Integrated analysis of miRNA-mRNA expression of newly emerging swine H3N2 influenza virus cross-species infection with tree shrews.

BACKGROUND: Cross-species transmission of zoonotic IAVs to humans is potentially widespread and lethal, posing a great threat to human health, and their cross-species transmission mechanism has attracted much attention. miRNAs have been shown to be involved in the regulation of IAVs infection and immunity, however, few studies have focused on the molecular mechanisms underlying miRNAs and mRNAs expression after IAVs cross-species infection. METHODS: We used tree shrews, a close relative of primates, as a model and used RNA-Seq and bioinformatics tools to analyze the expression profiles of DEMs and DEGs in the nasal turbinate tissue at different time points after the newly emerged swine influenza A virus SW2783 cross-species infection with tree shrews, and miRNA-mRNA interaction maps were constructed and verified by RT-qPCR, miRNA transfection and luciferase reporter assay. RESULTS: 14 DEMs were screened based on functional analysis and interaction map, miR-760-3p, miR-449b-2, miR-30e-3p, and miR-429 were involved in the signal transduction process of replication and proliferation after infection, miR-324-3p, miR-1301-1, miR-103-1, miR-134-5p, miR-29a, miR-31, miR-16b, miR-34a, and miR-125b participate in negative feedback regulation of genes related to the immune function of the body to activate the antiviral immune response, and miR-106b-3p may be related to the cross-species infection potential of SW2783, and the expression level of these miRNAs varies in different days after infection. CONCLUSIONS: The miRNA regulatory networks were constructed and 14 DEMs were identified, some of them can affect the replication and proliferation of viruses by regulating signal transduction, while others can play an antiviral role by regulating the immune response. It indicates that abnormal expression of miRNAs plays a crucial role in the regulation of cross-species IAVs infection, which lays a solid foundation for further exploration of the molecular regulatory mechanism of miRNAs in IAVs cross-species infection and anti-influenza virus targets.

Nanobody-based trispecific T cell engager (Nb-TriTE) enhances therapeutic efficacy by overcoming tumor-mediated immunosuppression.

BACKGROUND: T cell engagers (TCEs) have been established as an emerging modality for hematologic malignancies, but solid tumors remain refractory. However, the upregulation of programmed cell death 1 (PD-1) is correlated with T cell dysfunction that confer tumor-mediated immunosuppression. Developing a novel nanobody-based trispecific T cell engager (Nb-TriTE) would be a potential strategy to improve therapeutic efficacy. METHODS: Given the therapeutic potential of nanobodies (Nbs), we first screened Nb targeting fibroblast activation protein (FAP) and successfully generated a Nb-based bispecific T cell engager (Nb-BiTE) targeting FAP. Then, we developed a Nb-TriTE by fusing an anti-PD-1 Nb to the Nb-BiTE. The biological activity and antitumor efficacy of the Nb-TriTE were evaluated in vitro and in both cell line-derived and patient-derived xenograft mouse models. RESULTS: We had for the first time successfully selected a FAP Nb for the generation of novel Nb-BiTE and Nb-TriTE, which showed good binding ability to their targets. Nb-TriTE not only induced robust tumor antigen-specific killing, potent T cell activation and enhanced T cell function in vitro, but also suppressed tumor growth, improved survival and mediated more T cell infiltration than Nb-BiTE in mouse models of different solid tumors without toxicity. CONCLUSIONS: This novel Nb-TriTE provides a promising and universal platform to overcome tumor-mediated immunosuppression and improve patient outcomes in the future.

Pathogenicity and anti-infection immunity of animal H3N2 and H6N6 subtype influenza virus cross-species infection with tree shrews.

Animal influenza viruses can spread across species and pose a fatal threat to human health due to the high pathogenicity and mortality. Animal models are crucial for studying cross-species infection and the pathogenesis of influenza viruses. Tupaia belangeri (tree shrew) has been emerging as an animal model for multiple human virus infections recently because of the close genetic relationship and phylogeny with humans. So far, tree shrew has been reported to be susceptible to human influenza virus subtype H1N1, avian influenza viruses subtype H9N2, subtype H5N1, and subtype H7N9. However, the pathogenicity, infection, and immunity of swine and land avian influenza viruses with low pathogenicity and the potential to jump to humans remain largely unexplored in the tree shrew model. Previously, our team has successfully isolated the newly emerging swine influenza virus subtype H3N2 (A/Swine/GX/NS2783/2010, SW2783) and avian influenza virus subtype H6N6 (A/CK/ZZ/346/2014, ZZ346). In this study, we observed the pathogenicity, immune characteristics, and cross-species infection potential ability of SW2783 and ZZ346 strains in tree shrew model with 50% tissue culture infective dose (TCID50), hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), real-time quantitative PCR (qRT-PCR) and other experimental methods. Both animal-borne influenza viruses had a strong ability on tissue infection in the turbinate and the trachea of tree shrews in vitro, in which SW2783 showed stronger replication ability than in ZZ346. SW2783 and ZZ346 both showed pathogenic ability with infected tree shrews model in vivo without prior adaptive culture, which mainly happened in the upper respiratory tract. However, the infection ability was weak, the clinical symptoms were mild, and the histopathological changes in the respiratory tract were relatively light. Furthermore, innate immune responses and adaptive immunity were observed in the tree shrew model after the infection of SW2783 and ZZ346 strains. We observed that the unadapted SW2783 and ZZ346 virus could transmit among tree shrews by direct contact. We also observed that SW2783 virus could transmit from tree shrews to guinea pigs. These results indicated that both animal-borne influenza viruses could induce similar pathogenicity and immune response to those caused by human-common influenza viruses. Tree shrews may be an excellent animal model for studying the interaction between the influenza virus and the host and the cross-species infection mechanism of the animal influenza virus.

Endoglin-Aptamer-Functionalized Liposome-Equipped PD-1-Silenced T Cells Enhance Antitumoral Immunotherapeutic Effects.

BACKGROUND: The broader application of adoptive cell therapy (ACT) in cancer immunotherapies (particularly for solid tumors) has always been limited by the immunosuppressive tumor microenvironment (TME) and the insufficient targetability of effector T cells, resulting in unsatisfied therapeutic outcome. Here, we designed a new strategy by using aptamer-based immunoliposomes to modify PD-1-silencing T cells, which were activated by dendritic cell (DC)/tumor fusion cells (FCs) to improve the antitumor potency of cytotoxic T lymphocytes (CTLs/CD8+ T cells). METHODS: PD-1 gene was knocked out from CD8+ T cells using CRISPR/Cas9 system to liberate T cell activity from immunosuppression. The PD-1- T cells were stimulated with DC/tumor FCs, followed by further functional modification of tumor-specific nanoliposomes (hEnd-Apt/CD3-Lipo) to generate FC/PD-1- CTLs. The activation and proliferation and specificity of the modified FC/PD-1- CTLs were measured. The antitumor activity of these CTLs against HepG2-tumors was evaluated in xenograft NOD/SCID mice, and the antitumor mechanism was investigated based on tissue immunohistochemistry and serum ELISA. RESULTS: Our results indicated that the modification of hEnd-Apt/CD3-Lipo nanocomposites on the FC/PD-1- CTLs had a more substantial synergetic effect in inhibiting tumor growth and prolonging animal survival, rather than other control liposomes. Furthermore, the hEnd-Apt/CD3-Lipo-modified FC/PD-1- CTLs showed a stronger antitumor outcome in the tumor-bearing mouse model, through the mechanisms of suppressing tumor cell proliferation, promoting tumor apoptosis, reducing angiogenesis but increasing the infiltration of the FC/PD-1- CTLs in the tumor tissue, as well as upregulating the systemic levels of IFN-γ, IL-2, TNF-α and IL-6 cytokines, by comparison of the control settings. CONCLUSION: In sum, our investigation suggests an enhancement of antitumor effect by the surface modification of endoglin-targeting nanoliposomes upon DC/tumor FC-activated PD-1- CTLs, therefore, provides a new tumoral endoglin-targeted approach as a promising strategy to reduce immunosuppression of tumor microenvironment and improve the immunotherapeutic outcome of anticancer ACT.

Nanobody-based chimeric antigen receptor T cells designed by CRISPR/Cas9 technology for solid tumor immunotherapy.

Chimeric antigen receptor-based T-cell immunotherapy is a promising strategy for treatment of hematological malignant tumors; however, its efficacy towards solid cancer remains challenging. We therefore focused on developing nanobody-based CAR-T cells that treat the solid tumor. CD105 expression is upregulated on neoangiogenic endothelial and cancer cells. CD105 has been developed as a drug target. Here we show the generation of a CD105-specific nanobody, an anti-human CD105 CAR-T cells, by inserting the sequences for anti-CD105 nanobody-linked standard cassette genes into AAVS1 site using CRISPR/Cas9 technology. Co-culture with CD105+ target cells led to the activation of anti-CD105 CAR-T cells that displayed the typically activated cytotoxic T-cell characters, ability to proliferate, the production of pro-inflammatory cytokines, and the specific killing efficacy against CD105+ target cells in vitro. The in vivo treatment with anti-CD105 CAR-T cells significantly inhibited the growth of implanted CD105+ tumors, reduced tumor weight, and prolonged the survival time of tumor-bearing NOD/SCID mice. Nanobody-based CAR-T cells can therefore function as an antitumor agent in human tumor xenograft models. Our findings determined that the strategy of nanobody-based CAR-T cells engineered by CRISPR/Cas9 system has a certain potential to treat solid tumor through targeting CD105 antigen.

Recombinant Endoglin-Single-Chain Variable Fragment/ Induced Protein 10 Fusion Protein Potently Boosts the Anti-Tumor Efficacy of Adoptively Transferred TRP2-Specific CD8+ CD28+ Cytotoxic T Lymphocytes in Mice.

In this research, we studied the therapeutic efficacy of a newly designed fusion protein containing Endoglin single-chain variable fragment and IP10 (Endoglin-scFv/IP10), together with our recently generated TRP2-specific CD8+ CD28+ CTLs (CD8+ CD28+ CTLs) in controlling melanoma growth in mice. The recombinant Endoglin-scFv/IP10 was expressed in E. coli, purified by affinity chromatography, and characterized in vitro for its chemotactic movement and immunoreactivity with endoglin-expressing cells. In vivo, melanoma xenografts were established in mice (C57BL/6) using B16F10 cells. After that, mice were treated with intravenous injections of vehicle (PBS), Endoglin-scFv/IP10 alone, CD8+ CD28+ CTLs alone, or Endoglin-scFv/IP10+ CD8+ CD28+ CTLs. The therapeutic efficacy was assessed by monitoring tumor growth, mouse survival and cellular biomarkers. Endoglin-scFv/IP10 fusion protein combined with CD8+ CD28+ CTLs observed a reduction in tumor growth, resulting in improved survival. On the cellular level, the combination treatment dramatically reduced the number of systemic and tumor associated myeloid-derived suppressor cells or regulatory T cells, increased tumor-responsive interferon-γ-producing lymphocytes and tumor-associated CD8+ CXCR3+ T cells, and inhibited proliferation and angiogenesis but stimulated apoptosis within melanoma tissue. This study demonstrates the therapeutic potential of Endoglin-scFv/IP10 fusion protein in combination with CD8+ CD28+ CTLs in melanoma treatment.

In vivo Targeting of Liver Cancer with Tissue- and Nuclei-Specific Mesoporous Silica Nanoparticle-Based Nanocarriers in mice.

PURPOSE: Cancer tissue-specific and nuclei-targeted drug delivery is ideal for the delivery of chemotherapy. However, it has only been achieved in in vitro studies mainly due to low efficiency in vivo. In this study, we aimed to establish an efficient dual-targeted system that targets liver cancer tissue as well as the nuclei of cancer cells in vivo. METHODS: We first synthesized TAT peptide (TATp)-mesoporous silica nanoparticle (MSN) complex (TATp-MSN) and generated liposomes that carried liver cancer-specific aptamer TLS11a (TLS11a-LB). We then generated the drug TLS11a-LB@TATp-MSN/doxorubicin (DOX) by mixing TLS11a-LB and DOX-loaded TATp-MSN. After physical and chemical characterization of the nanoparticles, DOX release from these formulations was evaluated at pH 5.0 and 7.4. Furthermore, we also evaluated nuclear localization and cytotoxicity of the drug in H22 cells in vitro and investigated the liver cancer targeting and antitumor activities of the nano-drug in vivo using a H22 tumor-bearing mice model. RESULTS: TLS11a-LB@TATp-MSN/DOX and its controls were confirmed as nano-drugs (<100 nm) using transmission electron microscopy (TEM). The DOX release rate of TLS11a-LB@TATp-MSN/DOX was significantly faster at pH 5.0 than at pH 7.4. TLS11a-LB@TATp-MSN/DOX effectively targeted the nuclei of H22 cells and released DOX with a higher efficiency than that of the control groups. In addition, TLS11a-LB@TATp-MSN/DOX exhibited slight cytotoxicity, but not significantly more than controls. In vivo studies showed that TLS11a-LB@TATp-MSN accumulated in subcutaneous H22 tumors in the right axilla of BALB/c mice, reaching peak levels at 48 h after intravenous injection, respectively, and demonstrated that TLS11a-LB@TATp-MSN/DOX group enhanced tumor treatment efficacy while reducing systemic side effects. CONCLUSION: TLS11a-LB@TATp-MSN/DOX can efficiently deliver DOX to the nuclei of liver cancer cells by dual targeting liver cancer tissue and the nuclei of the cancer cells in mice. Thus, it is a promising nano-drug for the treatment of liver cancer.

Single-Domain Antibody-Based TCR-Like CAR-T: A Potential Cancer Therapy.

Retargeting the antigen-binding specificity of T cells to intracellular antigens that are degraded and presented on the tumor surface by engineering chimeric antigen receptor (CAR), also named TCR-like antibody CAR-T, remains limited. With the exception of the commercialized CD19 CAR-T for hematological malignancies and other CAR-T therapies aiming mostly at extracellular antigens achieving great success, the rareness and scarcity of TCR-like CAR-T therapies might be due to their current status and limitations. This review provides the probable optimized initiatives for improving TCR-like CAR-T reprogramming and discusses single-domain antibodies administered as an alternative to conventional scFvs and secreted by CAR-T cells, which might be of great value to the development of CAR-T immunotherapies for intracellular antigens.

A Nanobody Against Cytotoxic T-Lymphocyte Associated Antigen-4 Increases the Anti-Tumor Effects of Specific CD8+ T Cells.

Adoptive cell-based immunotherapy typically utilizes cytotoxic T lymphocytes (CTLs), expanding these cells ex vivo. Such expansion is traditionally accomplished through the use of autologous APCs that are capable of interactions with T cells. However, incidental inhibitory program such as CTLA-4 pathway can impair T cell proliferation. We therefore designed a nanobody which is specific for CTLA-4 (CTLA-4 Nb 16), and we then used this molecule to assess its ability to disrupt CTLA-4 signaling and thereby overcome negative costimulation of T cells. With CTLA-4 Nb16 stimulation, dendritic cell/hepatocellular carcinoma fusion cells (DC/HepG2-FCs) enhanced autologous CD8+ T cell proliferation and production of IFN-γ in vitro, thereby leading to enhanced killing of tumor cells. Using this approach in the context of adoptive CD8+ immunotherapy led to a marked suppression of tumor growth in murine NOD/SCID hepatocarcinoma or breast cancer xenograft models. We also observed significantly increased tumor cell apoptosis, and corresponding increases in murine survival. These findings thus demonstrate that in response to nanobody stimulation, DC/tumor cells-FC-induced specific CTLs exhibit superior anti-tumor efficacy, making this a potentially valuable means of achieving better adoptive immunotherapy outcomes in cancer patients.

Opportunities and Challenges for Antibodies against Intracellular Antigens.

Therapeutic antibodies are one most significant advances in immunotherapy, the development of antibodies against disease-associated MHC-peptide complexes led to the introduction of TCR-like antibodies. TCR-like antibodies combine the recognition of intracellular proteins with the therapeutic potency and versatility of monoclonal antibodies (mAb), offering an unparalleled opportunity to expand the repertoire of therapeutic antibodies available to treat diseases like cancer. This review details the current state of TCR-like antibodies and describes their production, mechanisms as well as their applications. In addition, it presents an insight on the challenges that they must overcome in order to become commercially and clinically validated.

Enhanced cytotoxic T lymphocytes recruitment targeting tumor vasculatures by endoglin aptamer and IP-10 plasmid presenting liposome-based nanocarriers.

Background: Adequate recruitment of highly active tumor antigen-specific cytotoxic T lymphocytes (CTLs) remains a major challenge in cancer immunotherapy. Objective: To construct liposome (LP)-based nanocapsules with surface endoglin aptamer (ENG-Apt) encapsulating mouse interferon-inducible protein-10 (mIP-10), with the ability to target mouse tumor vascular endothelial cells (mTECs) and enhance CTLs targeting and recruitment to the tumor vasculature. Methods: ENG-Apt/mIP-10-LP nanocapsules were prepared by grafting DSPE-PEG2000-ENG-Apt on the surface of liposomes containing mIP-10 plasmids, characterized and assessed for the cell binding specificity in vitro. The tumor-targeting ability of ENG-Apt/mIP-10-LP nanocapsules was evaluated in vivo. The anti-tumor efficacy of ENG-Apt/mIP-10-LP nanocapsules treatment, as well as the combination treatment of ENG-Apt/mIP-10-LP nanocapsules and adoptive TRP2CD8+ T cells, were both tested in melanoma-bearing mice, by evaluation of the tumor volume and the mouse survival time. To discuss the anti-tumoral mechanism of ENG-Apt/mIP-10-LP nanocapsules-based therapies, IFN-γ secretion, proportion of TRP2CD8+ T cells among TILs, MDSCs in the tumor microenvironment and Tregs in the spleen, were determined after the treatments. Proliferation and apoptosis of tumor cells, and tumor angiogenesis were also assessed. Results: The prepared ENG-Apt/mIP-10-LP nanocapsules possess an adequate nanometric size, good stability, high specificity to mTECs and tumor sites, along with the ability to induce mIP-10 expression in vitro and in vivo. Treatment of ENG-Apt/mIP-10-LP nanocapsules demonstrated CTLs enrichment into the tumor site, which inhibited tumor cell proliferation and angiogenesis, as well as promoted tumor-cell apoptosis, leading to a decrease in tumor progression and prolonged survival time in melanoma tumor-bearing mice. In addition, the proportion of MDSCs and Tregs was found to decrease. The combination of ENG-Apt/mIP-10-LP nanocapsules with adoptive TRP2CD8+ T cells, showed stronger abilities in inhibiting tumor growth and increasing animal survival time, thereby displayed an enhanced anti-melanoma tumor efficacy, due to the recruitment of both endogenous CD8+ T cells and exogenous TRP2CD8+ T cells in vivo. Conclusion: ENG-Apt/mIP-10-LP nanocapsules could enhance the recruitment of both endogenous and exogenous CTLs specifically targeting melanoma tumor vasculatures and exert anti-tumoral effect, therefore provides a potentially novel strategy for tumor immunotherapy.

Highly sensitive detection of CTLA-4-positive T-cell subgroups based on nanobody and fluorescent carbon quantum dots.

The detection of cytotoxic T-lymphocyte antigen-4-positive (CTLA-4+) T-cell subgroups in peripheral blood samples and tumor tissues is of great significance. In the present study, a rapid, succinct and efficient method was designed for the detection of CTLA-4+ human T cells using a CTLA-4-specific nanobody-fluorescent carbon quantum dots complex (QDs-Nb36). QDs-Nb36 was used for high sensitivity detection of CTLA-4+ T cells by flow cytometry or immumofluorescent staining. The present study demonstrated that the novel technique was more specific and effective in the detection of CTLA-4+ T-cell ratio in the peripheral blood and tumor tissues compared with a traditional monoclonal antibody approach. Furthermore, no significant toxicity was identified in vitro and in vivo, thus suggesting that the method may have broad applications for the detection of certain lowly expressed targets.

Anti-Tumor Activity of Mannose-CpG-Oligodeoxynucleotides-Conjugated and Hepatoma Lysate-Loaded Nanoliposomes for Targeting Dendritic Cells In Vivo.

Dendritic cell (DC)-based tumor vaccines are a promising immunotherapeutic method of cancer treatment. However, their therapeutic applications are significantly limited by their weak immunogenicity, costly culturing steps, and easily degradable properties. Thus, the anti-tumor activity for the vaccines should be improved. In this study, a novel lipid nanoparticle (M/CpG-ODN-H22-Lipo) was developed, which was conjugated with synthetic CpG oligodeoxynucleotides (CpG-ODN) and mannose and then loaded with H22 hepatoma lysate. Our data corroborate that M/CpG-ODN-H22-Lipo selectively targeted DCs and significantly increased their induced-maturation. Besides, the vaccine halted tumor growth and extended survival of mice with hepatocellular carcinoma. Moreover, M/CpG-ODN-H22-Lipo treatment reduced the percentages of myeloid-derived suppressor cells (in the tumor and bone marrow) and regulatory T cells (Treg) in the spleen. In contrast, the number of IFN-gamma-positive cells in the spleen along with the serum IgG levels were up-regulated. Moreover, tumor angiogenesis and tumor-cell proliferation were halted by the treatment of M/CpG-ODN-H22-Lipo, whereas tumor cell apoptosis was up-regulated. Our data revealed that CD8 + T cells and NK cells were vital to mediate the anti-tumor immunity of M/CpG-ODN-H22-Lipo treatment. In sum, the results here proved M/CpG-ODN-H22-Lipo vaccine a safe, specific and effective DC-based anti-tumor immunotherapy with great potential for clinical applications.

Novel fusion cells derived from tumor cells expressing the heterologous α-galactose epitope and dendritic cells effectively target cancer.

Tumor cells/dendritic cells (DCs) fusion cells (tumor/DC) represent a promising immunotherapeutic strategy but are still under performed in clinical trials for cancer treatment. To further boost their anticancer efficacy, here we developed a novel design for fusing dendritic cells with MDA-MB-231 cells expressing the heterologous α-galactose (α-gal) epitope and assessed its anticancer activities both in vitro and in vivo. The high expression of α-gal in MDA-MB-231 (Gal+)/DC correlated with enhanced DC activation. When applied to T cells, MDA-MB-231 (Gal+)/DC significantly stimulated T-cell proliferation and activation, promoted productions of cytokines IL-2 and IFN-γ, and enhanced T-cell-mediated cytotoxicity against MDA-MB-231 cells. MDA-MB-231 (Gal+)/DC inhibited proliferation and promoted apoptosis of tumor cells in vivo, prolonged mouse survival, and significantly boosted anticancer immunity by increasing CD4+ and CD8+ T cells systemically and elevating serum levels of cytokines and IgG. These results suggested that fusing dendritic cells with tumor cells expressing the heterologous α-gal epitope provides a novel therapeutic strategy for cancer treatment.

Fusion of Dendritic Cells and Cancer-Associated Fibroblasts for Activation of Anti-Tumor Cytotoxic T Lymphocytes.

Here we explored the fusion of dendritic cells (DCs), potent antigen-presenting cells that initiate primary immune responses, with cancer-associated fibroblasts (CAFs), which are a stromal component needed for tumor progression, with the aim of stimulating T cells to inhibit tumor growth. Dendritic cells from the bone marrow of BALB/c mice were co-cultured with CAFs from H22 mouse hepatoma cells. CAFs were found to express fibroblast activation protein and α-smooth muscle actin by flow cytometry, Western blotting and immunofluorescence. Polyethylene glycol was added to the co-culture medium to encourage fusion, and the ability of the resulting fusion cells to produce TNF-α, IL-1ß, IL-6, and IL-12p70 was confirmed using ELISA. These fusion cells efficiently stimulated T lymphocytes in vitro, causing them to generate IFN-α and IFN-γ. T cells activated by DC/CAF fusion cells led to strong CTL response against CAFs in vitro. The activated T cells also inhibited growth of H22 xenografts in vivo. These results indicate that DC/CAF fusion cells show potential for stimulating T cells as a novel anti-tumor vaccine.

The enhanced antitumor-specific immune response with mannose- and CpG-ODN-coated liposomes delivering TRP2 peptide.

PURPOSE: Dendritic cell (DC)-based cancer vaccines is a newly emerging and potent form of immune therapy. As for any new technology, there are still considerable challenges that need to be addressed. Here, we investigate the antitumor potential of a novel liposomal vaccine, M/CpG-ODN-TRP2-Lipo. METHODS: We developed a vaccination strategy by assembling the DC-targeting mannose and immune adjuvant CpG-ODN on the surface of liposomes, which were loaded with melanoma-specific TRP2180-188 peptide as liposomal vaccine. M/CpG-ODN-TRP2-Lipo treatment was used to intendedly induce activation of DCs and antitumor- specific immune response in vivo. RESULTS: Our results demonstrated in vitro that the prepared liposomal particles were efficiently taken up by DCs. This uptake led to an enhanced activation of DCs, as measured by the upregulation of MHC II, CD80, and CD86. Furthermore, M/CpG-ODN-TRP2-Lipo effectively inhibited the growth of implanted B16 melanoma and prolonged the survival of mice. This therapy significantly reduced the number of myeloid-derived suppressor cells (MDSCs) and regulatory T cells, while simultaneously increasing the number of activated T cells, tumor antigen-specific CD8+ cytotoxic T cells, and interferon-γ-producing cells. At the same time, it was found to suppress tumor angiogenesis and tumor cell proliferation, as well as up-regulate their apoptosis. Interestingly, MyD88-knockout mice had significantly shorter median survival times compared to wild-type mice following the administration of M/CpG-ODN-TRP2-Lipo. CONCLUSIONS: The results suggested that the antitumor activities of the vaccine partially rely on the Myd88 signaling pathway. Interestingly, compared to whole tumor cell lysate-based vaccine, M/CpG-ODN-TRP2-Lipo, tumor specific antigen peptide-based vaccine, improved survival of tumor-bearing mice as well as enhanced their antitumor responses. All in all, we describe a novel vaccine formulation, M/CpG-ODN-TRP2-Lipo, with the aim of improving antitumor responses by alleviating the immunosuppressive environment in tumors.

Screening and antitumor effect of an anti­CTLA­4 nanobody.

Cytotoxic T­lymphocyte antigen­4 (CTLA­4) is a critical negative regulator of immune responses. CTLA­4 is rapidly upregulated following T­cell activation, and then binds to B7 molecules with a higher affinity than CD28. CTLA­4 may abolish the initiation of the responses of T cells by raising the threshold of signals required for full activation of T cells, and it also may terminate ongoing T-cell responses. This regulatory role has led to the development of monoclonal antibodies (mAbs) designed to block CTLA­4 activity for enhancing immune responses against cancer. mAbs have several disadvantages including high production cost and unstable behavior. Nanobodies (Nbs) are single­domain antigen­binding fragments derived from the camelid heavy­chain antibodies, which are highly attractive in cancer immunotherapy due to their small size, high specificity, and stability. We selected CTLA­4­specific Nbs from a high quality dromedary camel immune library by phage display technology. Four positive colonies were sequenced and classified based on the amino acids sequences in the CDR3 region. These Nbs recognized unique epitopes on CTLA­4 and displayed high binding rates when used on PHA­stimulated human T cells. Treatment of B16 melanoma­bearing C57BL/6 mice with anti­CTLA­4 nanobody 16 (Nb16) delayed melanoma growth and prolonged the survival time of mice. These data indicate that anti­CTLA­4 Nbs selected from a high quality phage display library may be effective for the treatment of patients with tumors.

[Expression and significance of basic fibroblast growth factor in infant vascular anomalies].

PURPOSE: To investigate the expression of basic fibroblast growth factor (bFGF) in infant's peripheral blood and tumor blood of vascular malformation and peripheral venous blood of hemangioma, and the possibility to differentiate hemangioma from vascular malformations by detecting bFGF level in peripheral blood. METHODS: The level of bFGF in peripheral blood(49 vascular malformations ,32 hemangioma cases and 23 normal cases) and tumor blood(14 venous malformations) of infants was measured by ELISA.SPSS11.5 software package was used to analyze the data and student's t test and ANOVA were used to determine the difference between different groups. RESULTS: The expression of bFGF in tumor blood from infants with venous malformations was significantly higher than that in peripheral blood (P<0.05); No significant difference of bFGF level in peripheral blood was found between hemangioma,vascular malformation and normal infants(P>0.05). CONCLUSIONS: The level of bFGF in tumor blood of from infants with venous malformation was higher than that in peripheral blood; Examination of bFGF in peripheral blood can not differentiate hemangioma from vascular malformations.