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CD40 (TNFRSF5) és una glicoproteïna de membrana que pertany a la família de receptors del factor de necrosi tumoral (TNF-R). La interacció amb el seu lligand fisiològic (CD40L o CD154) regula una àmplia varietat de funcions biològiques, des de l'activació del sistema immune, tant humoral com cel·lular, a diferents aspectes de la resposta inflamatòria. Els principals objectius d'aquesta tesi han estat 1) l'estudi a nivell molecular del paper de CD40 en l'activació de les cèl·lules endotelials, i 2) les conseqüències del bloqueig d'aquesta via de senyalització en la resposta immunoinflamatòria. S'ha obtingut un siRNA potent i específic capaç de reduir l'expressió del receptor CD40 humà en un 85%, tant a nivell de mRNA com a nivell de proteïna. Aquest siRNA expressat en forma de shRNA en un vector lentiviral permet assolir un silenciament gènic eficient i estable de CD40 al llarg del temps. S'ha validat l'activitat antiinflamatòria d'aquest siRNA analitzant les conseqüències funcionals del silenciament d'aquest receptor en cèl·lules endotelials. Aquest siRNA bloqueja l'activació endotelial via CD40L impedint la inducció de l'expressió de les molècules d'adhesió ICAM-1, VCAM-1 i E-selectina, i inhibint l'adhesió leucocitària en aquestes cèl·lules en un 87%. S'ha utilitzat el potencial antiinflamatori del siRNA dirigit contra CD40 humà per estudiar el paper d'aquest receptor en cèl·lules endotelials en inflamació. Utilitzant microarrays, s'ha realitzat una anàlisi comparativa del perfil global d'expressió gènica en cèl·lules endotelials humanes en les quals s'inactiva específicament aquesta via de senyalització mitjançant RNAi, en el context de la interacció cèl·lula endotelial i limfòcit T activat (CD40L+), com a primer pas en l'inici de la resposta immunoinflamatòria. Aquest estudi d'expressió gènica ha permès identificar nous gens i noves vies de senyalització implicades en la inducció de la resposta inflamatòria desencadenada per l'activació de CD40 en endoteli.Per altra banda, es volia avaluar l'eficàcia del silenciament gènic de CD40 per a la inducció de tolerància immunològica en un model experimental d'al·lotrasplantament renal. Amb aquesta finalitat s'ha obtingut un siRNA anti-CD40 de rata amb una potent eficiència silenciadora i s'ha optimitzat la transferència d'aquest siRNA en el ronyó a trasplantar mitjançant electroporació in vivo de l'òrgan. L'objectiu d'aquest estudi era silenciar temporalment l'expressió d'aquest receptor en les cèl·lules renals i bloquejar la interacció CD40-CD40L essencial per a la inducció de procés inflamatori que dóna lloc al rebuig del ronyó trasplantat. Resultats preliminars mostren que la teràpia gènica local amb siRNA redueix la resposta inflamatòria posttrasplantament. El siRNA anti-CD40 causa una reducció significativa de l'expressió del mRNA de CD40, disminuint l'aparició de rebuig agut humoral i allargant el temps mig de supervivència dels animals trasplantats amb els ronyons tractats amb siRNA en comparació amb els animals control., CD40 (TNFRSF5) belongs to the tumor necrosis factor receptor family (TNF-R). Its interaction with its physiological ligand (CD40L or CD154) regulates a wide variety of biological functions, from the activation of the immune system, to different aspects of the inflammatory response.The main goals of this work have been 1) the molecular study of the role of CD40 in the activation of the endothelial cells, and 2) the consequences of blocking this signaling in the immunoinflammatory response. We have obtained a powerful and specific siRNA able to reduce human CD40 expression by 85%. The anti-inflammatory activity of this siRNA has been validated by analyzing the functional consequences of CD40 silencing in endothelial cells activated via CD40L. This siRNA blocks the induction of ICAM-1, VCAM-1 and E-selectin expression, and inhibits leukocyte adhesion in these cells by 87%.We have used the anti-inflammatory potential of this siRNA directed against human CD40 to carry out a global gene expression profiling analysis in order to study the role of CD40 in endothelial cells during inflammation. Using microarrays, we have performed a comparative transcriptome analysis in human endothelial cells, in the context of the endotelial cell interaction with activated T lymphocyte (CD40L+), as the first step of the immunoinflammatory response. This study has allowed us to identify new genes and signaling pathways involved in the induction of the inflammatory response by CD40 activation in endothelium.On the other hand, we have obtained an siRNA against rat CD40 with a powerful silencing activity and we have optimized its transfer to the kidney through in vivo electroporation. The purpose of this study was to determine the CD40 gene silencing effectiveness in inducing immunological tolerance in an experimental model of renal allotransplantation. Preliminary results show that local gene therapy with siRNA reduces the inflammatory response post-transplantation. The anti-CD40 siRNA treatment causes a significant reduction of the incidence of acute humoral rejection and increases the survival half life of animals transplanted with kidneys treated with siRNA compared to control animals.

Cellular immunity is crucial for an efficient host immune response against rabies virus (RABV) infection. But the T cell receptor (TCR) repertoire in human after RABV vaccine immunization remained unclear. In this study, we conducted high-throughput sequencing of TCR β chain complementarity determining region 3(CDR3) repertoires in 4 healthy volunteers before and after immunization with RABV vaccine. Our data showed that RABV vaccination changed the TCR diversity and the usage of V/J gene segments, as well as V-J pairing. The high-frequency clonotypes that altered after vaccination were identified. These results may provide us with new insights into T cell receptor condition after RABV vaccination.

Specific miRNA in immune thrombocytopenia (ITP) was screened to explore its intervention effects and mechanisms in ITP. MTT assay and CFSE staining were used to detect the effects of gradient concentrations of thrombopoietin (TPO) on cell proliferation. Expressions of differentially expressed miRNAs were analysed via qRT-PCR in TPO-induced megakaryocytes and ITP plasma. Effects of miR-557 on cell physiological functions were examined by MTT and flow cytometry. Expressions of miR-557, apoptosis-associated genes and Akt/ERK pathways were detected by qRT-PCR and Western blot as needed. Multinucleation of TPO-induced megakaryocytes was determined by megakaryocyte colonies. The toe skin and intestinal bleeding of the ITP rat model were observed and evaluated. Effects of miR-557 on the numbers of platelets, megakaryocytes, and peripheral blood platelets and the expressions of CD4(+) T cells, Treg cells, TGF-β, IL-6 and miR-557 in the ITP rats were detected by Giemsa staining, flow cytometry, ELISA and qRT-PCR. MiR-557 was identified as an specific miRNA associated with both ITP and TPO treatment. MiR-557 inhibitor enhanced the physiological functions of TPO-induced megakaryocytes, while miR-557 mimic had the opposite effect. At the molecular level, the expressions of miR-557, cleaved Caspase-3 and Bax were further silenced by inhibitor, on the contrary, the expressions of bcl-2, p-Akt and p-ERK were upregulated. Animal experiments showed that, miR-557 inhibitor increased the numbers of platelets and megakaryocytes, and improved the symptoms of ITP model rats. Our results indicated that miR-557 inhibitor improved ITP by regulating apoptosis-related genes and cellular immunity and activating the Akt/ERK pathway.

Accurate biomarkers to predict the genesis and progression of pancreatic adenocarcinoma (PAAD) are needed in the fight against this deadly disease. Here, we combined multiple datasets (GEO, TCGA and GTEx) to conduct a comprehensive analysis of pancreatic cancer. Through an in-depth analysis, we discovered that the expression of the gene encoding interferon alpha-inducible protein 27 (IFI27) was significantly higher in pancreatic cancer tissues than that in normal tissues, and that higher expression of IFI27 was negatively correlated with the overall survival rate of pancreatic cancer patients. The functional annotation of IFI27 demonstrated relationships to cellular immunity and metabolism, especially glycolysis. Analysis of infiltrating immune cells displayed that higher expression of IFI27 expression correlates with decreased CD8 + T cells and increased M2 macrophages in the tumor immune microenvironment (TIME), then biochemical analyses of a mouse model and immunohistochemical (IHC) staining verified that glycolytic enzymes and M2 macrophages increased significantly in pancreatic cancer tissues. We speculate that IFI27 may affect the tumor microenvironment (TME) of PAAD by regulating cellular immunity and metabolism, thereby promoting the progression of pancreatic carcinoma and worsening the prognosis. These findings of our present study are solid evidence that IFI27 is a potential prognostic biomarker of pancreatic cancer and that it affects the tumor immune microenvironment.

Entomopathogenic fungi naturally infect insect hosts in environment. Fungal invasion and host immune defense are still in the progress of co-evolution. In this study, entomopathogenic fungus Beauveria bassiana and lepidopteran insect Galleria mellonella were used to investigate host cellular immunity and fungal strategy to evade host defense. First of all, genome-wide expression revealed the transcriptomic responses of hemocytes to insect mycopathogen, which dynamically varied during infection process. Enrichment analysis indicated that differentially expressed genes were primarily involved in metabolism, cellular process and immune system. Notably, cellular response involved a series of hydrolytic enzyme and antimicrobial peptide genes which were sorted together in clustering analysis. In B. bassiana, a cell-wall protein gene (BbCwp) contributes to fungal development in host hemocoel and virulence. RT-qPCR analyses indicated that infection by ΔBbCwp mutant strain caused the up-regulated expression of a series of immunity-related genes, including β-1, 3-glucan recognition protein, hydrolytic enzyme and antimicrobial peptide genes. Disruption of BbCwp resulted in a significant change in conidial lectin-binding feature and the enhanced encapsulation by the host hemocytes. After being treated with hydrolytic enzymes, ΔBbCwp mutant displayed a significantly enhanced sensitivity to osmotic and oxidative stresses. In conclusion, fungal invasion initiates comprehensive physiological responses in the host hemocytes. For mycopathogen, cell-wall protein plays an important role in fungal evasion of immunity defense and colonization in host. Our studies provide an initial framework for exploring more mechanistic details about the fungus–host interaction.

Chicken coccidiosis is a protozoan parasitic disease that leads to considerable economic losses in the poultry industry. In this study, we used invasive Lactobacillus plantarum (L.P) expressing the FnBPA protein as a novel bacterial carrier for DNA delivery into epithelial cells to develop a live oral DNA vaccine. A fusion DNA vaccine co-expressing EtMIC2 and chicken IL-18 (chIL-18) was constructed and then delivered to the host by invasive L.P. Its efficacy against Eimeria tenella challenge was evaluated in chickens by examining the relative weight gain rate; caecal lesion score; OPG; anti-coccidial index (ACI); levels of EtMIC2 antibody, FnBPA, IL-4, IL-18, IFN-γ and SIgA; and proliferation ability and percentages of CD4+ and CD8+ splenocytes. The experimental results showed that chickens immunized with invasive L.P carrying the eukaryotic expression vector pValac-EtMIC2 (pValac-EtMIC2/pSIP409-FnBPA) had markedly improved immune protection against challenge compared with that of chickens immunized with non-invasive L.P (pValac-EtMIC2/pSIP409). However, invasive L.P co-expressing EtMIC2 with the chIL-18 vector exhibited the highest protection efficiency against E. tenella. These results indicate that invasive Lactobacillus-expressing FnBPA improved humoural and cellular immunity and enhanced resistance to E. tenella. The DNA vaccine delivered by invasive Lactobacillus provides a new concept and method for the prevention of E. tenella.

The most widely used influenza vaccines are prepared by chemical inactivation. However, chemical, especially formalin, treatment-induced modifications of the antigenic structure of the virus are frequently associated with adverse effects including low efficacy of protection, unexpected immune responses, or exacerbation of disease. Gamma-irradiation was suggested as an alternative influenza virus inactivation method due to its great features of completely inactivating virus while not damaging the structures of protein antigens, and cross-protective ability against heterologous strains. However, immunological features of gamma radiation-inactivated influenza vaccine have not been fully understood. In this study, we aimed to investigate the humoral and cellular immune responses of gamma radiation-inactivated influenza vaccine. The gamma irradiation-inactivated influenza vaccine (RADVAX(FluA)) showed complete viral inactivation but retained normal viral structure with functional activities of viral protein antigens. Intranasal immunization of RADVAX(FluA) provided better protection against influenza virus infection than formalin-inactivated influenza virus (FIV) in mice. RADVAX(FluA) greatly enhanced the production of virus-specific serum IgG and alveolar mucosal IgA, which effectively neutralized HA (hemagglutinin) and NA (neuraminidase) activities, and blocked viral binding to the cells, respectively. Further analysis of IgG subclasses showed RADVAX(FluA)-immunized sera had higher levels of IgG1 and IgG2a than those of FIV-immunized sera. In addition, analysis of cellular immunity found RADVAX(FluA) induced strong dendritic cells (DC) activation resulting in higher DC-mediated activation of CD8(+) T cells than FIV. The results support improved immunogenicity by RADVAX(FluA).

Objectives Enhancement of the potential ability of biomacromolecules to cross cell membranes is a critical step for development of effective therapeutic vaccine especially DNA vaccine against human immunodeficiency virus-1 (HIV-1) infection. The supercharged proteins were known as powerful weapons for delivery of different types of cargoes such as DNA and protein. Hence, we applied B1 protein with + 43 net charges obtained from a single frameshift in the gene encoding enhanced green fluorescent protein (eGFP) for delivery of two multi-epitope DNA constructs (nef-vpu-gp160-p24 and nef-vif-gp160-p24) in vitro and in vivo for the first time. For this purpose, B1 protein was generated in bacterial expression system under native conditions, and used to interact with both DNA constructs. Results Our data indicated that B1 protein (~ 27 kDa) was able to form a stable nanoparticle (~ 80–110 nm) with both DNA constructs at nitrogen: phosphate (N: P) ratio of 1:1. Moreover, the transfection efficiency of B1 protein for DNA delivery into HEK-293T cell line indicated that the cellular uptake of nef-vif-gp160-p24 DNA/ B1 and nef-vpu-gp160-p24 DNA/ B1 nanoparticles was about 32–35% with lower intensity as compared to TurboFect commercial reagent. On the other hand, immunization of BALB/c mice with different modalities demonstrated that B1 protein could enhance the levels of antibody, IFN-gamma and Granzyme B eliciting potent and strong Th1-directed cellular immunity. Conclusion Generally, our findings showed the potency of B1 protein as a promising gene delivery system to improve an effective therapeutic vaccine against HIV-1 infection.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrosis (TEN) are rare but life-threatening severe cutaneous adverse reactions. Current studies have suggested that the pathobiology of drug-mediated SJS/TEN involves a dysregulation of cellular immunity with overwhelming activation of cytotoxic T lymphocytes. The canonical Wnt signaling pathway plays important roles in T cell development and activation, which may provide potential avenues for alleviating dysregulated immunity in SJS/TEN. In this study, we aimed to assess the implication of Wnt signaling in drug-reactive T cells in SJS/TEN. We showed downregulation of Wnt signaling components, including T cell factor 1 (TCF-1)/lymphoid enhancer binding factor 1 (LEF-1) transcription factors, in SJS/TEN patients, suggesting that canonical Wnt signaling is regulated during cytotoxic T cell responses in SJS/TEN. Further analyses demonstrated that engagement of the T cell receptor by antigen encounter and treatment of a prognostic marker of SJS/TEN, IL-15, in vitro led to the downregulation of LEF-1 and TCF-1 expression in CD8+ T cells. Enhancement of Wnt signaling by adding the Wnt activators attenuated ex vivo activation of drug-specific T cells from SJS/TEN patients, indicating a functional involvement of Wnt signaling in the pathomechanism of SJS/TEN. These findings provide additional insight into the immunopathogenesis and therapeutic intervention of this devastating condition.

Chronic hepatitis B virus (HBV) infection causes hepatitis, liver cirrhosis and hepatocellular carcinoma. Covalently closed circular DNA (cccDNA) is the sole viral transcription template and not affected by current treatment options, constituting a key determinant of HBV persistence. Novel therapeutics with demonstrable effectiveness against cccDNA are required. Methods: Previously, we established an HBV persistence mouse model using replicon plasmid derived from a clinical isolate (termed BPS) and identified IL-21 as a potent clearance-inducer. We also described another persistence mouse model based on cccDNA mimics produced in vivo termed recombinant cccDNA (rcccDNA). In this work, effectiveness of IL-21-based gene and cellular therapies was tested using these models. Results: In both models of HBV persistence, single injections with adeno-associated virus (AAV) expressing murine IL-21 highly efficiently induced clearance of both HBV markers from serum, and more importantly, BPS DNA and rcccDNA from mouse liver. Mechanistically, IL-21-induced clearance was associated with activation and liver infiltration of CD8+ T cells, and CD8 antibody injections negatively affected AAV-IL-21 effectiveness. More notably, adoptive transfer of CD8+ T cells from AAV-IL-21-cured mice engendered clearance in acceptor HBV persistence mice. Furthermore, cured mice were protected against re-challenge with long-lived memory. Most significantly, infusion of splenocytes from treatment-naive mice stimulated ex vivo with IL-21 protein and HBV antigen could also induce clearance in treatment-naive mice. Conclusion: These data demonstrate IL-21-based gene and cellular therapies as valid candidates for treating chronic HBV infections, with potential in removing cccDNA-harboring hepatocytes via activated CD8+ T cells accompanied by long-term protective memory.

The following was emphasised in an informative, educational issued on the American College of Rheumatology website in April 2017: "About one child in every 1000 develops some type of chronic arthritis. These disorders can affect children at any age, although rarely in the first six months of life. It is estimated that around 300,000 children in the United States have been diagnosed with the condition". Therefore, knowledge of immunological investigations in patients with juvenile idiopathic arthritis is important for finding new treatment pathways. Our aim was to assess the immunological investigations and immune system implications in juvenile idiopathic arthritis. We will discuss: a) the specifically targeted proteins - the citrullinated peptide antibodies; b) non-specifically targeted proteins - heat-shock proteins (anti-HSP60, -65, and -70 antibodies), CLEC16A, inflammasomes, and phagocyte-derived S100; c) interleukins - IL-1, IL-6, IL-10, IL-17, and IL-18; d) innate immunity - macrophage activation syndrome, natural killer cells, complement activity, and immune complexes; and e) therapeutic targets - monoclonal antibodies, JAK inhibitors, and intravenous immune globulin.

Clostridium perfringens α-toxin is one of the major virulence factors during C. perfringens infection, causing hemolysis of erythrocytes in various species. Here, genetically engineered Lactobacillus casei (pPG-α/L. casei 393) constitutively expressing the toxoid of C. perfringens α-toxin was generated and its immunogenicity in mice for induction of protective immunity against the α-toxin was evaluated via oral immunization. The α-toxoid was constitutively expressed by pPG-α/L. casei 393 without a specific inducer, as confirmed by western blotting, laser confocal microscopy, and flow cytometry. In an experiment on BALB/c mice to evaluate the oral immunogenicity of pPG-α/L. casei 393, significant levels of a specific secretory IgA (sIgA) antibody in the intestinal mucus and feces and an IgG antibody in the serum of the probiotic vaccine group were detected after booster immunization (p

6 figures, 6 tables.-- Supplementary material available., Coronavirus disease 2019 (COVID19), caused by SARS-CoV-2, is a complex disease, with a variety of clinical manifestations ranging from asymptomatic infection or mild cold-like symptoms to more severe cases requiring hospitalization and critical care. The most severe presentations seem to be related with a delayed, deregulated immune response leading to exacerbated inflammation and organ damage with close similarities to sepsis., [Methods]: In order to improve the understanding on the relation between host immune response and disease course, we have studied the differences in the cellular (monocytes, CD8+ T and NK cells) and soluble (cytokines, chemokines and immunoregulatory ligands) immune response in blood between Healthy Donors (HD), COVID19 and a group of patients with non-COVID19 respiratory tract infections (NON-COV-RTI). In addition, the immune response profile has been analyzed in COVID19 patients according to disease severity., [Results]: In comparison to HDs and patients with NON-COV-RTI, COVID19 patients show a heterogeneous immune response with the presence of both activated and exhausted CD8+ T and NK cells characterised by the expression of the immune checkpoint LAG3 and the presence of the adaptive NK cell subset. An increased frequency of adaptive NK cells and a reduction of NK cells expressing the activating receptors NKp30 and NKp46 correlated with disease severity. Although both activated and exhausted NK cells expressing LAG3 were increased in moderate/severe cases, unsupervised cell clustering analyses revealed a more complex scenario with single NK cells expressing more than one immune checkpoint (PD1, TIM3 and/or LAG3). A general increased level of inflammatory cytokines and chemokines was found in COVID19 patients, some of which like IL18, IL1RA, IL36B and IL31, IL2, IFNα and TNFα, CXCL10, CCL2 and CCL8 were able to differentiate between COVID19 and NON-COV-RTI and correlated with bad prognosis (IL2, TNFα, IL1RA, CCL2, CXCL10 and CXCL9). Notably, we found that soluble NKG2D ligands from the MIC and ULBPs families were increased in COVID19 compared to NON-COV-RTI and correlated with disease severity., [Conclusions]: Our results provide a detailed comprehensive analysis of the presence of activated and exhausted CD8+T, NK and monocyte cell subsets as well as extracellular inflammatory factors beyond cytokines/chemokines, specifically associated to COVID19. Importantly, multivariate analysis including clinical, demographical and immunological experimental variables have allowed us to reveal specific immune signatures to i) differentiate COVID19 from other infections and ii) predict disease severity and the risk of death., The authors would like to thank the Biobank of the Aragon Health System integrated in the Spanish National Biobanks Network and the Servicios Científico Técnicos de Citometria de Flujo del CIBA for their collaboration. Work in the JP laboratory is funded by the FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón, Group B29_17R), Health National Institute Carlos III (COV20-00308), Aragón Government (Fondo COVID-19), Fundación Santander-Universidad de Zaragoza (Programa COVID-19), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RBI00), Fundación Inocente, ASPANOA and Carrera de la Mujer de Monzón. EMG is funded by Agencia Estatal de Investigación (SAF2017-83120-C2-1-R and PID2020-113963RB-I00). IUM and SH are supported by a PhD fellowship from Aragon Government, CP by a PhD fellowship from AECC, LS by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. DDM is supported by a postdoctoral fellowship 'Sara Borrell', and MA is supported by a postdoctoral fellowship 'Juan de la Cierva-incorporacion' from the Ministry of Science, Innovation and Universities. EM and BGT are supported by Rio Hortega contract. JP is supported by the ARAID Foundation.

Background The long-term course of immunity among individuals with a history of COVID-19, in particular among those who received a booster vaccination, has not been well defined so far. Methods SARS-CoV-2-specific antibody levels were measured by ELISA over 1 year among 136 health care workers infected during the first COVID-19 wave and in a subgroup after booster vaccination approximately 1 year later. Furthermore, spike-protein-reactive memory T cells were quantified approximately 7 months after the infection and after booster vaccination. Thirty healthy individuals without history of COVID-19 who were routinely vaccinated served as controls. Results Levels of SARS-CoV-2-specific IgM- and IgA-antibodies showed a rapid decay over time, whereas IgG-antibody levels decreased more slowly. Among individuals with history of COVID-19, booster vaccination induced very high IgG- and to a lesser degree IgA-antibodies. Antibody levels were significantly higher after booster vaccination than after recovery from COVID-19. After vaccination with a two-dose schedule, healthy control subjects developed similar antibody levels as compared to individuals with history of COVID-19 and booster vaccination. SARS-CoV-2-specific memory T cell counts did not correlate with antibody levels. None of the study participants suffered from a reinfection. Conclusions Booster vaccination induces high antibody levels in individuals with a history of COVID-19 that exceeds by far levels observed after recovery. SARS-CoV-2-specific antibody levels of similar magnitude were achieved in healthy, COVID-19-naïve individuals after routine two-dose vaccination.

BACKGROUND: The emergence of Zika virus (ZIKV) as an important cause of congenital and childhood developmental disorders presents another challenge to global health. Efforts to develop a Zika vaccine have begun although vaccine development against flaviviruses, of which ZIKV belongs to, has proven to be time-consuming and challenging. Defining the vaccine attributes that elicit adaptive immune response necessary for preventing ZIKV infection could provide an evidence-based guide to Zika vaccine development.METHODS: We used a previously described attenuated ZIKV DN-2 strain in a type-I interferon receptor deficient mouse model and tested the hypothesis that duration of vaccine burden rather than peak level of infection, is a determinant of immunogenicity. We quantified both humoral and cellular responses against ZIKV using plaque reduction neutralisation test and flow cytometry with ELISPOT assays, respectively. Vaccinated mice were challenged with wild-type ZIKV (H/PF/2013 strain) to determine the level of protection against infection.FINDINGS: We found that the overall vaccine burden is directly correlated with neutralising antibody titres. Reduced duration of vaccine burden lowered neutralising antibody titres that resulted in subclinical infection, despite unchanged peak vaccine viraemia levels. We also found that sterilising immunity is dependant on both neutralising antibody and CD8+T cell responses; depletion of CD8+T cells in vaccinated animals led to wild-type ZIKV infection, especially in the male reproductive tract.INTERPRETATION: Our findings indicate that duration of attenuated virus vaccine burden is a determinant of humoral and cellular immunity and also suggest that vaccines that elicit both arms of the adaptive immune response are needed to fully prevent ZIKV transmission.FUNDING: This study was supported by the National Medical Research Council through the Clinician-Scientist Award (Senior Investigator) to E.E.O. Salary support for S.W. was from a Competitive Research Programme grant awarded by the National Research Foundation of Singapore.

Abstract Filter-feeding organisms accumulate xenobiotics and other substances in their tissues. They can be useful as sentinel organisms in biomonitoring of the marine compartment. Bivalve cellular immunity is ensured by phagocytosis and cytotoxic reactions carried out by hemocytes in a network with humoral responses. These can be affected by chemical contaminants in water that can be immunosuppressors also at a low concentration increasing the sensibility to pathogens. This work is an attempt to individuate cellular markers for pollution detection, investigating the effect of methylmercury (CH3HgCl) at different concentrations on the activity and hemocyte morphology of the Mediterranean mussel, Mytilus galloprovincialis. We assessed the effect of three sub-lethal concentrations of the organometal on the cellular morphology, the efficacy of phagocytosis toward yeast cells, the alteration of the lysosomal membrane and the ability to release cytotoxic molecules. The results provide information on the alteration of hemocyte viability, modification of the morphological and cytoskeletal features and besides the cellular spreading, intrinsic ability of motile cells was used as a complementary investigation method. Exposure to the contaminant affected the percentage of phagocytosis and the phagocytosis index. Moreover, morphological and cytoskeleton alteration, caused by the pollutant, leads to reduced ability to incorporate the target and adhere to the substrate and the low ability of cells to retain neutral red could depend on the effects of methylmercury on membrane permeability. These results reinforce the use of the Mediterranean mussel as model for the evaluation of environmental quality in aquatic ecosystems integrating the novel information about hemocyte functions and morphology sensibility to organic mercury. Graphic abstract

Although immune-related adverse events (irAEs) of treatment with immune-checkpoint inhibitors may be due to cellular immunity mediated by T lymphocytes, their pathogenesis has remained unknown. Here we collected bronchoalveolar lavage fluid (BALF) from a cancer patient with nivolumab-induced pneumonitis and isolated mononuclear cells for next-generation sequencing of the complementarity-determining region of the T cell receptor (TCR) β chain. Mononuclear cells in peritumoral pleural effusion isolated from the patient were similarly analyzed, and the results obtained for the two specimens were compared. A substantial number of TCRβ clones in BALF were also identified among lymphocytes in the peritumoral pleural effusion. Such a correlation was not apparent between TCRβ clones in BALF and those in peripheral blood. Moreover, many tumor-associated clones with a read frequency of ≥0.10% were also present in BALF. Our data suggest that irAEs might be induced by drug-activated lymphocytes originating from tumor tissue. Deep sequencing will thus be indispensable for investigations of the immune-based pathogenesis of, and the development of optimal treatments for, irAEs.

Systemic lupus erythematosus (SLE) patients are at high risk of herpes zoster. Previously, we found increased immunoglobulin (Ig)G levels against varicella-zoster virus (VZV) in SLE patients compared to controls, while antibody levels against diphtheria and cellular immunity to VZV were decreased. We aimed to test our hypothesis that increased VZV-IgG levels in SLE result from subclinical VZV reactivations, caused by stress because of lupus disease activity or immunosuppressive drug use. Methods Antibody levels to VZV (IgG, IgA, IgM), total IgG and VZV-DNA were longitudinally determined in the serum of 34 SLE patients, using enzyme-linked immunosorbent assay and polymerase chain reaction. Clinical data were retrieved from medical records. Reactivation of VZV was defined as an at least fivefold rise in VZV-IgG or presence of VZV-IgM or VZV-DNA. Generalized estimating equations (GEE) were used to longitudinally analyse associations between antibody levels, lupus disease activity and medication use. Systemic Lupus Erythematosus Disease Activity Index, anti-double-stranded DNA and complement levels were used as indicators of lupus disease activity. Results A VZV reactivation was determined in 11 patients (33%). In at least five of them, herpes zoster was clinically overt. No association between SLE disease activity or medication use and VZV-specific antibody levels was found. There was a weak association between total IgG and VZV-IgG. Conclusions Our results indicate that increased VZV-IgG levels in SLE do not result from frequent subclinical VZV reactivations, and are not associated with lupus disease activity. Increased VZV-IgG can only partially be explained by hypergammaglobulinaemia.

// Irina N. Mikhaylova 1 , Irina Zh. Shubina 1 , George Z. Chkadua 1 , Natalia N. Petenko 1 , Lidia F. Morozova 1 , Olga S. Burova 1 , Robert Sh. Beabelashvili 2 , Kermen A. Parsunkova 3 , Natalia V. Balatskaya 4 , Dmitrii K. Chebanov 5 , Vadim I. Pospelov 6 , Valeria V. Nazarova 1 , Anastasia S. Vihrova 1 , Evgeny A. Cheremushkin 1 , Alvina A. Molodyk 1 , Mikhail V. Kiselevsky 1 and Lev V. Demidov 1 1 N.N. Blokhin Russian Cancer Research Center, Moscow, Russia 2 Laboratory of Genetic Engineering, Institute of Experimental Cardiology, Russian Cardiological Research and Production Complex, Moscow, Russia 3 Diagnostic Laboratory Clinical Center “Optimum”, Sochi, Russia 4 Department of Immunology and Virology, Moscow Helmholtz Research Institute of Eye Diseases, Moscow, Russia 5 RUDN University, Moscow, Russia 6 “Genetic Technologies and Analyses”, Moscow, Russia Correspondence to: Irina Zh. Shubina, email: irinashubina@mail.ru Keywords: cancer vaccine therapy; melanoma; predictive markers; B2-microglobulin Received: September 04, 2017 Accepted: March 22, 2018 Published: May 11, 2018 ABSTRACT Immunotherapy has shown promising results in a variety of cancers, including melanoma. However, the responses to therapy are usually heterogeneous, and understanding the factors affecting clinical outcome is still not achieved. Here, we show that immunological monitoring of the vaccine therapy for melanoma patients may help to predict the clinical course of the disease. We studied cytokine profile of cellular Th1 (IL-2, IL-12, IFN-γ) and humoral Th2 (IL-4, IL-10) immune response, vascular endothelial growth factor (VEGFA), transforming growth factor-β 2 (TGF-β 2), S100 protein (S100A1B and S100BB), adhesion molecule CD44 and serum cytokines β2-microglobulin to analyze different peripheral blood mononuclear cell subpopuations of patients treated with dendritic vaccines and/or cyclophosphamide in melanoma patients in the course of adjuvant treatment. The obtained data indicate predominance of cellular immunity in the first adjuvant group of patients with durable time to progression and shift to humoral with low cellular immunity in patients with short-term period to progression (increased levels of IL-4 and IL- 10). Beta-2 microglobulin was differentially expressed in adjuvant subgroups: its higher levels correlated with shorter progression-free survival and the total follow-up time. Immunoregulatory index was overall higher in patients with disease progression compared to the group of patients with no signs of disease progression.

Background T-cell responses against highly conserved influenza antigens have been previously associated with protection. However, these immune responses are poorly maintained following recovery from influenza infection and are not boosted by inactivated influenza vaccines. We have previously demonstrated the safety and immunogenicity of two viral vectored vaccines, modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAdOx1 expressing conserved influenza virus antigens, nucleoprotein (NP) and matrix protein-1 (M1). We now report on the safety and long-term immunogenicity of multiple combination regimes of these vaccines in young and older adults. Methods We conducted a Phase I open-label, randomized, multi-center study in 49 subjects aged 18–46 years and 24 subjects aged 50 years or over. Following vaccination, adverse events were recorded and the kinetics of the T cell response determined at multiple time points for up to 18 months. Findings Both vaccines were well tolerated. A two dose heterologous vaccination regimen significantly increased the magnitude of pre-existing T-cell responses to NP and M1 after both doses in young and older adults. The fold-increase and peak immune responses after a single MVA-NP + M1 vaccination was significantly higher compared to ChAdOx1 NP + M1. In a mixed regression model, T-cell responses over 18 months were significantly higher following the two dose vaccination regimen of MVA/ChAdOx1 NP + M1. Interpretation A two dose heterologous vaccination regimen of MVA/ChAdOx1 NP + M1 was safe and immunogenic in young and older adults, offering a promising vaccination strategy for inducing long-term broadly cross-reactive protection against influenza A. Funding Source Medical Research Council UK, NIHR BMRC Oxford., Highlights • Heterologous prime-boost vaccination regimens of MVA/ChAdOx1 NP+M1 are safe and immunogenic in young and older adults • All MVA/ChAdOx1 NP+M1 regimens tested significantly increased cross-reactive T-cells • Responses were durable and were maintained 18 months after vaccination • The fold-increase after a single MVA-NP+M1 vaccination was significantly higher compared to ChAdOx1 NP+M1 Current seasonal influenza vaccines induce antibody responses to external glycoproteins, which are highly susceptible to the accumulation of mutations within antigenic sites, allowing escape from serological immunity conferred by prior infection or vaccination. In this phase I clinical study, we present the results of using two replication-deficient viral vectors expressing conserved influenza A antigens in four different vaccination regimens, administered at intervals of either 8 weeks or one year. We found that vaccination was safe and boosted T cell responses to influenza antigens substantially, with a further increase after the second vaccination, both in young and older adults.

Chronic obstructive pulmonary disease (COPD) that includes multiple mechanisms such as inflammation, infection, smoking, hypoxia, and lack of antioxidant response can cause oxidative stress. In our study, we aimed to determine the changes in some oxidative stress [malondialdehyde and glutathione] and some cellular immunity markers (neopterin and TGF-b) in patients diagnosed with COPD and determine the damage to the organism.While the high-performance liquid chromatography (HPLC) method (Immuchrom kit, Germany) was utilized to determine MDA, GSH and NP levels, the ELISA method was used for TGF-b levels.All obtained data regarding each parameter were compared with both COPD and healthy individuals and between parameters. There was a statistically significant difference between the control group of healthy subjects and COPD group in all parameters (p0.05). A negative and correlation between oxidant MDA and antioxidant GSH parameters was determined (p=-0.394).As a result, it was seen that oxidative balance changed in the patient group and cellular immunity increased. When the obtained data and literature are taken into account, these changes occurring in oxidative balance and cellular immunity are of importance in determining the development in the pathogenesis of COPD, treatment op - tions and their risks for heart disease in advance.Hronićna opstruktivna bolest pluča (HOBP) koja ukljućuje više mehanizama kao što su inflamacija, infekcija, pušenje, hipoksija i odsustvo antioksidantnog odgovora može izazvati oksidativni stres. U našoj studiji, cilj je bio da se utvrde promene u nekim markerima oksidativnog stresa (malondialdehid i glutation) i čelijskog imuniteta (neopterin i TGF-b) kod pacijenata sa dijagnozom HOBP i procene oštečenja organizma.Dok je za određivanje nivoa MDA, GSH i NP korišćena metoda HPLC (Immuchrom kit, Nemačka), za nivoe TGF-β upotrebljen je metod ELISA.Svi dobijeni podaci vezani za svaki parametar upoređeni su kod obolelih od HOBP i zdravih subjekata kao i međusobno. Pronađena je statistički značajna razlika između kontrolne grupe zdravih osoba i grupe sa HOBP za sve parametre (p0,05). Negativna i jaka korelacija utvrđena je između oksidantnog parametra MDA i antioksidantnog parametra GSH (p=-0,394).Kao rezultat, uočeno je da je u grupi obolelih izmenjena oksidativna ravnoteža i da je pojačan ćelijski imunitet. Kad se uzmu u obzir dobijeni podaci i literatura, ove promene koje nastaju u oksidativnoj ravnoteži i ćelijskom imunitetu od značaja su za određivanje razvoja u patogenezi HOBP, opcija lečenja i budućih rizika od srčanih bolesti.

Live attenuated vaccines are critical in the control of avian infectious bronchitis. It is necessary to know the protection conferred by commonly used commercial live vaccines. In this study, specific pathogen-free chicks were vaccinated with the commercial live vaccines H120, 4/91 and LDT3-A. Blood samples were collected at weekly intervals for the detection of IBV-specific antibodies and quantification of CD4+ and CD8+ T lymphocytes. At 21 days post-inoculation the vaccinated birds were challenged with the IBV prevalent local strains GX-YL5, GX-GL11079 and GX-NN09032, respectively. Trachea and kidney samples were collected at 5 days post-challenge for the detection of the virus. The results showed that the H120 group exhibited medium antibody levels, the lowest percentages of CD4+, CD8+ T lymphocytes and the highest viral loads. The 4/91 group showed the lowest antibody levels, but the highest percentages of CD4+, CD8+ T lymphocytes and the lowest viral loads. The LDT3-A group showed the highest antibody levels, the medium percentages of CD4+, CD8+ T lymphocytes and the medium viral loads. The protection rates of H120, 4/91 and LDT3-A groups were 41.7-58.3%, 75.0-83.7% and 66.7-75.0%, respectively. The present study demonstrated that the vaccines H120, 4/91 and LDT3-A could stimulate the immunized chicks to produce different levels of humoral and cellular immunity to resist the infection of IBV, but couldn't provide complete protection against the prevalent local strains of IBV in southern China. Also, the vaccine 4/91 offered the best immune protection among the three vaccines.

Background: Dialysis and kidney transplant patients are vulnerable populations for COVID-19 related disease and mortality. Methods: We conducted a prospective study exploring the eight week time course of specific cellular (interferon-I³ release assay and flow cytometry) or/and humoral immune responses (ELISA) to SARS-CoV-2 boost vaccination in more than 3100 participants including medical personnel, dialysis patients and kidney transplant recipients using mRNA vaccines BNT162b2 or mRNA-1273. Results: SARS-CoV-2-vaccination induced seroconversion efficacy in dialysis patients was similar to medical personnel (> 95%), but markedly impaired in kidney transplant recipients (42%). T-cellular immunity largely mimicked humoral results. Major risk factors of seroconversion failure were immunosuppressive drug number and type (belatacept, MMF-MPA, calcineurin-inhibitors) as well as vaccine type (BNT162b2 mRNA). Seroconversion rates induced by mRNA-1273 compared to BNT162b2 vaccine were 97% to 88% (p

// Hongrui Guo 1, * , Ping Kuang 1, * , Qin Luo 1, * , Hengmin Cui 1, 2 , Huidan Deng 1 , Huan Liu 1 , Yujiao Lu 1 , Jing Fang 1, 2 , Zhicai Zuo 1, 2 , Junliang Deng 1, 2 , Yinglun Li 1, 2 , Xun Wang 1, 2 and Ling Zhao 1, 2 1 College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China 2 Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, Sichuan Agriculture University, Ya’an 625014, China * These authors have contributed equally to this work Correspondence to: Hengmin Cui, email: cui580420@sicau.edu.cn Keywords: NaF, blood, cellular immunity, humoral immunity, mice Received: March 21, 2017 Accepted: July 19, 2017 Published: August 10, 2017 ABSTRACT Exposure to high fluorine can cause toxicity in human and animals. Currently, there are no systematic studies on effects of high fluorine on blood cellular immunity and humoral immunity in mice. We evaluated the alterations of blood cellular immunity and humoral immunity in mice by using flow cytometry and ELISA. In the cellular immunity, we found that sodium fluoride (NaF) in excess of 12 mg/Kg resulted in a significant decrease in the percentages of CD3 + , CD3 + CD4 + , CD3 + CD8 + T lymphocytes in the peripheral blood. Meanwhile, serum T helper type 1 (Th1) cytokines including interleukin (IL)-2, interferon (IFN)-γ, tumor necrosis factor (TNF), and Th2 cytokines including IL-4, IL-6, IL-10, and Th17 cytokine (IL-17A) contents were decreased. In the humoral immunity, NaF reduced the peripheral blood percentages of CD19 + B lymphocytes and serum immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM). The above results show that NaF can reduce blood cellular and humoral immune function in mice, providing an excellent animal model for clinical studies on immunotoxicity-related fluorosis.

Cyclic di-nucleotides (CDN) are potent stimulators of innate and adaptive immune responses. Cyclic di-AMP (CDA) is a promising adjuvant that generates humoral and cellular immunity. The strong STING-dependent stimulation of type I IFN represents a key feature of CDA. However, recent studies suggested that this is dispensable for adjuvanticity. Here we demonstrate that stimulation of IFN-γ-secreting CD8+ cytotoxic T lymphocytes (CTL) is significantly decreased after vaccination in the absence of type I IFN signaling. The biological significance of this CTL response was confirmed by the stimulation of MHC class I-restricted protection against influenza virus challenge. We show here that type I IFN (and not TNF-α) is essential for CDA-mediated cross-presentation by a cathepsin independent, TAP and proteosome dependent cytosolic antigen processing pathway, which promotes effective cross-priming and further CTL induction. Our data clearly demonstrate that type I IFN signaling is critical for CDN-mediated cross-presentation., Highlights • Cyclic di-AMP is a potent adjuvant with a proven capacity to promote cellular and humoral immunity. • It was postulated that type I IFN is irrelevant for CDA-generated immunity. • We show that CDA-mediated IFN-α/β signaling is essential for cytosolic cross-presentation, generating CTL by cross-priming. Antigen cross-presentation generates cross-priming of CD8 cytotoxic T cells (CTL), which kill intracellular pathogens and cancer cells. The generation of CTL is the main feature of cyclic di- nucleotide adjuvants (CDN). The production of type I IFN has been shown to be essential for the generation of CTL by other adjuvants, but not for cyclic di-AMP, a paramount CDN. Surprisingly, it was recently claimed that type I IFN is dispensable for the immunogenicity of CDN. We show here that type I IFN (and not TNF) is necessary for the cytosolic cross-presentation pathway activated by CDN, which results in CTL generation. This finding and the knowledge of the mechanism are of crucial importance for the safe development of CDN-adjuvanted vaccines.

// Mingrong Cheng 1, 2, * , Weiping Zhu 3, 5, * , Qing Li 4, * , Dejian Dai 2 and Yiming Hou 5 1 Department of General Surgery, Pudong New Area District Zhoupu Hospital, Shanghai 201318, China 2 Department of General Surgery, Shanghai Tianyou Hospital, Tongji University, Shanghai 200000, China 3 Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, Shanghai Medical College, Fudan University, Shanghai 200032, China 4 Department of Medical Clinic, Pujiang Community Health Service Center, Shanghai 201112, China 5 Department of General Surgery, Shanghai Fifth People’s Hospital, Fudan University, Shanghai 200240, China * These authors have contributed equally to this work Correspondence to: Weiping Zhu, email: wpzhush@hotmail.com Dejian Dai, email: daidejian@hotmail.com Keywords: biotin chitosan, curative effect, drug delivery system, liver cancer, nanoparticles Received: August 08, 2016 Accepted: June 02, 2017 Published: July 10, 2017 ABSTRACT The present study investigated the synthesis of biotinylated chitosan (Bio-CS) from chitosan using a nanomaterial skeleton with biotin and the successful targeting of the formulation in liver cancer cells. Bio-CS was validated by fourier transformed infrared spectroscopy and hydrogen -1 nuclear magnetic resonance spectroscopy. Bio-CS and plasmid DNA were used to construct Bio-CS/plasmid DNA nanoparticles according to the optimal molar ratio of 1:1 and the optimal pH-value of 5.5. Under these conditions, the parameters mean particle size, potential, encapsulation rate and drug loading, were 82.9 nm, +21.8 mV, 85.7% and 35.4%, respectively. Bio-CS exhibited an apparent liver cancer targeting effect in vitro and in vivo , as demonstrated by confocal laser scanning, green fluorescent protein transfection, and in vivo imaging assays. In addition, the Bio-CS/plasmid DNA nanoparticles significantly increased the survival period of the orthotropic liver cancer mouse model compared with the plasmid DNA, with no apparent side effects on the cells. Bio-CS nanomaterials stimulated an immune response in hepatoma cells via increased expression of GM-CSF, IL-21 and Rae-1 markers. The data suggest that Bio-CS increased the inhibition of liver cancer cell proliferation in vitro and the activation of the cellular immunity in vivo .

Cellular immunity is important for protection against the serious complications of influenza in older adults. As it is unclear if newer influenza vaccines elicit greater cellular responses than standard vaccines, we compared responses to 2 standard and 2 newer licensed trivalent inactivated vaccines (TIVs) in a randomized trial in older adults. Non-frail adults ≥ 65 y old were randomly assigned to receive standard subunit, MF59-adjuvanted subunit, standard split-virus or intradermal split-virus TIV. Peripheral blood mononuclear cells (PBMC) harvested pre- and 3-weeks post-vaccination were stimulated with live A/H3N2 virus. PBMC supernatants were tested for interleukin 10 (IL-10) and interferon gamma (IFN-γ), and lysates for granzyme B (GrB). Flow cytometry identified CD4+ and CD8+ T- cells expressing intracellular IL-2, IL-10, IFN-γ, GrB, or perforin. Differences following immunization were assessed for paired subject samples and among vaccines. 120 seniors participated, 29-31 per group, which were well matched demographically. Virus-stimulated PBMCs were GrB-rich before and after vaccination, with minimal increases evident. Immunization did not increase secretion of IFN-γ or IL-10. However, cytolytic effector T-cells (CD8+GrB+perforin+) increased significantly in percentage post-vaccination in all groups, to similar mean values across groups. CD4+GrB+perforin+ T-cells also increased significantly after each vaccine, to similar mean values among vaccines. Vaccination did not increase the low baseline percentages of CD4+ or CD8+ T-cells expressing IFN-γ, IL-2 or IL-10 . In conclusion, participants had pre-existing cellular immunity to H3N2 virus. All 4 vaccines boosted cellular responses to a similar but limited extent, particularly cytolytic effector CD8+ T-cells associated with clinical protection against influenza.

// Wei Wang 1 , Ge Shen 1 , Shikai Wu 1 , Shiping Song 1 , Yanli Ni 1 , Zhuoyao Suo 1 , Xiangying Meng 1 , Dan Li 1 , Lin Zhou 1 , Rimin Hao 1 , Yaowei Zhao 1 , Li Bai 1 , Lili Hou 1 , Bing Liu 1 and Guangxian Liu 1 1 Cancer Therapy Center, Affiliated Hospital of The Academy of Military Medical Sciences, Beijing 100071, China Correspondence to: Guangxian Liu, email: liugx270@hotmail.com Bing Liu, email: bingliu17@yahoo.com Keywords: PD-1, peripheral immune cells, immunomodulation, radiation therapy, cancer Received: June 15, 2016 Accepted: January 16, 2017 Published: February 02, 2017 ABSTRACT Immune checkpoint inhibitors that block the PD-1/PD-L1 signaling pathway have been used to treat a wide variety of cancers. Although results have been promising, significant inter-individual and inter-tumor variability has been observed. It is believed that better clinical outcome could be achieved if the treatment was individually designed based on the functional status of the PD-1/PD-L1 signaling and the cellular immunity. In this study, we analyzed the mRNA expression of PD-1 and other immunomodulatory genes in peripheral blood from cancer patients, and immunomodulatory gene expression during radiotherapy and immunomodulation therapy with cytokines. Our results show that the PD-1 mRNA expression is significantly increased in peripheral blood in cancer patients. Anti-cancer treatments can significantly modulate the PD-1 expression, but this is largely dependent on the initial immune status. Moreover, the PD-1 expression on peripheral lymphocytes can be immunoactivation-derived. These results suggest that the regulation and expression pattern of PD-1/PD-L1 signal is complicated which will influence the effect of blockade of the PD-1/PD-L1 signaling pathway for cancer treatment. Through combined analysis of PD-1, CTLA-4, and other immune markers in peripheral blood, we may accurately evaluate the functional status of PD-1/PD-L1 signaling and cellular immunity, thereby providing clues for guiding anti-PD-1 or anti-PD-L1 treatment.

// So Young Kim 1, * , Dongxu Kang 1, 2, * , Hye Jin Choi 3 , Yeonsoo Joo 1, 4 , Joo-Hang Kim 5 , Jae J. Song 1, 4 1 Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Korea 2 Department of Oncology, Affiliated Hospital of Yanbian University, Yanji, Jilin Province, P.R. China 3 Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea 4 Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea 5 CHA Bundang Medical Center, CHA University, Seongnam, Korea * These authors have contributed equally to this work Correspondence to: Joo-Hang Kim, email: kim123@cha.ac.kr Jae J. Song, email: jjs109@yuhs.ac Keywords: oncolytic adenovirus, MART1, DNA vaccine, GM-CSF, TGF-β2 Received: June 20, 2016 Accepted: December 31, 2016 Published: February 02, 2017 ABSTRACT A successful DNA vaccine for the treatment of tumors should break established immune tolerance to tumor antigen. However, due to the relatively low immunogenicity of DNA vaccines, compared to other kinds of vaccines using live virus or protein, a recombinant viral vector was used to enhance humoral and cellular immunity. In the current study, we sought to develop a novel anti-cancer agent as a complex of DNA and oncolytic adenovirus for the treatment of malignant melanoma in the C57BL/6 mouse model. MART1, a human melanoma-specific tumor antigen, was used to induce an increased immune reaction, since a MART1-protective response is required to overcome immune tolerance to the melanoma antigen MelanA. Because GM-CSF is a potent inducer of anti-tumor immunity and TGF-β2 is involved in tumor survival and host immune suppression, mouse GM-CSF (mGM-CSF) and shRNA of mouse TGF-β2 (shmTGF-β2) genes were delivered together with MART1 via oncolytic adenovirus. MART1 plasmid was also used for antigen-priming. To compare the anti-tumor effect of oncolytic adenovirus expressing both mGM-CSF and shmTGF-β2 (Ad GshT ) with that of oncolytic adenovirus expressing mGM-CSF only (Ad G ), each virus was intratumorally injected into melanoma-bearing C57BL/6 mice. As a result, mice that received Ad GshT showed delayed tumor growth than those that received Ad G . Heterologous prime-boost immunization was combined with oncolytic Ad GshT and MART1 expression to result in further delayed tumor growth. This regression is likely due to the following 4 combinations: MART1-derived mouse melanoma antigen-specific immune reaction, immune stimulation by mGM-CSF/shmTGF-β2, tumor growth inhibition by shmTGF-β2, and tumor cell-specific lysis via an oncolytic adenovirus. Immune activation was mainly induced by mature tumor-infiltrating dendritic cell (TIDC) and lowered regulatory T cells in tumor-infiltrating lymphocytes (TIL). Taken together, these findings demonstrate that human MART1 induces a mouse melanoma antigen-specific immune reaction. In addition, the results also indicate that combination therapy of MART1 plasmid, together with an oncolytic adenovirus expressing MART1, mGM-CSF, and shmTGF-β2, is a promising candidate for the treatment of malignant melanoma.

// Chao Liu 1 , Shikai Wu 1 , Xiangying Meng 1 , Guangxian Liu 2 , Dongmei Chen 1 , Yang Cong 1 , Ge Shen 1 , Bing Sun 1 , Wei Wang 2 , Qian Wang 1 , Hongjun Gao 3 and Xiaoqing Liu 3 1 Department of Radiation Oncology, Affiliated Hospital of Academy of Military Medical Sciences, Beijing, China 2 Cancer Therapy Center, Affiliated Hospital of Academy of Military Medical Sciences, Beijing, China 3 Department of Lung Cancer, Affiliated Hospital of Academy of Military Medical Sciences, Beijing, China Correspondence to: Shikai Wu, email: // Keywords : T cells; lymphocyte subsets; stereotactic body radiation therapy; non-small cell lung cancer; immunotherapy Received : December 19, 2016 Accepted : January 27, 2017 Published : February 09, 2017 Abstract Background: Studies increasingly focus on the impact of radiotherapy on immunity; however, the role of peripheral cellular immunity prior to radiotherapy in cancer patients remains largely unknown. In this study, we investigated the predictive roles of lymphocyte subsets on tumor progression in non-small cell lung cancer (NSCLC) patients undergoing radiotherapy, and their expression in NSCLC patients at first relapse. Methods: We enrolled 70 NSCLC patients and 14 age- and sex-matched healthy donors and tested the lymphocyte subsets in their peripheral blood by flow cytometry. Among them, 40 newly diagnosed patients received radiotherapy and were enrolled to investigate the predictive value of lymphocyte subsets on tumor progression after radiotherapy by uni- and multivariate analyses; 30 patients at first relapse were included to evaluate the differences of lymphocyte subsets between them and first diagnosed patients and healthy volunteers. Results: Increased proportions of regulatory T cells, CD8+ T cells, and CD8+CD28- T cells and decreased CD4+ T cells and CD4/CD8 ratios were observed in NSCLC patients at first relapse compared to newly diagnosed patients. In the 40 first diagnosed patients undergoing radiotherapy, uni- and multivariate analyses showed that increased level of regulatory T cells correlated with poor progression-free survival (hazard ratio = 2.55 and 3.76, P = 0.022 and 0.010, respectively). Conclusions: Peripheral regulatory T cells were increased and independently predict tumor progression in NSCLC patients undergoing radiotherapy, suggesting the promising combination of radiotherapy and immunotherapy.

// Ping Kuang 1, * , Huidan Deng 1, * , Hengmin Cui 1, 2 , Lian Chen 1 , Jing Fang 1, 2 , Zhicai Zuo 1, 2 , Junliang Deng 1, 2 , Xun Wang 1, 2 , Ling Zhao 1, 2 1 College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China 2 Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, Sichuan Agricultural University, Ya’an 625014, China * These authors contributed equally to this work Correspondence to: Hengmin Cui, email: cui580420@sicau.edu.cn Keywords: NaF, growth index, T and B-cell, cytokine, cell cycle Received: November 07, 2016 Accepted: December 07, 2016 Published: December 16, 2016 ABSTRACT At present, very limited studies focus on the toxic effect of sodium fluoride (NaF) on splenic development of human and animals in vivo . This study was firstly designed to evaluate the toxic effects of NaF on the splenic development of mice in vivo by observing histopathological lesions, changes of splenic growth index (GI), T and B cells, immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) contents, cytokine protein expression levels, and cell cycle and cyclins/cdks protein expression levels using the methods of pathology, flow cytometry (FCM), western blot (WB), and enzyme-linked immunosorbent assay (ELISA). A total of 240 ICR mice were equally allocated into four groups with intragastric administration of distilled water in the control group and 12, 24, 48 mg/kg NaF solution in the experimental groups for 42 days. The results showed that NaF in 12 mg/kg and over caused the toxic effects on splenic development, which was characterized by reducing growth index and lymphocytes in the white and red pulp histopathologically, increasing cell percentages of the G0/G1 phase and decreasing cell percentages of the S phase, and reducing T cells and B cells as well as IgA, IgG, and IgM contents when compared with those in the control group. Concurrently, cytokines including interleukin-2 (IL-2), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and cyclin (E/D and CDK2/4) protein expression levels were markedly decreased ( P < 0.05 or P < 0.01), and interleukin-10 (IL-10) protein expression levels were significantly increased ( P < 0.05 and P < 0.01) in the three NaF-treated groups. Toxic effects finally impaired the splenic cellular immunity and humoral immunity due to the reduction of T and B cell population and activity. Cell cycle arrest is the molecular basis of NaF-caused toxic effects on the splenic development.

// Erlong Wang 1,* , Bo Long 1,* , Kaiyu Wang 1,2 , Jun Wang 1 , Yang He 1 , Xingli Wang 1 , Qian Yang 1 , Tao Liu 1 , Defang Chen 3 , Yi Geng 1,2 , Xiaoli Huang 3 , Ping Ouyang 1 and Weimin Lai 1 1 Department of Basic Veterinary, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, China 2 Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, China 3 Department of Aquaculture, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, China * These authors have contributed equally to this work Correspondence to: Kaiyu Wang, email: // Keywords : aquaculture, bacterial diseases, DNA vaccine, interleukin-8, molecular adjuvant, Immunology and Microbiology Section, Immune response, Immunity Received : August 20, 2016 Accepted : November 15, 2016 Published : November 30, 2016 Abstract DNA vaccines had been widely used in animal models against various viral infections, while it was not so convincing for many infectious diseases especially bacterial disease in aquaculture. Interleukin-8(IL-8) as one of the CXC chemokines, its immunological role and adjuvant potential which had been proved in mammals were rarely reported in fish species. In this study, recombination plasmid pcDNA3.1/IL-8(pcIL-8) was conducted and the capacity of IL-8 as molecular adjuvant was explored from several aspects by co-injecting with a DNA vaccine encoding α-enolase(pcENO) against Streptococcus iniae infection in channel catfish. The results suggested that co-injection of pcIL-8 with DNA vaccine increased the innate immunity and specific antibody levels, as well as increased the immune-related genes involving in pro-inflammatory response, humoral and cellular immunity. Moreover, pcIL-8 enhanced the immunoprotection of pcENO with the relative percent survival(RPS) of 60% to 80% against S.iniae infection at 4 week post vaccination(p.v.), with the significantly higher RPS of 73.33% in pcENO+pcIL-8 group compared with that of pcENO alone(53.33%) at challenge test of 8 weeks p.v. Taken together, these results indicate pcIL-8 as a molecular adjuvant co-injected with DNA vaccine not only improves the immunoprotection but also maintains long period of immunity for channel catfish against S.iniae infection. Our study signifies that IL-8 holds promise to serve as a potential adjuvant in DNA vaccines against bacterial infections for long time.

IL-33 is an important member of the IL-1 family which has pleiotropic activities in innate and adaptive immune responses. Recently, some researchers have focused on the function of cellular immunity in the development of tumor. The biological role of IL-33 in glioma is poorly understood. In this study, we showed that glioma cells and tissues expressed higher levels of IL-33 and its receptor ST2 compared to normal brain. Clinically, IL-33 expression was associated with poor survival in patients with glioma. Administration of human IL-33 enhanced cell migration, invasion, epithelial to mesenchymal transition and stemness. Anti-ST2 blocked these effects of IL-33 on tumor. Mechanistically, IL-33 activated JNK signaling pathway via ST2 and increased the expression of key transcription factors that controlled the process of EMT and stemness. Moreover, IL-33 prevented temozolomide induced tumor apoptosis. Anti-ST2 or knockdown IL-33 increased the sensitivity of tumor to temozolomide. Thus, targeting the IL-33/ST2 axis may offer an opportunity to the treatment of glioma patients.

Natural killer cells lacking expression of CD56 (CD56neg NK cells) have been described in chronic HIV and hepatitis C virus infection. Features and functions of CD56neg NK cells in the context of latent infection with CMV and / or EBV with age are not known. In a cohort of healthy donors >60 years of age, we found that co-infection with CMV and EBV drives expansion of CD56neg NK cells. Functionally, CD56neg NK cells displayed reduced cytotoxic capacity and IFN-γ production, a feature that was enhanced with CMV / EBV co-infection. Further, the frequency of CD56neg NK cells correlated with accumulation of end-stage-differentiated T cells and a reduced CD4 / CD8 T cell ratio, reflecting an immune risk profile. CD56neg NK cells had a mature phenotype characterized by low CD57 and KIR expression and lacked characteristics of cell senescence. No changes in their activating NK cell receptor expression, and no upregulation of the negative co-stimulation receptors PD-1 or TIM-3 were observed. In all, our data identify expansion of dysfunctional CD56neg NK cells in CMV+EBV+ elderly individuals suggesting that these cells may function as shape-shifters of cellular immunity and argue for a previously unrecognized role of EBV in mediating immune risk in the elderly.

// Yanxia Lu 1 , Crystal Tze Ying Tan 2 , Ma Shwe Zin Nyunt 3 , Esther Wing Hei Mok 2 , Xavier Camous 2 , Hassen Kared 2 , Tamas Fulop 4 , Liang Feng 5 , Tze Pin Ng 3,* and Anis Larbi 2,4* 1 Department of Clinical Psychology and Psychiatry/School of Public Health, Zhejiang University College of Medicine, Hangzhou, China 2 Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore 3 Gerontology Research Programme, Department of Psychological Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 4 Geriatrics Division, Department of Medicine, Research Center on Aging, University of Sherbrooke, Sherbrooke, Quebec, Canada 5 Graduate Medical School, Duke-National University of Singapore, Singapore * These authors have contributted equally to this work Correspondence to: Anis Larbi, email: // Keywords : inflammation; immunosenescence; frailty risk; T cell subsets; B cells; Gerotarget Received : November 18, 2015 Accepted : April 10, 2016 Published : April 22, 2016 Abstract Chronic systematic inflammation and reduced immune system fitness are considered potential contributing factors to the development of age-related frailty, but the underlying mechanisms are poorly defined. This exploratory study aimed to identify frailty-related inflammatory markers and immunological phenotypes in a cohort of community-dwelling adults aged ≥ 55 years. Frailty was assessed using two models, a Frailty Index and a categorical phenotype, and correlated with levels of circulating immune biomarkers and markers of senescence in immune cell subsets. We identified eight serological biomarkers that were associated with frailty, including sgp130, IL-2Rα, I-309, MCP-1, BCA-1, RANTES, leptin, and IL-6R. Frailty Index was inversely predicted by the frequency of CD3+, CD45RA+, and central memory CD4 cells, and positively predicted by the loss of CD28 expression, especially in CD8+ T cells, while frailty status was predicted by the frequency of terminal effector CD8+ T cells. In γ/δ T cells, frailty was negatively associated with CD27, and positively associated with IFNγ+TNFα- secretion by γ/δ2+ cells and IFNγ-TNFα+ secretion by γ/δ2- cells. Increased numbers of exhausted and CD38+ B cells, as well as CD14+CD16+ inflammatory monocytes, were also identified as frailty-associated phenotypes. This pilot study supports an association between inflammation, cellular immunity, and the process of frailty. These findings have significance for the early identification of frailty using circulating biomarkers prior to clinical manifestations of severe functional decline in the elderly.

Tumor necrosis factor-α induced protein 8 like-2 (TNFAIP8L2, TIPE2) is a lately discovered negative regulator of innate immunity and cellular immunity. The present study was designed to investigate whether naturally occurring dendritic cells (DCs) could express TIPE2 mRNA/protein and its potential significance. Expressions of co-stimulatory molecules on DC surface and cytokines were analyzed to assess the functional role of TIPE2 in controlling DC maturation as well as activation. The activated DCs were assessed for their capacity to stimulate the proliferation and differentiation of T cells. It was found that TIPE2 was a cytoplasmic protein expressed in DCs, and the percentage of DCs which expressed co-stimulatory molecules and cytokines were obviously up-regulated when TIPE2 gene silenced by siRNA in vitro and in vivo. DCs undergone TIPE2 knockdown were found to promote the maturation of DCs, T-cell proliferation as well as differentiation, and they were significantly elevated IL-2 level and intranuclear NF-AT activation. Conversely, in over-expressing TIPE2 DC cells, it could inhibit T-cell proliferation and differentiation, and markedly down-regulate IL-2 expression and intranuclear NF-AT activation after scald injury. The results suggested that TIPE2 appeared to be a critical immunoregulatory molecule which affected DC maturation and subsequent T-cell mediated immunity.

UNLABELLED Echinacea purpurea-containing remedies are herbal medicines used in respiratory tract infections and several inflammatory conditions as enhancers of non-specific and modulators of specific cellular immunity. They also exert anti-inflammatory, anti-viral, and anti-microbial activity. The aim of the present study was to compare the in vivo influence of orally administered three Echinacea purpurea-based remedies (IMMUNAL drops, ECHINACEA FORTE drops, IMMUNAL FORTE tablets) on some parameters of cellular and humoral immunity in mice. RESULTS Feeding mice for seven days with IMMUNAL drops resulted in enhanced anti-SRBC antibody production and modulatory effect on proliferative response to PHA of their splenic lymphocytes. No stimulatory effect was observed on splenocytes chemokinesis. Mice fed with ECHINACEA FORTE drops presented enhanced response to PHA of their splenocytes. However, contrary to the previous group, no enhancement of antibody production was observed. In this group, lymphocyte-induced immunological angiogenesis (LIA) and chemokinesis (spontaneous migration - SM) of spleen lymphocytes was diminished after feeding mice with both doses (LIA) or with a higher dose (SM) of remedy. Lymphocyte-induced immunological angiogenesis activity of splenocytes collected from animals fed with prophylactic and therapeutic IMMUNAL FORTE tablet doses did not differ from the controls.

In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP(+) microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo.

// Qing-Kun Song 1, * , Jun Ren 1, 2, 3, * , Xin-Na Zhou 1, 2 , Xiao-Li Wang 1, 2 , Guo-Hong Song 2 , Li-Jun Di 2 , Jing Yu 2 , Amy Hobeika 3 , Michael A. Morse 4 , Yan-Hua Yuan 1 , Hua-Bing Yang 1 , Herbert Kim Lyerly 3 1 Beijing Key Laboratory of Therapeutic Cancer Vaccines, Capital Medical University Cancer Center, Beijing Shijitan Hospital, Beijing 10038, China 2 Department of Medical Oncology, Peking University Cancer Hospital & Institute, Beijing 100142, China 3 Department of Surgery, Duke University Medical Center, Durham, NC 27710, United States 4 Department of Medicine, Duke University Medical Center, Durham, NC 27710, United States * These authors have contributed equally to this work Correspondence to: Jun Ren, e-mail: jun.ren@duke.edu Herbert Kim Lyerly, e-mail: kim.lyerly@duke.edu Keywords: CD4 + CD25 + T lymphocyte, early stage breast cancer, triple negative breast cancer, prognosis, adoptive cell therapy Received: June 16, 2015 Accepted: September 07, 2015 Published: October 05, 2015 ABSTRACT Objective: This study aimed to assess the prognostic value of CD4 + CD25 + T lymphocyte in peripheral blood among breast cancer patients treated with adoptive T lymphocytes immunotherapy. Methods: 217 patients participated in the follow-up study. CD4 + CD25 + proportion was measured by flow cytometry in peripheral T cells. The median survival was estimated by Kaplan-Meier curve, Log-rank test and Cox hazard proportion regression model, between groups of CD4 + CD25 + proportion more than 5% and less than or equal to 5% in peripheral T cells. Results: Peripheral CD4 + CD25 + T lymphocytes had not a relationship with progression-free survival. It was featured that above 5% peripheral CD4 + CD25 + proportion of T cells was related with the median overall survival by a shorten of 51 months ( p < 0.05) with the HR 1.65 (95%CI 1.04, 2.62). Above 5% CD4 + CD25 + proportion of T cells produced the HR to be 1.76 (95%CI 1.07, 2.87) In stage 0-II patients, and 3.59 (95%CI 1.05, 12.29) in triple negative breast cancer patients. Conclusion: Cellular immunity restoration recovered by adoptive T cell infusions which resulted in less proportion of peripheral CD4 + CD25 + T lymphocytes could be a potential prognostic indicator among early stage and triple negative patients.

Granulysin is a recently discovered cytolytic protein of natural killer (NK) cells and cytotoxic T lymphocytes. Studies of healthy and immunocompromised patients with primary or recurrent varicella-zoster infections demonstrate the importance of virus-specific cellular immunity in controlling viral replication, but also some studies presented granulysin as a molecule that can play a role in chickenpox immunopathogenesis. This study investigated possible correlation between serum granulysin levels and clinical course of chickenpox. A total of 69 patients with chickenpox were included in the study. We measured the levels of granulysin and percentage count for CD4+, CD8+ and NK cells in serum for all patients and healthy controls. For detection and quantification of granulysin in sera, we performed ELISA test and flow cytometry for detection, identification and percentage measurement of T and B lymphocytes. Descriptive methods, analysis of variance and multivariate logistic regression were used for statistical data analysis. We found respective correlation between serum granulysin level and severity of clinical presentation. These findings can be a good input for further studies, since there is no relevant prognostic parameter of chickenpox in everyday clinical practice. Granulysin, as a therapeutic, also deserves to be a point of interests in the future. If we prove its potential to stop dissemination of human herpes viruses, possibilities to use it in some life-threatening forms of viral disease can be very valuable.

Cross-talk by pattern recognition receptors may facilitate the maturation of dendritic cells and fine tune the immune response. Thus, the inclusion of ligands agonistic to multiple receptors in a vaccine formula may be an effective strategy to elicit robust antitumor cellular immunity. We tested the adjuvant effects and possible synergy of CpG (CpG oligodeoxynucleotide), Poly I:C (polyinosinic-polycytidylic acid) and the cationic peptide Cramp (cathelicidin-related antimicrobial peptide) formulated in a DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) liposomal HPV E7 epitope vaccine on a TC-1 grafted mouse model. The vaccine formulations were administered both preventively and therapeutically. Based on our results, both CpG and Poly I:C-adjuvanted vaccines abolished tumor development in a preventive trial and significantly suppressed tumor growth in a therapeutic trial. Increased interferon (IFN)-γ expression and potent memory T cells in splenocytes as well as elevated CD8+IFN-γ+ cells in both spleen and tumor tissue indicated an elevated E744-62-specific cellular immune response. Although synergistic effects were detected between CpG and Poly I:C, their adjuvant effects were not enhanced further when combined with Cramp. Because the enhancement of tumor antigen-specific cellular immune responses is vital for the clearance of infected and cancerous cells, our results contribute a potential adjuvant combination for cancer vaccines.