Fusion of Bacterial Flagellin to a Dendritic Cell-Targeting αCD40 Antibody Construct Coupled With Viral or Leukemia-Specific Antigens Enhances Dendritic Cell Maturation and Activates Peptide-Responsive T Cells.

Conventional dendritic cell (DC) vaccine strategies, in which DCs are loaded with antigens ex vivo, suffer biological issues such as impaired DC migration capacity and laborious GMP production procedures. In a promising alternative, antigens are targeted to DC-associated endocytic receptors in vivo with antibody-antigen conjugates co-administered with toll-like receptor (TLR) agonists as adjuvants. To combine the potential advantages of in vivo targeting of DCs with those of conjugated TLR agonists, we generated a multifunctional antibody construct integrating the DC-specific delivery of viral- or tumor-associated antigens and DC activation by TLR ligation in one molecule. We validated its functionality in vitro and determined if TLR ligation might improve the efficacy of such a molecule. In proof-of-principle studies, an αCD40 antibody containing a CMV pp65-derived peptide as an antigen domain (αCD40CMV) was genetically fused to the TLR5-binding D0/D1 domain of bacterial flagellin (αCD40.FlgCMV). The analysis of surface maturation markers on immature DCs revealed that fusion of flagellin to αCD40CMV highly increased DC maturation (3.4-fold elevation of CD80 expression compared to αCD40CMV alone) by specifically interacting with TLR5. Immature DCs loaded with αCD40.FlgCMV induced significantly higher CMVNLV-specific T cell activation and proliferation compared to αCD40CMV in co-culture experiments with allogeneic and autologous T cells (1.8-fold increase in % IFN-γ/TNF-α+ CD8+ T cells and 3.9-fold increase in % CMVNLV-specific dextramer+ CD8+ T cells). More importantly, we confirmed the beneficial effects of flagellin-dependent DC stimulation using a tumor-specific neoantigen as the antigen domain. Specifically, the acute myeloid leukemia (AML)-specific mutated NPM1 (mNPM1)-derived neoantigen CLAVEEVSL was delivered to DCs in the form of αCD40mNPM1 and αCD40.FlgmNPM1 antibody constructs, making this study the first to investigate mNPM1 in a DC vaccination context. Again, αCD40.FlgmNPM1-loaded DCs more potently activated allogeneic mNPM1CLA-specific T cells compared to αCD40mNPM1. These in vitro results confirmed the functionality of our multifunctional antibody construct and demonstrated that TLR5 ligation improved the efficacy of the molecule. Future mouse studies are required to examine the T cell-activating potential of αCD40.FlgmNPM1 after targeting of dendritic cells in vivo using AML xenograft models.

Effect of Cytomegalovirus Recombinant Phosphoprotein 150 (pp150) on Maturation and Function of Murine Dendritic Cells: an In-Vitro Study.

BACKGROUND: Tegument protein pp150 of cytomegaloviruses (CMVs) plays a vital role in all stages of viral life cycle, representing the most important tegument protein candidate for HCMV treatment. However, the exact role of pp150 in immune regulation is yet to be elucidated. OBJECTIVE: To examine the effects of pp150 on the maturity and function of murine dendritic cells (DCs). METHODS: Maturity status (CD40, CD86, and MHC-II expression) and phagocytic capacity of DCs (dextran uptake assay) were characterized. Gene expression profiles of ROR-γ, GATA-3, T-bet, and FOXP-3 as well as the protein expression of INF-γ (Th1), IL-4 (Th2), IL-35 (Treg), IL-17A (Th17), IL-22, TNF-α, IL-6, and IL-2 were evaluated in T cells co-cultured with DCs. RESULTS: A significant increase in CD40, CD86, and CCR7 expression and a reduction in the phagocytosis rate were observed in pp150-stimulated DCs compared with unstimulated DCs. T cells co-cultured with stimulated DCs showed higher expressions of ROR-γ, IL-6, IL-2, IL-17A, IL-22, and TNF-α. CONCLUSION: Despite improvements in maturity status, pp150-stimulated DCs did not seem to be able to induce Th1 or Th2 immunity. In fact, Th17 and its mediators, IL-17A and IL-22, might be the main inflammatory factors involved in pp150-stimulated DC's mechanism of action. However, it is necessary to conduct further investigations to corroborate these observations.

Human antigen presenting cells stimulated with Salmonella delivered influenza antigens induce cytokine production and proliferation of human CD4+ T cells in vitro.

This study aimed to investigate whether the human antigen presenting cells (APCs) can process and present Salmonella expressing H7N9 hemagglutinin (Sal-HA), neuraminidase (Sal-NA) or M2 ectodomain (Sal-M2e) to T cells and subsequently activate CD4+ T cell responses in vitro. In this study, APCs generated from human peripheral blood mononuclear cells (PBMCs) were first treated with mitomycin-C, followed by stimulation with Sal-HA, Sal-M2e, Sal-NA or Salmonella alone for 24 h. Subsequently, stimulated APCs were coincubated with untreated PBMCs (1:10) of the same individual for 24 or 72 h and then analysed for cytokine induction and T cell proliferations by qRT-PCR assay and flow cytometry, respectively. Our results demonstrated that APCs stimulated with Sal-HA, Sal-M2e or Sal-NA induced significantly (p < .05) higher CD3+CD4+ T cell proliferations compared to the APCs treated with Salmonella alone. Our data further revealved that APCs treated with Sal-HA induced significantly (p < .05) higher CD3+CD4+ T cell responses compared to the APCs treated with either Sal-M2e or Sal-NA, which both induced almost comparable levels. The T cell proliferation responses were further measured by lymphocyte proliferation assay and the results showed that Sal-HA and Sal-M2e stimulated APCs induced significantly (p < .05) higher proliferations in T cells compared to the APCs stimulated with either Sal-NA or Salmonella alone. With respect to cytokine inductions, APCs treated with either Sal-HA or Sal-M2e induced significantly (p < .05) higher mRNA transcription levels of proinflammatory (IL-1ß, IL-6, IL-12 and IL-23), Th1 (IFN-γ), Th17 (IL-17 and IL-21) and Th2 (IL-10 and TGF-ß) cytokines in T cells compared to Sal-NA or Salmonella alone treated APCs. In conclusion, we show that Salmonella system can efficiently deliver vaccine antigens to APCs and is, thus, capable to elicit heterologous antigen-specific adaptive immunity.

Porous Nanoparticles With Self-Adjuvanting M2e-Fusion Protein and Recombinant Hemagglutinin Provide Strong and Broadly Protective Immunity Against Influenza Virus Infections.

Due to the high risk of an outbreak of pandemic influenza, the development of a broadly protective universal influenza vaccine is highly warranted. The design of such a vaccine has attracted attention and much focus has been given to nanoparticle-based influenza vaccines which can be administered intranasally. This is particularly interesting since, contrary to injectable vaccines, mucosal vaccines elicit local IgA and lung resident T cell immunity, which have been found to correlate with stronger protection in experimental models of influenza virus infections. Also, studies in human volunteers have indicated that pre-existing CD4+ T cells correlate well to increased resistance against infection. We have previously developed a fusion protein with 3 copies of the ectodomain of matrix protein 2 (M2e), which is one of the most explored conserved influenza A virus antigens for a broadly protective vaccine known today. To improve the protective ability of the self-adjuvanting fusion protein, CTA1-3M2e-DD, we incorporated it into porous maltodextrin nanoparticles (NPLs). This proof-of-principle study demonstrates that the combined vaccine vector given intranasally enhanced immune protection against a live challenge infection and reduced the risk of virus transmission between immunized and unimmunized individuals. Most importantly, immune responses to NPLs that also contained recombinant hemagglutinin (HA) were strongly enhanced in a CTA1-enzyme dependent manner and we achieved broadly protective immunity against a lethal infection with heterosubtypic influenza virus. Immune protection was mediated by enhanced levels of lung resident CD4+ T cells as well as anti-HA and -M2e serum IgG and local IgA antibodies.

Porcine monocyte-derived dendritic cells can be differentially activated by vesicular stomatitis virus and its matrix protein mutants.

Vesicular stomatitis virus (VSV) can cause serious vesicular lesions in pigs, and the matrix (M) protein is its predominant virulence factor. Dendritic cells (DCs) act as the bridge between innate and adaptive immune responses. However, the susceptibility of porcine DCs to VSV infection and the role of M protein in modulating the function of infected DCs are still poorly defined. Thus, this study aimed to determine the ability of virulent wild-type VSV(wtVSV) and two attenuated M protein variants (VSVΔM51 and VSVMT) to induce maturation of porcine monocyte-derived DCs (MoDCs) in vitro. It was found that both wtVSV and the M protein mutant VSVs could productively replicate in porcine MoDCs. Infection with wtVSV resulted in weak proinflammatory cytokine responses and interfered with DC maturation via downregulation of the costimulatory molecule complex CD80/86. Whilst VSVΔM51 could activate porcine MoDCs, VSVMT, a highly attenuated recombinant VSV with triple mutations in the M protein, induced a potent maturation of MoDCs, as evidenced by efficient cytokine induction, and upregulation of CD80/86 and MHC class II. Overall, our findings reveal that porcine MoDCs are differentially activated by VSV, dependent on the presence of a functional M protein. M protein plays a crucial role in modulating porcine DC-VSV interactions. The data further support the potential use of VSVMT as a vaccine vector for pigs.

Electrospray Synthesis of Poly(lactide-co-glycolide) Nanoparticles Encapsulating Peptides to Enhance Proliferation of Antigen-Specific CD8+ T Cells.

Polymer nanoparticles (NP) are of escalating interest for their application as immune stimulatory pharmaceutics. The production of nanosized carrier systems is currently being widely investigated, but commonly used techniques, such as the double emulsion technique, are limited by shortcomings of low encapsulation efficiency and poor control over size distribution. In this study, the electrospray technique was successfully implemented and optimized to produce monodisperse 200-nm poly(lactide-co-glycolide) (PLGA) NP. For cytomegalovirus (CMV) pp65 and IE-1 peptides, a consistent encapsulation efficiency of approximately 85% was achieved. In vitro stimulation of peripheral blood mononuclear cells (PBMCs) from CMV+ donors using electrosprayed pp65489-503 peptide-loaded NP revealed a significantly increased proliferation rate and frequency of antigen-specific CD8+ T cells as compared to the soluble peptide. The results of this study demonstrate the suitability of the electrospray technique for production of monodisperse PLGA NP with high drug encapsulation efficiency as promising peptide-based vaccine carriers.

Immune responses to highly conserved influenza A virus matrix 1 peptides.

Influenza vaccine development is considered to be complicated and challenging. Constantly evolving influenza viruses require continuous global monitoring and reformulation of the vaccine strains. Peptides that are conserved among different strains and subtypes of influenza A virus are strongly considered to be attractive targets for development of cross protective influenza vaccines that stimulate cellular responses. In this study, three highly conserved (>90%) matrix 1 peptides that contain multiple T cell epitopes, ILGFVFTLTVPSERGLQRRRF (PM 1), LIRHENRMVLASTTAKA (PM 2) and LQAYQKRMGVQMQR (PM 3), were assessed for their immunogenic potential in vitro by subjecting peripheral blood mononuclear cells from healthy volunteers to repetitive stimulation with these chemically synthesised peptides and measuring their IFN-γ concentrations, proliferation by ELISA, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Seven samples were screened for immunogenicity of PM 1 and PM 2, and six for that of PM 3. All six samples had positive responses (IFN-γ secretion) to PM 3 stimulation, as did five and three for PM 2 and PM 1 respectively. In contrast, seven (PM 1 and PM 2) and four (PM 3) samples showed proliferative response as compared with unstimulated cells. The encouraging immunogenic response generated by these highly conserved matrix 1 peptides indicates they are prospective candidates for development of broadly reactive influenza vaccines.

S100A4 hypomethylation affects epithelial-mesenchymal transition partially induced by LMP2A in nasopharyngeal carcinoma.

To identify cellular target genes involved in NPC cell invasion and metastasis, gene expression profiles of CNE-1 cells with or without ectopic LMP2A expression were compared by using the metastatic gene array. S100 calcium binding protein A4 (S100A4) was the highest increased one among these genes both in mRNA and protein levels of NPC cells. Moreover, S100A4 was upregulated in LMP2A-positive NPC tissues. We found that CNE-1-S100A4 showed significantly increased invasion ability as compared to the controls both in vitro and in vivo, which indicated that S100A4 induced EMT occurrence and promoted metastasis. Notably, the DNA hypomethylation of S100A4 was found in LMP2A-positive NPC tissues. Besides, inhibition of DNA methyltransferases via 5-Aza-dC stimulated the expression of S100A4 in the cells without ectopic LMP2A expression. The methylation changes were confirmed by methylation specific PCR (MSP), suggesting that LMP2A ectopic expression led to the demethylation of S100A4 promoter. These results demonstrated that LMP2A-induced hypomethylation participated in regulating S100A4 expression in NPC. Our findings provide an evidence for the emerging notion that hypomethylation and activation of correlated genes are crucial for metastasis progression in cancer. © 2015 Wiley Periodicals, Inc.

Immunotherapy with Donor T Cells Sensitized with Overlapping Pentadecapeptides for Treatment of Persistent Cytomegalovirus Infection or Viremia.

We conducted a phase I trial of allogeneic T cells sensitized in vitro against a pool of pentadecapeptides (15-mer peptides) spanning the sequence of CMVpp65 for adoptive therapy of 17 allogeneic hematopoietic cell transplant recipients with cytomegalovirus (CMV) viremia or clinical infection persisting despite prolonged treatment with antiviral drugs. All but 3 of the patients had received T cell-depleted transplants without graft-versus-host disease (GVHD) prophylaxis with immunosuppressive drugs after transplantation. The CMVpp65-specific T cells (CMVpp65CTLs) generated were oligoclonal and specific for only 1 to 3 epitopes, presented by a limited set of HLA class I or II alleles. T cell infusions were well tolerated without toxicity or GVHD. Of 17 patients treated with transplant donor (n = 16) or third-party (n = 1) CMVpp65CTLs, 15 cleared viremia, including 3 of 5 with overt disease. In responding patients, the CMVpp65CTLs infused consistently proliferated and could be detected by T cell receptor Vß usage in CMVpp65/HLA tetramer + populations for period of 120 days to up to 2 years after infusion. Thus, CMVpp65CTLs generated in response to synthetic 15-mer peptides of CMVpp65 are safe and can clear persistent CMV infections in the post-transplantation period.

Targeting ß2 adrenergic receptors regulate human T cell function directly and indirectly.

It is well-established that central nervous system activation affects peripheral blood mononuclear cell (PBMCs) function through the release of the catecholamines (Epi) and norepinephrine (NE), which act on ß2-adrenergic receptors (ß2AR). However, most studies have used non-specific stimulation of cells rather than antigen-specific responses. Likewise, few studies have parsed out the direct effects of ß2AR stimulation on T cells versus indirect effects via adrenergic stimulation of antigen presenting cells (APC). Here we report the effect of salmeterol (Sal), a selective ß2AR agonist, on IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells following stimulation with Cytomegalovirus lysate (CMVL-strain AD169) or individual peptides spanning the entire region of the HCMV pp65 protein (pp65). Cells were also stimulated with Staphylococcal enterotoxin B. Additionally, we investigated the effect of Epi and Sal on cytotoxic cell killing of transfected target cells at the single cell level using the CD107a assay. The results show that Sal reduced the percentage of IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells both when applied directly to isolated T cells, and indirectly via treatment of APC. These inhibitory effects were mediated via a ß2 adrenergic-dependent pathway and were stronger for CD8 as compared to CD4 T cells. Similarly, the results show that Sal suppressed cytotoxicity of both CD8 T and NK cells in vitro following stimulation with Chinese hamster ovary cell line transfected with MICA(*009) (T-CHO) and the human erythromyeloblastoid leukemic (K562) cell line. The inhibitory effect on cytotoxicity following stimulation with T-CHO was stronger in NK cells compared with CD8 T cells. Thus, targeting the ß2AR on lymphocytes and on APC leads to inhibition of inflammatory cytokine production and target cell killing. Moreover, there is a hierarchy of responses, with CD8 T cells and NK cells inhibited more effectively than CD4 T cells.

NetFCM: a semi-automated web-based method for flow cytometry data analysis.

Multi-parametric flow cytometry (FCM) represents an invaluable instrument to conduct single cell analysis and has significantly increased our understanding of the immune system. However, due to new techniques allowing us to measure an increased number of phenotypes within the immune system, FCM data analysis has become more complex and labor-intensive than previously. We have therefore developed a semi-automatic gating strategy (NetFCM) that uses clustering and principal component analysis (PCA) together with other statistical methods to mimic manual gating approaches. NetFCM is an online tool both for subset identification as well as for quantification of differences between samples. Additionally, NetFCM can classify and cluster samples based on multidimensional data. We tested the method using a data set of peripheral blood mononuclear cells collected from 23 HIV-infected individuals, which were stimulated with overlapping HIV Gag-p55 and CMV-pp65 peptides or medium alone (negative control). NetFCM clustered the virus-specific CD8+ T cells based on IFNγ and TNF responses into distinct compartments. Additionally, NetFCM was capable of identifying HIV- and CMV-specific responses corresponding to those obtained by manual gating strategies. These data demonstrate that NetFCM has the potential to identify relevant T cell populations by mimicking classical FCM data analysis and reduce the subjectivity and amount of time associated with such analysis. © 2014 International Society for Advancement of Cytometry.

Molecular determinants of susceptibility to oncolytic vesicular stomatitis virus in pancreatic adenocarcinoma.

BACKGROUND: M protein mutant vesicular stomatitis virus (M51R-VSV) has oncolytic properties against many cancers. However, some cancer cells are resistant to M51R-VSV. Herein, we evaluate the molecular determinants of vesicular stomatitis virus (VSV) resistance in pancreatic adenocarcinoma cells. METHODS: Cell viability and the effect of ß-interferon (IFN) were analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Gene expression was evaluated via microarray analysis. Cell infectability was measured by flow cytometry. Xenografts were established in athymic nude mice and treated with intratumoral M51R-VSV. RESULTS: Four of five pancreatic cancer cell lines were sensitive to M51R-VSV, whereas Panc 03.27 cells remained resistant (81 ± 3% viability 72 h after single-cycle infection). Comparing sensitive MiaPaCa2 cells with resistant Panc 03.27 cells, significant differences in gene expression were found relating to IFN signaling (P = 2 × 10(-5)), viral entry (P = 3 × 10(-4)), and endocytosis (P = 7 × 10(-4)). MiaPaCa2 cells permitted high levels of VSV infection, whereas Panc 03.27 cells were capable of resisting VSV cell entry even at high multiplicities of infection. Extrinsic ß-IFN overcame apparent defects in IFN-mediated pathways in MiaPaCa2 cells conferring VSV resistance. In contrast, ß-IFN decreased cell viability in Panc 3.27 cells, suggesting intact antiviral mechanisms. VSV-treated xenografts exhibited reduced tumor growth relative to controls in both MiaPaCa2 (1423 ± 345% versus 164 ± 136%; P < 0.001) and Panc 3.27 (979 ± 153% versus 50 ± 56%; P = 0.002) tumors. Significant lymphocytic infiltration was seen in M51R-VSV-treated Panc 03.27 xenografts. CONCLUSIONS: Inhibition of VSV endocytosis and intact IFN-mediated defenses are responsible for M51R-VSV resistance in pancreatic adenocarcinoma cells. M51R-VSV treatment appears to induce antitumor cellular immunity in vivo, which may expand its clinical efficacy.

Epstein-Barr Virus_Encoded LMP1 upregulates microRNA-21 to promote the resistance of nasopharyngeal carcinoma cells to cisplatin-induced Apoptosis by suppressing PDCD4 and Fas-L.

Approximately 30% of patients with Epstein-Barr virus (EBV)-positive advanced nasopharyngeal carcinoma (NPC) display chemoresistance to cisplatin-based regimens, but the underlying mechanisms are unclear. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, contributes substantially to the oncogenic potential of EBV through the activation of multiple signaling pathways, and it is closely associated with a poorer prognosis for NPC. Recent studies show that EBV infection can induce the expression of many cellular miRNAs, including microRNA-21, a biomarker for chemoresistance. However, neither a link between LMP1 expression and miR-21 upregulation nor their cross talk in affecting chemoresistance to cisplatin have been reported. Here, we observed that stable LMP1-transformed NPC cells were less sensitive to cisplatin treatment based on their proliferation, colony formation, the IC50 value of cisplatin and the apoptosis index. Higher levels of miR-21 were found in EBV-carrying and LMP1-positive cell lines, suggesting that LMP1 may be linked to miR-21 upregulation. These data were confirmed by our results that exogenous LMP1 increased miR-21 in both transiently and stably LMP1-transfected cells, and the knock down of miR-21 substantially reversed the resistance of the NPC cells to cisplatin treatment. Moreover, the proapoptotic factors programmed cell death 4 (PDCD4) and Fas ligand (Fas-L), which were negatively regulated by miR-21, were found to play an important role in the program of LMP1-dependent cisplatin resistance. Finally, we demonstrated that LMP1 induced miR-21 expression primarily by modulating the PI3K/AKT/FOXO3a signaling pathway. Taken together, we revealed for the first time that viral LMP1 triggers the PI3K/Akt/FOXO3a pathway to induce human miR-21 expression, which subsequently decreases the expression of PDCD4 and Fas-L, and results in chemoresistance in NPC cells.

Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine.

We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.

Matrix protein of vesicular stomatitis virus: a potent inhibitor of vascular endothelial growth factor and malignant ascites formation.

Malignant ascites is common in various types of cancers and is difficult to manage. Vascular endothelial growth factor (VEGF) has a pivotal role in malignant ascites. The matrix protein of vesicular stomatitis virus (VSVMP) has been shown to inhibit host gene expression and induce the apoptosis of cancer cells. The present study was designed to determine whether VSVMP suppresses the formation of ascites in ascites-producing peritoneal carcinomatosis. BALB/c female mice, 6-8 weeks old, bearing peritoneal tumors of H22 or MethA cells received an intraperitoneal administration of 50 µg VSVMP/250 µg liposome complexes, 50 µg empty plasmid/250 µg liposome complexes or 0.9% NaCl solution, respectively, every 2 days for 3 weeks. Administration of VSVMP resulted in a significant inhibition in ascites formation, improvement in health condition and prolonged survival of the treated mice. Decreased peritoneum osmolarity and reduced tumor vascularity coincided with dramatic reductions in the VEGF level in ascites fluid and plasma. Examination of floating tumor cells collected from the peritoneal wash revealed an apparently increased number of apoptotic cells and profound downregulation of VEGF mRNA in the VSVMP-treated mice. Our data indicate for the first time that in BALB/c mice bearing H22 or MethA cell peritoneal tumors, VSVMP may inhibit VEGF production and suppress angiogenesis, consequently abolishing ascites formation.

Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity.

The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

Successful isolation and expansion of CMV-reactive T cells from G-CSF mobilized donors that retain a strong cytotoxic effector function.

Cytomegalovirus (CMV) infections post-haematopoietic stem cell transplantation (HSCT) can be effectively controlled through the adoptive transfer of donor-derived CMV-specific T cells (CMV-T). Current strategies involve a second leukapheresis collection from the original donor to manufacture CMV-T, which is often not possible in the unrelated donor setting. To overcome these limitations we have investigated the use of a small aliquot of the original granulocyte-colony stimulating factor (G-CSF) mobilized HSCT graft to manufacture CMV-T. We explored the T cell response to CMVpp65 peptide stimulation in G-CSF mobilized peripheral blood mononuclear cells (PBMC) and subsequently examined isolation of CMV-T based on the activation markers CD154 and CD25. CD25(+) enriched CMV-T from G-CSF mobilized PBMC contained a higher proportion of FoxP3 expression than non-mobilized PBMC and showed superior suppression of T cell proliferation. Expanded CMV-T enriched through CD154 were CD4(+) and CD8(+) , demonstrated a high specificity for CMV, secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. These data provide the first known evidence that CMV-T can be effectively manufactured from G-CSF mobilized PBMC and that they share the same characteristics as CMV-T isolated in an identical manner from conventional non-mobilized PBMC. This provides a novel strategy for adoptive immunotherapy that abrogates the need for successive donation.

Fcγ receptor antigen targeting potentiates cross-presentation by human blood and lymphoid tissue BDCA-3+ dendritic cells.

The reactivation of human cytomegalovirus (HCMV) poses a serious health threat to immune compromised individuals. As a treatment strategy, dendritic cell (DC) vaccination trials are ongoing. Recent work suggests that BDCA-3(+) (CD141(+)) subset DCs may be particularly effective in DC vaccination trials. BDCA-3(+) DCs had however been mostly characterized for their ability to cross-present antigen from necrotic cells. We here describe our study of human BDCA-3(+) DCs in elicitation of HCMV-specific CD8(+) T-cell clones. We show that Fcgamma-receptor (FcγR) antigen targeting facilitates antigen cross-presentation in several DC subsets, including BDCA-3(+) DCs. FcγR antigen targeting stimulates antigen uptake by BDCA-1(+) rather than BDCA-3(+) DCs. Conversely, BDCA-3(+) DCs and not BDCA-1(+) DCs show improved cross-presentation by FcγR targeting, as measured by induced release of IFNγ and TNF by antigen-specific CD8(+) T cells. FcγR-facilitated cross-presentation requires antigen processing in both an acidic endosomal compartment and by the proteasome, and did not induce substantial DC maturation. FcγRII is the most abundantly expressed FcγR on both BDCA-1(+) and BDCA-3(+) DCs. Furthermore we show that BDCA-3(+) DCs express relatively more stimulatory FcγRIIa than inhibitory FcγRIIb in comparison with BDCA-1(+) DCs. These studies support the exploration of FcγR antigen targeting to BDCA-3(+) DCs for human vaccination purposes.

Viral oncoprotein LMP1 disrupts p53-induced cell cycle arrest and apoptosis through modulating K63-linked ubiquitination of p53.

Disruption of the gatekeeper p53 tumor suppressor is involved in various virus-associated tumorigeneses, with aberrant ubiquitination as the major cause of p53 abnormalities in virus-associated tumors. Of note, wild-type p53 is accumulated in Epstein-Barr virus (EBV)-associated tumors, especially in nasopharyngeal carcinoma (NPC). We have previously identified that p53 is accumulated and phosphorylated by EBV oncoprotein latent membrane protein 1 (LMP1) in NPC. Here, we further found that LMP1 promoted p53 accumulation via two distinct ubiquitin modifications. LMP1 promoted p53 stability and accumulation by suppressing K48-linked ubiquitination of p53 mediated by E3 ligase MDM2, which is associated with its phosphorylation at Ser20, while increasing the levels of total cellular ubiquitinated p53. LMP1 also induced K63-linked ubiquitination of p53 by interacting with tumor necrosis factor receptor-associated factor 2 (TRAF2), thus contributing to p53 accumulation. Furthermore, LMP1 rescued tumor cell apoptosis and cell cycle arrest mediated by K63-linked ubiquitination of p53. Collectively, these results demonstrate aberrant ubiquitin modifications of p53 and its biological functions by viral protein LMP1, which has broad implications to the pathogenesis of multiple EBV-associated tumors.