNαβ, compared to C57BL/6 or BALB/C mice. Ablation of IFNαβR signalling in 129 mice markedly reduced mortality, levels of proinflammatory cytokines, inflammatory cell recruitment and AEC apoptosis. Susceptibility to IAV-induced disease is influenced by the number of functional alleles for the IFNαβR subunit, IFNAR1, within the genome. IFNAR1+/-(129) mice were less susceptible than wild type 129s yet more so than IFNαβR-/-(129) mice. Conversely, triplication of a section of murine chromosome 16 that includes IFNAR1 in C57BL/6 mice enhanced IFNα response to IAV infection and downstream immunopathology. Finally, IFNα therapy of infected B6. A2G-Mxl mice reduced IAV titers, yet increased secretion of proinflammatory cytokines, innate cell recruitment and AEC apoptosis in the lung, due to the potent immunostimulatory capability to IFNα. In contrast, treatment with IFNλ, whose receptor is largely restricted to AECs, promoted IAV control without exacerbating IAV-induced inflammation. Thus, by manipulating the IFNαβ signal in various ways, we demonstrate that excessive IFNαβ in IAV infection can increase AEC death and enhance proinflammatory responses that ultimately increase disease severity. Our findings have important implications for prediction and treatment of severe influenza