Dual-targeting CD33/CD123 NANOBODY T-cell engager with potent anti-AML activity and good safety profile.

ABSTRACT: Novel therapies are needed for effective treatment of acute myeloid leukemia (AML). Relapse is common and salvage treatment with cytotoxic chemotherapy is rarely curative. CD123 and CD33, 2 clinically validated targets in AML, are jointly expressed on blasts and leukemic stem cells in >95% of patients with AML. However, their expression is heterogenous between subclones and between patients, which may affect the efficacy of single-targeting agents in certain patient populations. We present here a dual-targeting CD33/CD123 NANOBODY T-cell engager (CD33/CD123-TCE) that was designed to decrease the risk of relapse from possible single antigen-negative clones and to increase coverage within and across patients. CD33/CD123-TCE killed AML tumor cells expressing 1 or both antigens in vitro. Compared with single-targeting control compounds, CD33/CD123-TCE conferred equal or better ex vivo killing of AML blasts in most primary AML samples tested, suggesting a broader effectiveness across patients. In a disseminated cell-line-derived xenograft mouse model of AML, CD33/CD123-TCE cleared cancer cells in long bones and in soft tissues. As cytokine release syndrome is a well-documented adverse effect of TCE, the compound was tested in a cytokine release assay and shown to induce less cytokines compared to a CD123 single-targeting control. In an exploratory single-dose nonhuman primate study, CD33/CD123-TCE revealed a favorable PK profile. Depletion of CD123 and CD33 expressing cells was observed, but there were neither signs of cytokine release syndrome nor clinical signs of toxicity. Taken together, the CD33/CD123 dual-targeting NANOBODY TCE exhibits potent and safe anti-AML activity and promises a broad patient coverage.

A Computational Pipeline for the Diagnosis of CVID Patients.

Common variable immunodeficiency (CVID) is one of the most frequently diagnosed primary antibody deficiencies (PADs), a group of disorders characterized by a decrease in one or more immunoglobulin (sub)classes and/or impaired antibody responses caused by inborn defects in B cells in the absence of other major immune defects. CVID patients suffer from recurrent infections and disease-related, non-infectious, complications such as autoimmune manifestations, lymphoproliferation, and malignancies. A timely diagnosis is essential for optimal follow-up and treatment. However, CVID is by definition a diagnosis of exclusion, thereby covering a heterogeneous patient population and making it difficult to establish a definite diagnosis. To aid the diagnosis of CVID patients, and distinguish them from other PADs, we developed an automated machine learning pipeline which performs automated diagnosis based on flow cytometric immunophenotyping. Using this pipeline, we analyzed the immunophenotypic profile in a pediatric and adult cohort of 28 patients with CVID, 23 patients with idiopathic primary hypogammaglobulinemia, 21 patients with IgG subclass deficiency, six patients with isolated IgA deficiency, one patient with isolated IgM deficiency, and 100 unrelated healthy controls. Flow cytometry analysis is traditionally done by manual identification of the cell populations of interest. Yet, this approach has severe limitations including subjectivity of the manual gating and bias toward known populations. To overcome these limitations, we here propose an automated computational flow cytometry pipeline that successfully distinguishes CVID phenotypes from other PADs and healthy controls. Compared to the traditional, manual analysis, our pipeline is fully automated, performing automated quality control and data pre-processing, automated population identification (gating) and deriving features from these populations to build a machine learning classifier to distinguish CVID from other PADs and healthy controls. This results in a more reproducible flow cytometry analysis, and improves the diagnosis compared to manual analysis: our pipelines achieve on average a balanced accuracy score of 0.93 (±0.07), whereas using the manually extracted populations, an averaged balanced accuracy score of 0.72 (±0.23) is achieved.

A CARD9 Founder Mutation Disrupts NF-κB Signaling by Inhibiting BCL10 and MALT1 Recruitment and Signalosome Formation.

Background: Inherited CARD9 deficiency constitutes a primary immunodeficiency predisposing uniquely to chronic and invasive fungal infections. Certain mutations are shown to negatively impact CARD9 protein expression and/or NF-κB activation, but the underlying biochemical mechanism remains to be fully understood. Objectives: To investigate a possible founder origin of a known CARD9 R70W mutation in five families of Turkish origin. To explore the biochemical mechanism of immunodeficiency by R70W CARD9. Methods: We performed haplotype analysis using microsatellite markers and SNPs. We designed a model system exploiting a gain-of-function (GOF) CARD9 L213LI mutant that triggers constitutive NF-κB activation, analogous to an oncogenic CARD11 mutant, to study NF-κB signaling and signalosome formation. We performed reporter assays, immunoprecipitation and confocal imaging on HEK cells overexpressing different CARD9 variants. Results: We identified a common haplotype, thus providing evidence for a common Turkish founder. CARD9 R70W failed to activate NF-κB and abrogated NF-κB activation by WT CARD9 and by GOF CARD9. Notably, R70W CARD9 also exerted negative effects on NF-κB activation by CARD10, CARD11, and CARD14. Consistent with the NF-κB results, the R70W mutation prevented GOF CARD9 to pull down the signalosome partner proteins BCL10 and MALT1. This reflected into drastic reduction of BCL10 filamentous assemblies in a cellular context. Indeed, structural analysis revealed that position R70 in CARD9 maps at the putative interface between successive CARD domains in CARD9 filaments. Conclusions: The R70W mutation in CARD9 prevents NF-κB activation by inhibiting productive interactions with downstream BCL10 and MALT1, necessary for assembly of the filamentous CARD9-BCL10-MALT1 signalosome.

IL-21 Is Increased in Nasal Polyposis and after Stimulation with Staphylococcus aureus Enterotoxin B.

BACKGROUND: Chronic rhinosinusitis with nasal polyposis (CRSwNP) is an inflammatory disease associated with lymphoid aggregates and local IgE production related to Staphylococcus aureus enterotoxins. T-follicular helper cells and their effector cytokine interleukin (IL)-21 play an important role in germinal center proliferation. METHODS: IL-21 was determined on the mRNA level by qPCR in nasal tissue of 3 groups of patients: control (n = 17), chronic rhinosinusitis without nasal polyposis (CRSsNP; n = 23), and CRSwNP (n = 35). The expression of IL-21 by CD4+ T cells was analyzed in tissue at baseline and after 24-h stimulation of tissue fragments with S. aureus enterotoxin B (SEB) using flow cytometry. Finally, human nasal IL-21+CXCR5+CD4+ T cells were isolated and coincubated with human blood naive B cells to investigate their functionality. RESULTS: IL-21 mRNA expression was increased in the CRSwNP group (p < 0.05) compared to the control group, and B-cell lymphoma-6 and B-lymphocyte-induced maturation protein-1 were upregulated in CRSwNP versus CRSsNP. Furthermore, SEB was able to increase IL-21 mRNA expression significantly (p < 0.01) in nasal polyps. Flow cytometry revealed that the source of IL-21 was predominantly CD4+ T cells and that IL-21+CD4+ T cells were significantly increased in polyp tissue and further increased after SEB stimulation. Finally, tissue CXCR5+CD4+ T cells derived from nasal polyp tissue were able to induce maturation of human naive B cells. CONCLUSIONS: IL-21- and IL-21-producing CD4+ T cells were increased in CRSwNP. In addition, SEB induced an increase in IL-21 and IL-21+CD4+ T cells, suggesting that S. aureus can modulate the function of Tfh cells in nasal polyps. We speculate that T-follicular helper cells and IL-21 are important in the pathophysiology of CRSwNP.

Early-onset primary antibody deficiency resembling common variable immunodeficiency challenges the diagnosis of Wiedeman-Steiner and Roifman syndromes.

Syndromic primary immunodeficiencies are rare genetic disorders that affect both the immune system and other organ systems. More often, the immune defect is not the major clinical problem and is sometimes only recognized after a diagnosis has been made based on extra-immunological abnormalities. Here, we report two sibling pairs with syndromic primary immunodeficiencies that exceptionally presented with a phenotype resembling early-onset common variable immunodeficiency, while extra-immunological characteristics were not apparent at that time. Additional features not typically associated with common variable immunodeficiency were diagnosed only later, including skeletal and organ anomalies and mild facial dysmorphism. Whole exome sequencing revealed KMT2A-associated Wiedemann-Steiner syndrome in one sibling pair and their mother. In the other sibling pair, targeted testing of the known disease gene for Roifman syndrome (RNU4ATAC) provided a definite diagnosis. With this study, we underline the importance of an early-stage and thorough genetic assessment in paediatric patients with a common variable immunodeficiency phenotype, to establish a conclusive diagnosis and guide patient management. In addition, this study extends the mutational and immunophenotypical spectrum of Wiedemann-Steiner and Roifman syndromes and highlights potential directions for future pathophysiological research.

Treatment of Intestinal Fibrosis in Experimental Inflammatory Bowel Disease by the Pleiotropic Actions of a Local Rho Kinase Inhibitor.

BACKGROUND: Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFß-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. METHODS: Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. RESULTS: ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFß1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. CONCLUSIONS: Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.

The transcriptome of lung tumor-infiltrating dendritic cells reveals a tumor-supporting phenotype and a microRNA signature with negative impact on clinical outcome.

Targeting immunomodulatory pathways has ushered a new era in lung cancer therapy. Further progress requires deeper insights into the biology of immune cells in the lung cancer micro-environment. Dendritic cells (DCs) represent a heterogeneous and highly plastic immune cell system with a central role in controlling immune responses. The intratumoral infiltration and activation status of DCs are emerging as clinically relevant parameters in lung cancer. In this study, we used an orthotopic preclinical model of lung cancer to dissect how the lung tumor micro-environment affects tissue-resident DCs and extract novel biologically and clinically relevant information. Lung tumor-infiltrating leukocytes expressing generic DC markers were found to predominantly consist of CD11b+ cells that, compare with peritumoral lung DC counterparts, strongly overexpress the T-cell inhibitory molecule PD-L1 and acquire classical surface markers of tumor-associated macrophages (TAMs). Transcriptome analysis of these CD11b+ tumor-infiltrating DCs (TIDCs) indicates impaired antitumoral immunogenicity, confirms the skewing toward TAM-related features, and indicates exposure to a hypoxic environment. In parallel, TIDCs display a specific microRNA (miRNA) signature dominated by the prototypical lung cancer oncomir miR-31. In vitro, hypoxia drives intrinsic miR-31 expression in CD11b+ DCs. Conditioned medium of miR-31 overexpressing CD11b+ DCs induces pro-invasive lung cancer cell shape changes and is enriched with pro-metastatic soluble factors. Finally, analysis of TCGA datasets reveals that the TIDC-associated miRNA signature has a negative prognostic impact in non-small cell lung cancer. Together, these data suggest a novel mechanism through which the lung cancer micro-environment exploits the plasticity of the DC system to support tumoral progression.

Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+ Tie2:cre and Phd3f/f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1ß production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The immunophenotypic fingerprint of patients with primary antibody deficiencies is partially present in their asymptomatic first-degree relatives.

The etiology of primary antibody deficiencies is largely unknown. Beside rare monogenic forms, the majority of cases seem to have a more complex genetic basis. Whereas common variable immunodeficiency has been investigated in depth, there are only a few reports on milder primary antibody deficiencies such as idiopathic primary hypogammaglobulinemia and IgG subclass deficiency. We performed flow cytometric immunophenotyping in 33 patients with common variable immunodeficiency, 23 with idiopathic primary hypogammaglobulinemia and 21 with IgG subclass deficiency, as well as in 47 asymptomatic first-degree family members of patients and 101 unrelated healthy controls. All three groups of patients showed decreased memory B- and naïve T-cell subsets and decreased B-cell activating factor receptor expression. In contrast, circulating follicular helper T-cell frequency and expression of inducible T-cell co-stimulator and chemokine receptors were only significantly altered in patients with common variable immunodeficiency. Asymptomatic first-degree family members of patients demonstrated similar, albeit intermediate, alterations in naïve and memory B- and T-cell subsets. About 13% of asymptomatic relatives had an abnormal peripheral B-cell composition. Furthermore, asymptomatic relatives showed decreased levels of CD4+ recent thymic emigrants and increased central memory T cells. Serum IgG and IgM levels were also significantly lower in asymptomatic relatives than in healthy controls. We conclude that, in our cohort, the immunophenotypic landscape of primary antibody deficiencies comprises a spectrum, in which some alterations are shared between all primary antibody deficiencies whereas others are only associated with common variable immunodeficiency. Importantly, asymptomatic first-degree family members of patients were found to have an intermediate phenotype for peripheral B- and T-cell subsets.

Chronic and Invasive Fungal Infections in a Family with CARD9 Deficiency.

Chronic mucocutaneous or invasive fungal infections are generally the result of primary or secondary immune dysfunction. Patients with autosomal recessive CARD9 mutations are also predisposed to recurrent mucocutaneous and invasive fungal infections with Candida spp., dermatophytes (e.g., Trichophyton spp.) and phaeohyphomycetes (Exophiala spp., Phialophora verrucosa). We study a consanguineous family of Turkish origin in which three members present with distinct clinical phenotypes of chronic mucocutaneous and invasive fungal infections, ranging from chronic mucocutaneous candidiasis (CMC) in one patient, treatment-resistant cutaneous dermatophytosis and deep dermatophytosis in a second patient, to CMC with Candida encephalitis and endocrinopathy in a third patient. Two patients consented to genetic testing and were found to have a previously reported homozygous R70W CARD9 mutation. Circulating IL-17 and IL-22 producing T cells were decreased as was IL-6 and granulocyte/macrophage colony-stimulating factor (GM-CSF) secretion upon stimulation with Candida albicans. Patients with recurrent fungal infections in the absence of known immunodeficiencies should be analyzed for CARD9 gene mutations as the cause of fungal infection predisposition.

Local immunoglobulin e in the nasal mucosa: clinical implications.

Immunoglobulin E (IgE) can be highly elevated in the airway mucosa independently of IgE serum levels and atopic status. Mostly, systemic markers are assessed to investigate inflammation in airway disease for research or clinical practice. A more accurate but more cumbersome approach to determine inflammation at the target organ would be to evaluate markers locally. We review evidence for local production of IgE in allergic rhinitis (AR) and chronic rhinosinusitis with nasal polyps (CRSwNP). Diagnostic and therapeutic consequences in clinical practice are discussed. We describe that the airway mucosa has the intrinsic capability to produce IgE. Moreover, not only do IgE-positive B cells reside within the mucosa, but all tools are present locally for affinity maturation by somatic hypermutation (SHM), clonal expansion, and class switch recombination to IgE. Recognizing local IgE in the absence of systemic IgE has diagnostic and therapeutic consequences. Therefore, we emphasize the importance of local IgE in patients with a history of AR or CRSwNP.

C-type lectin-like receptor LOX-1 promotes dendritic cell-mediated class-switched B cell responses.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern-recognition receptor for a variety of endogenous and exogenous ligands. However, LOX-1 function in the host immune response is not fully understood. Here, we report that LOX-1 expressed on dendritic cells (DCs) and B cells promotes humoral responses. On B cells LOX-1 signaling upregulated CCR7, promoting cellular migration toward lymphoid tissues. LOX-1 signaling on DCs licensed the cells to promote B cell differentiation into class-switched plasmablasts and led to downregulation of chemokine receptor CXCR5 and upregulation of chemokine receptor CCR10 on plasmablasts, enabling their exit from germinal centers and migration toward local mucosa and skin. Finally, we found that targeting influenza hemagglutinin 1 (HA1) subunit to LOX-1 elicited HA1-specific protective antibody responses in rhesus macaques. Thus, LOX-1 expressed on B cells and DC cells has complementary functions to promote humoral immune responses.

Lambert-Eaton myasthenic syndrome in a 13-year-old girl with Xp11.22-p11.23 duplication.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the presynaptic neuromuscular junction, typically occurring in adults as a paraneoplastic syndrome. Only rare cases have been reported in childhood. In most childhood cases, malignancies have not been detected but a propensity to autoimmune disease was noticed. Nevertheless, little is known about genetic factors that may contribute to the susceptibility of an individual to develop LEMS. We report on a 13-year-old girl, known with the Xp11.22-p11.23 duplication syndrome, who presented with severe non-paraneoplastic LEMS. The potential role of this microduplication syndrome in the development of LEMS is explored. Previous literature review of twelve Xp11.2 duplication syndrome patients showed that three of them suffered from various autoimmune diseases. The common duplicated region in those three patients and the presented case comprises 12 disease-associated genes including the FOXP3 (Forkhead Box P3) and WAS (Wiskott-Aldrich syndrome) gene, both implicated in immune function. However, it is unclear whether increased gene dosage of one or both of these genes can cause susceptibility to autoimmune diseases. In conclusion, the presented case emphasizes that autoimmune disease is a recurrent feature of the Xp11.2 duplication syndrome, which should be considered in the follow-up of these patients. The exact mechanism underlying this autoimmune propensity remains to be elucidated.

Macrophages induce differentiation of plasma cells through CXCL10/IP-10.

In tonsils, CD138(+) plasma cells (PCs) are surrounded by CD163(+) resident macrophages (Ms). We show here that human Ms (isolated from tonsils or generated from monocytes in vitro) drive activated B cells to differentiate into CD138(+)CD38(++) PCs through secreted CXCL10/IP-10 and VCAM-1 contact. IP-10 production by Ms is induced by B cell-derived IL-6 and depends on STAT3 phosphorylation. Furthermore, IP-10 amplifies the production of IL-6 by B cells, which sustains the STAT3 signals that lead to PC differentiation. IP-10-deficient mice challenged with NP-Ficoll show a decreased frequency of NP-specific PCs and lower titers of antibodies. Thus, our results reveal a novel dialog between Ms and B cells, in which IP-10 acts as a PC differentiation factor.

Local inflammation in chronic upper airway disease.

Chronic Rhinosinusitis (CRS), a chronic upper airway inflammation, is an inflammation of the nose and the paranasal cavities and is highly prevalent. Chronic rhinosinusitis is currently classified as CRS with nasal polyps or CRS without nasal polyps. This review highlights the pathophysiological differences in CRS on remodeling and on T-cell patterns. Nasal polyps have a high co-morbidity with the lower airway inflammatory disease, asthma. Evidence is accumulating for the role of superantigens, Staphylococcus aureus enterotoxins, in CRS with nasal polyps and asthma, both T helper 2 -biased diseases. Until today there are no biomarkers involved in the diagnosis of CRS or the treatment follow-up. Further differentiation of the phenotype of the disease is needed, which will reflect in the development of new biomarkers and in new innovative treatment options. Defining and predicting response to therapy in individual CRS patients is a challenge for future research.

Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion.

Although a fraction of human blood memory CD4(+) T cells expresses chemokine (C-X-C motif) receptor 5 (CXCR5), their relationship to T follicular helper (Tfh) cells is not well established. Here we show that human blood CXCR5(+)CD4(+) T cells share functional properties with Tfh cells and appear to represent their circulating memory compartment. Blood CXCR5(+)CD4(+) T cells comprised three subsets: T helper 1 (Th1), Th2, and Th17 cells. Th2 and Th17 cells within CXCR5(+), but not within CXCR5(-), compartment efficiently induced naive B cells to produce immunoglobulins via interleukin-21 (IL-21). In contrast, Th1 cells from both CXCR5(+) and CXCR5(-) compartments lacked the capacity to help B cells. Patients with juvenile dermatomyositis, a systemic autoimmune disease, displayed a profound skewing of blood CXCR5(+) Th cell subsets toward Th2 and Th17 cells. Importantly, the skewing of subsets correlated with disease activity and frequency of blood plasmablasts. Collectively, our study suggests that an altered balance of Tfh cell subsets contributes to human autoimmunity.

A T cell-dependent mechanism for the induction of human mucosal homing immunoglobulin A-secreting plasmablasts.

Mucosal immunoglobulin A (IgA) secreted by local plasma cells (PCs) is a critical component of mucosal immunity. Although IgA class switching can occur at mucosal sites, high-affinity PCs are optimally generated in germinal centers (GCs) in a T cell-dependent fashion. However, how CD4(+) helper T cells induce mucosal-homing IgA-PCs remains unclear. Here, we show that transforming growth factor beta1 (TGFbeta1) and interleukin 21 (IL-21), produced by follicular helper T cells (Tfh), synergized to generate abundant IgA-plasmablasts (PBs). In the presence of IL-21, TGFbeta1 promoted naive B cell proliferation and differentiation and overrode IL-21-induced IgG class switching in favor of IgA. Furthermore, TGFbeta1 and IL-21 downregulated CXCR5 while upregulating CCR10 on plasmablasts, enabling their exit from GCs and migration toward local mucosa. This was supported by the presence of CCR10(+)IgA(+)PBs in tonsil GCs. These findings show that Tfh contribute to mucosal IgA. Thus, mucosal vaccines should aim to induce robust Tfh responses.

Activation of immature monocyte-derived dendritic cells after transduction with high doses of lentiviral vectors.

Dendritic cells (DCs) are an attractive tool for immunomodulation, targeting mature DCs (mDCs) for immunization or immature/semimature DCs (iDCs) for tolerization. Therefore, introducing antigens into DCs has become a prime topic in various immunological disciplines. Numerous studies have shown that lentiviruses are an efficient vehicle for this purpose. This study evaluates the effects of lentiviral transduction on iDC activation. Immature DCs are efficiently transduced with increasing doses of lentivirus without affecting cell viability. Transduction at low multiplicities of infection (MOIs) did not result in phenotypical or functional maturation. Higher doses of lentivirus, however, resulted in upregulation of adhesion, costimulatory, and HLA molecules, as well as in increased allostimulatory capacity and secretion of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha. Production of IL-12 p70, IL-10, and interferon-alpha was observed only at extremely high doses. Protein kinase R phosphorylation on transduction at an MOI of 150 was demonstrated by Western blotting. A Toll-like receptor (TLR)-driven luciferase reporter assay showed dose-dependent activation of TLR2, TLR3, and TLR8, which was independent of the pseudotype, production, or transduction protocol and was abrogated on heat inactivation. These data show that lentiviral vectors provide not only the antigen but also appropriate activation signals to iDCs, favoring their use for immunotherapy and vaccine development.

From pathogen to medicine: HIV-1-derived lentiviral vectors as vehicles for dendritic cell based cancer immunotherapy.

Over the years, the unique capacity of dendritic cells (DC) for efficient activation of naive T cells has led to their extensive use in cancer immunotherapy protocols. In order to be able to fulfil their role as antigen-presenting cells, the antigen of interest needs to be efficiently introduced and subsequently correctly processed and presented by the DC. For this purpose, a variety of both viral and non-viral antigen-delivery systems have been evaluated. Amongst those, HIV-1-derived lentiviral vectors have been used successfully to transduce DC. This review considers the use of HIV-1-derived lentiviral vectors to transduce human and murine DC for cancer immunotherapy. Lentivirally transduced DC have been shown to present antigenic peptides, prime transgene-specific T cells in vitro and elicit a protective cytotoxic T-lymphocyte (CTL) response in animal models. Different parameters determining the efficacy of transduction are considered. The influence of lentiviral transduction on the DC phenotype and function is described and the induction of immune responses by lentivirally transduced DC in vitro and in vivo is discussed in detail. In addition, direct in vivo administration of lentiviral vectors aiming at the induction of antigen-specific immunity is reviewed. This strategy might overcome the need for ex vivo generation and antigen loading of DC. Finally, future perspectives towards the use of lentiviral vectors in cancer immunotherapy are presented.