Tumor Site-Specific Cleavage Improves the Antitumor Efficacy of Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) play important roles in tumor therapy. However, traditional ADCs are limited by the extremely large molecular weight of the antibody molecules, which results in low permeability into solid tumors. The use of small ADCs may be expected to alleviate this problem, but this switch brings the new limitation of a greatly shortened blood circulation half-life. Here, we propose a new cleavable ADC design with excellent tumor tissue permeability and a long circulation half-life by fusing the small ADC ZHER2-MMAE with the Fc domain of the antibody for circulation half-life extension, and inserting a digestion sequence between them to release the small ADC inside tumors for better tumor penetration. The experimental results showed that the designed molecule Fc-U-ZHER2-MMAE has a significantly increased blood circulation half-life (7.1 h, 59-fold longer) compared to the small ADC ZHER2-MMAE, and significantly improved drug accumulation ability at tumor sites compared to the conventional full-length antibody-coupled ADC Herceptin-MMAE. These combined effects led to Fc-U-ZHER2-MMAE having significantly enhanced tumor treatment ability, as shown in mouse models of NCI-N87 gastric cancer and SK-OV-3 ovarian cancer, where Fc-U-ZHER2-MMAE treatment achieved complete regression of tumors in all or a portion of animals with no obvious side effects and an MTD exceeding 90 mg/kg. These data demonstrate the therapeutic advantages of this cleavable ADC strategy, which could provide a new approach for ADC design.

PEG Linker Improves Antitumor Efficacy and Safety of Affibody-Based Drug Conjugates.

Antibody drug conjugates (ADCs) have become an important modality of clinical cancer treatment. However, traditional ADCs have some limitations, such as reduced permeability in solid tumors due to the high molecular weight of monoclonal antibodies, difficulty in preparation and heterogeneity of products due to the high drug/antibody ratio (4-8 small molecules per antibody). Miniaturized ADCs may be a potential solution, although their short circulation half-life may lead to new problems. In this study, we propose a novel design strategy for miniaturized ADCs in which drug molecules and small ligand proteins are site-specifically coupled via a bifunctional poly(ethylene glycol) (PEG) chain. The results showed that the inserted PEG chains significantly prolonged the circulation half-life but also obviously reduced the cytotoxicity of the conjugates. Compared with the conjugate ZHER2-SMCC-MMAE (HM), which has no PEG insertion, ZHER2-PEG4K-MMAE (HP4KM) and ZHER2-PEG10K-MMAE (HP10KM) with 4 or 10 kDa PEG insertions have 2.5- and 11.2-fold half-life extensions and 4.5- and 22-fold in vitro cytotoxicity reductions, respectively. The combined effect leads to HP10KM having the most ideal tumor therapeutic ability at the same dosages in the animal model, and its off-target toxicity was also reduced by more than 4 times compared with that of HM. These results may indicate that prolonging the half-life is very helpful in improving the therapeutic capacity of miniaturized ADCs. In the future, the design of better strategies that can prolong half-life without affecting cytotoxicity may be useful for further improving the therapeutic potential of these molecules.

A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy.

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Anti-HER2 Affibody-Conjugated Photosensitizer for Tumor Targeting Photodynamic Therapy.

Antibody-coupled photosensitive molecules can achieve an ideal tumor-specific photodynamic therapy (PDT) and show strong clinical application potential. However, some inherent disadvantages, such as long circulation half-life, poor permeation into solid tumors, and difficulty in obtaining uniform coupling products, present potential problems to clinical applications. In this study, we propose a novel design of targeting photosensitizers, based on a very small targeting protein (an affibody molecule) coupled with photosensitive compounds, to address these problems. In the synthesis, photosensitive pyropheophorbide-a (Pyro) is modified with a PEG linker (molecular weight of 727 Da) and then site specifically coupled to the anti-HER2 ZHER2:2891 affibody protein to provide a homogeneous protein-coupled photosensitizer via a convenient process. In vitro and in vivo experiments show that this molecule has an ideal selectivity for binding and photocytotoxicity against HER2-positive cells (more than 50-fold selectivity between HER2-high expression and HER2-low expression cells) and highly specific tumor accumulation; at a relatively low dose, it effectively eliminated HER2-high expression NCI-N87 tumors in a mouse model. It is worth noting that Pyro only has a moderate photodynamic activity; however, the affibody-coupled Pyro molecule (Pyro-Linker-ZHER2) still shows excellent tumor therapeutic function. The more ideal tumor permeability of small ligands may be helpful to enhance the drug concentration in the tumor site and the ability to penetrate deeply inside the tumor. Coupling photosensitive compounds with affibody proteins may provide a new way for targeting PDT of tumors.

Lysine Mutation of the Claw-Arm-Like Loop Accelerates Catalysis by Cellobiohydrolases.

Searching for viable strategies to accelerate the catalytic cycle of glycoside hydrolase family 7 (GH7) cellobiohydrolase I (CBHI)-the workhorse cellulose-degrading enzymes, we have performed a total of 12-µs molecular dynamics simulations on GH7 CBHI, which brought to light a new mechanism for cellobiose expulsion, coined "claw-arm" action. The loop flanking the product binding site plays the role of a flexible "arm" extending toward cellobiose, and residue Thr389 of this loop acts as a "claw" that captures cellobiose. Five mutations of residue Thr389 were considered to enhance the loop-cellobiose interaction. The lysine mutant was found to significantly accelerate cellobiose expulsion and facilitate polysaccharide-chain translocation. Lysine mutation of Thr393 in Talaromyces emersonii CBHI (TeCel7A) performed similarly. Lysine approaches the catalytic area and stabilizes the Michaelis complex, potentially affecting glycosylation, the rate-limiting step of the catalytic cycle. QM/MM calculations indicate that lysine replacement diminishes the barrier against proton transfer, the crucial step of glycosylation, by 2.3 kcal/mol. Experimental validation was performed using the full-length wild-type (WT) of TeCel7A and its mutants, recombinantly expressed in Pichia pastoris, to degrade the substrates. Compared with the WT, the lysine mutant revealed an associated higher enzymatic reaction rate. Furthermore, cellobiose yield was also increased by lysine mutation, indicating that dissociation of the enzyme from cellulose was accelerated, which largely stems from the enhanced flexibility of the "arm". The present work is envisioned to help design strategies for improving enzymatic activity, while decreasing enzyme cost.

Targeting HIV/HCV Coinfection Using a Machine Learning-Based Multiple Quantitative Structure-Activity Relationships (Multiple QSAR) Method.

Human immunodeficiency virus type-1 and hepatitis C virus (HIV/HCV) coinfection occurs when a patient is simultaneously infected with both human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV), which is common today in certain populations. However, the treatment of coinfection is a challenge because of the special considerations needed to ensure hepatic safety and avoid drug-drug interactions. Multitarget inhibitors with less toxicity may provide a promising therapeutic strategy for HIV/HCV coinfection. However, the identification of one molecule that acts on multiple targets simultaneously by experimental evaluation is costly and time-consuming. In silico target prediction tools provide more opportunities for the development of multitarget inhibitors. In this study, by combining Naïve Bayes (NB) and support vector machine (SVM) algorithms with two types of molecular fingerprints, MACCS and extended connectivity fingerprints 6 (ECFP6), 60 classification models were constructed to predict compounds that were active against 11 HIV-1 targets and four HCV targets based on a multiple quantitative structure-activity relationships (multiple QSAR) method. Five-fold cross-validation and test set validation were performed to measure the performance of the 60 classification models. Our results show that the 60 multiple QSAR models appeared to have high classification accuracy in terms of the area under the ROC curve (AUC) values, which ranged from 0.83 to 1 with a mean value of 0.97 for the HIV-1 models and from 0.84 to 1 with a mean value of 0.96 for the HCV models. Furthermore, the 60 models were used to comprehensively predict the potential targets of an additional 46 compounds, including 27 approved HIV-1 drugs, 10 approved HCV drugs and nine selected compounds known to be active against one or more targets of HIV-1 or HCV. Finally, 20 hits, including seven approved HIV-1 drugs, four approved HCV drugs, and nine other compounds, were predicted to be HIV/HCV coinfection multitarget inhibitors. The reported bioactivity data confirmed that seven out of nine compounds actually interacted with HIV-1 and HCV targets simultaneously with diverse binding affinities. The remaining predicted hits and chemical-protein interaction pairs with the potential ability to suppress HIV/HCV coinfection are worthy of further experimental investigation. This investigation shows that the multiple QSAR method is useful in predicting chemical-protein interactions for the discovery of multitarget inhibitors and provides a unique strategy for the treatment of HIV/HCV coinfection.

Multimerization Increases Tumor Enrichment of Peptide⁻Photosensitizer Conjugates.

Photodynamic therapy (PDT) is an established therapeutic modality for the management of cancers. Conjugation with tumor-specific small molecule ligands (e.g., short peptides or peptidomimetics) could increase the tumor targeting of PDT agents, which is very important for improving the outcome of PDT. However, compared with antibody molecules, small molecule ligands have a much weaker affinity to their receptors, which means that their tumor enrichment is not always ideal. In this work, we synthesized multimeric RGD ligand-coupled conjugates of pyropheophorbide-a (Pyro) to increase the affinity through multivalent and cluster effects to improve the tumor enrichment of the conjugates. Thus, the dimeric and trimeric RGD peptide-coupled Pyro conjugates and the monomeric one for comparison were efficiently synthesized via a convergent strategy. A short polyethylene glycol spacer was introduced between two RGD motifs to increase the distance required for multivalence. A subsequent binding affinity assay verified the improvement of the binding towards integrin αvß3 receptors after the increase in the valence, with an approximately 20-fold improvement in the binding affinity of the trimeric conjugate compared with that of the monomeric conjugate. In vivo experiments performed in tumor-bearing mice also confirmed a significant increase in the distribution of the conjugates in the tumor site via multimerization, in which the trimeric conjugate had the best tumor enrichment compared with the other two conjugates. These results indicated that the multivalence interaction can obviously increase the tumor enrichment of RGD peptide-conjugated Pyro photosensitizers, and the prepared trimeric conjugate can be used as a novel antitumor photodynamic agent with high tumor enrichment.

cRGD Peptide-Conjugated Pyropheophorbide-a Photosensitizers for Tumor Targeting in Photodynamic Therapy.

Pyropheophorbide-a (Pyro) is a highly promising photosensitizer for tumor photodynamic therapy (PDT), although its very limited tumor-accumulation ability seriously restricts its clinical applications. A higher accumulation of photosensitizers is very important for the treatment of deeply seated and larger tumors. The conjugation of Pyro with tumor-homing peptide ligands could be a very useful strategy to optimize the physical properties of Pyro. Herein, we reported our studies on the conjugation of Pyro with a cyclic cRGDfK (cRGD) peptide, an integrin binding sequence, to develop highly tumor-specific photosensitizers for PDT application. To further reduce the nonspecific uptake and, thus, reduce the background distribution of the conjugates in normal tissues, we opted to add a highly hydrophilic polyethylene glycol (PEG) chain and an extra strongly hydrophilic carboxylic acid group as the linker to avoid the direct connection of the strongly hydrophobic Pyro macrocycle and cRGD ligand. We reported here the synthesis and characterization of these conjugates, and the influence of the hydrophilic modification on the biological function of the conjugates was carefully studied. The tumor-accumulation ability and photodynamic-induced cell-killing ability of these conjugates were evaluated through both in vitro cell-based experiment and in vivo distribution and tumor therapy experiments with tumor-bearing mice. Thus, the synthesized conjugate significantly improved the tumor enrichment and tumor selectivity of Pyro, as well as abolished the xenograft tumors in the murine model through a one-time PDT treatment.

Targeted Delivery of CRISPR/Cas9-Mediated Cancer Gene Therapy via Liposome-Templated Hydrogel Nanoparticles.

Due to its simplicity, versatility, and high efficiency, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has emerged as one of the most promising approaches for treatment of a variety of genetic diseases, including human cancers. However, further translation of CRISPR/Cas9 for cancer gene therapy requires development of safe approaches for efficient, highly specific delivery of both Cas9 and single guide RNA to tumors. Here, novel core-shell nanostructure, liposome-templated hydrogel nanoparticles (LHNPs) that are optimized for efficient codelivery of Cas9 protein and nucleic acids is reported. It is demonstrated that, when coupled with the minicircle DNA technology, LHNPs deliver CRISPR/Cas9 with efficiency greater than commercial agent Lipofectamine 2000 in cell culture and can be engineered for targeted inhibition of genes in tumors, including tumors the brain. When CRISPR/Cas9 targeting a model therapeutic gene, polo-like kinase 1 (PLK1), is delivered, LHNPs effectively inhibit tumor growth and improve tumor-bearing mouse survival. The results suggest LHNPs as versatile CRISPR/Cas9-delivery tool that can be adapted for experimentally studying the biology of cancer as well as for clinically translating cancer gene therapy.

HER2-targeted immunotoxins with low nonspecific toxicity and immunogenicity.

Immunotoxins have efficient anti-tumor activity due to their extreme potency. However, dose-limiting off-target toxicity and immunogenicity are the critical barriers for these immunotoxins to be used in a clinical setting. In this study, we designed a Pseudomonas exotoxin A (PE)-based human epidermal growth factor receptor-2 (HER2)-specific immunotoxin HER2-PE25-X7 by deleting most of domain II and introducing seven point mutations into domain III of the PE38 toxin. The anti-cancer activity, off-target toxicity and immunogenicity of this immunotoxin were carefully evaluated in vitro and in vivo. This new construct maintained the therapeutic potency of the original PE38-based immunotoxin HER2-PE38, with a greatly reduced off-target toxicity and immunogenicity. To compare with HER2-PE38, which resulted in the death of most of the mice after a single dose of 1.0 mg/kg, the new construct was completely tolerated at a dose of 10 mg/kg by the mice and almost completely depleted the tumor after treatment with five doses of 5 mg/kg of the immunotoxin. This work demonstrates a potentially attractive therapeutic modality for HER2-specific cancer treatment.

Synthesis and biological evaluation of peptide-conjugated phthalocyanine photosensitizers with highly hydrophilic modifications.

The selectivity of targeted photosensitizer delivery is a constant challenge for photodynamic therapy (PDT). Herein, with the aim of optimizing the affinity, selectivity and activity of peptide-conjugated photosensitizers for PDT therapy and fluorescence imaging, we designed a solid-phase strategy for the efficient synthesis of peptide-phthalocyanine (Pc) conjugates with two types of highly hydrophilic modifications to the Pc rings. The peptide conjugation clearly increased the photodynamic efficacy and selectivity of Pcs against cancer cells with different receptor expression levels. A highly hydrophilic modification to the Pc rings can block non-specific interactions between the Pcs and biological molecules while minimally influencing peptide ligand affinity with target receptors. Compared with the triethyleneglycol monomethyl ether group modification, the glycerol group modification exhibited a stronger capability to decrease the Pc aggregation tendency in aqueous medium, reduce non-specific binding and non-specific, light-induced cytotoxicity towards cells with low receptor expression in vitro; increased targeting selectivity was observed in the in vivo distribution experiments via the obvious reduction of background signals. Highly hydrophilic modifications to the Pc rings may be very useful for improving targeting selectivity for PDT with the construction of peptide-conjugated photosensitizers.

Magnetic Separation-Assistant Fluorescence Resonance Energy Transfer Inhibition for Highly Sensitive Probing of Nucleolin.

For the widely used "off-on" fluorescence (or phosphorescence) resonance energy transfer (FRET or PRET) system, the separation of donors and acceptors species was vital for enhancing the sensitivity. To date, separation of free donors from FRET/PRET inhibition systems was somewhat not convenient, whereas separation of the target-induced far-between acceptors has hardly been reported yet. We presented here a novel magnetic separation-assistant fluorescence resonance energy transfer (MS-FRET) inhibition strategy for highly sensitive detection of nucleolin using Cy5.5-AS1411 as the donor and Fe3O4-polypyrrole core-shell (Fe3O4@PPY) nanoparticles as the NIR quenching acceptor. Due to hydrophobic interaction and π-π stacking of AS1411 and PPY, Cy5.5-AS1411 was bound onto the surface of Fe3O4@PPY, resulting in 90% of fluorescence quenching of Cy5.5-AS1411. Owing to the much stronger specific interaction of AS1411 and nucleolin, the presence of nucleolin could take Cy5.5-AS1411 apart from Fe3O4@PPY and restore the fluorescence of Cy5.5-AS1411. The superparamagnetism of Fe3O4@PPY enabled all separations and fluorescence measurements complete in the same quartz cell, and thus allowed the convenient but accurate comparison of the sensitivity and fluorescence recovery in the cases of separation or nonseparation. Compared to nonseparation FRET inhibition, the separation of free Cy5.5-AS1411 from Cy5.5-AS1411-Fe3O4@PPY solution (the first magnetic separation, MS-1) had as high as 25-fold enhancement of the sensitivity, whereas further separation of the nucleolin-inducing far-between Fe3O4@PPY from the FRET inhibition solution (the second magnetic separation, MS-2) could further enhance the sensitivity to 35-fold. Finally, the MS-FRET inhibition assay displayed the linear range of 0.625-27.5 µg L(-1) (8.1-359 pM) and detection limit of 0.04 µg L(-1) (0.05 pM) of nucleolin. The fluorescence intensity recovery (the percentage ratio of the final restoring fluorescence intensity to the quenched fluorescence intensity of Cy5.5-AS1411 solution by 0.09 g L(-1) Fe3O4@PPY) was enhanced from 36% (for nonseparation) to 56% (for two magnetic separations). This is the first accurate evaluation for the effect of separating donor/acceptor species on the FRET inhibition assay.

Highly water-soluble and tumor-targeted photosensitizers for photodynamic therapy.

Biological uses of photosensitizers in photodynamic therapy (PDT) often suffer from a lack of tumor selectivity; a strategy based on molecule-targeted cancer therapies could provide a promising solution. To synthesize new water-soluble phthalocyanines (Pcs) for bio-conjugation with peptides or antibodies, we developed a method to synthesize asymmetrically substituted Pcs with both high water solubility and one monoamino group for conjugation with biological agents for tumor homing, using folic acid as the ligand model to direct the modified Pcs into target cells. Here, we report studies on the syntheses and characterization of these Pcs. In vitro and in vivo assays prove that the high solubility characteristic can greatly increase the tumor targeting capability of Pcs by reducing non-specific uptake. This newly designed photosensitizer accumulated almost completely in tumor regions, with a negligible signal found in other tissues in the xenograft tumor model. These initial data provide strong evidence of the high specificity tumor targeting of Pcs with folate and tri-glycerol substitutions. Theoretically, the synthesized Pcs could be conveniently conjugated to many other ligands, endorsing the broad applicability of this method for tumor-targeted PDT.

Peptide o-aminoanilides as crypto-thioesters for protein chemical synthesis.

Fully unprotected peptide o-aminoanilides can be efficiently activated by NaNO2 in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o-aminoanilides as a new type of crypto-thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o-aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q.

An intracellularly activatable, fluorogenic probe for cancer imaging.

A newly designed, dual-functional probe based on intracellular activation has been successfully developed for the detection of cancer cells. The probe is nearly non-fluorescent in buffer due to its highly efficient FRET quenching, but it can be specifically activated with dramatic fluorescence enhancement upon intracellular cathepsin B cleavage in target cancer cells after selective internalization via folate receptor-dependent endocytosis. Therefore, this probe enables "turn-on" visualization of cancer cells with desirable specificity and contrast enhancement. This targeted, intracellularly activatable probe exhibits low fluorescence-quenched background when compared with "always-on" probes and avoids non-specific activation by non-specifically expressed enzymes in normal tissue, which normally occurs when using common "turn on" probe design strategies. Therefore, this probe can be potentially applied in intraoperative inspection during clinical cancer surgery with higher contrast and sensitivity.

Irreversible site-specific hydrazinolysis of proteins by use of sortase.

Sortase-mediated hydrazinolysis of proteins with hydrazine or its derivatives was developed for the production of recombinant protein hydrazides. This process provides an alternative approach for protein semisynthesis through the use of recombinant protein hydrazides as thioester surrogates. It also provides an alternative method for C-terminal modification of proteins with functional units as well as for the preparation of C-to-C fusion proteins.

Therapeutic effects of a novel tylophorine analog, NK-007, on collagen-induced arthritis through suppressing tumor necrosis factor α production and Th17 cell differentiation.

OBJECTIVE: To analyze the effects of a novel compound, NK-007, on the prevention and treatment of collagen-induced arthritis (CIA) and the underlying mechanisms. METHODS: We determined the effect of NK-007 on lipopolysaccharide (LPS)-triggered tumor necrosis factor α (TNFα) production by murine splenocytes and a macrophage cell line (RAW 264.7) by enzyme-linked immunosorbent assay, intracellular cytokine staining, and Western blotting. The LPS-boosted CIA model was adopted, and NK-007 or vehicle was administered at different time points after immunization. Mice were monitored for clinical severity of arthritis, and joint tissues were used for histologic examination, cytokine detection, and immunohistochemical staining. Finally, stability of TNFα production and Th17 cell differentiation were studied using quantitative polymerase chain reaction and flow cytometry. RESULTS: NK-007 significantly suppressed LPS-induced TNFα production in vitro. Administration of NK-007 completely blocked CIA development and delayed its progression. Furthermore, treatment with NK-007 at the onset of arthritis significantly inhibited the progress of joint inflammation. Administration of NK-007 also suppressed production of TNFα, interleukin-6 (IL-6), and IL-17A in the joint and reduced percentages of IL-17+ cells among CD4+ and γ/δ T cells in draining lymph nodes. We further demonstrated that NK-007 acted on the stability of TNFα messenger RNA and reduced Th17 cell differentiation. In addition, it significantly inhibited levels of IL-6 and IL-17A in human coculture assay. CONCLUSION: For its effects on the development and progression of CIA and for its therapeutic effect on CIA, NK-007 has great potential to be a therapeutic agent for human rheumatoid arthritis.

Regulatory role of Vγ1 γδ T cells in tumor immunity through IL-4 production.

It has been demonstrated that the two main subsets of peripheral γδ T cells, Vγ1 and Vγ4, have divergent functions in many diseases models. Recently, we reported that Vγ4 γδ T cells played a protective role in tumor immunity through eomesodermin-controlled mechanisms. However, the precise roles of Vγ1 γδ T cells in tumor immunity, especially whether Vγ1 γδ T cells have any interaction with Vγ4 γδ T cells, remain unknown. We demonstrated in this paper that Vγ1 γδ T cells suppressed Vγ4 γδ T cell-mediated antitumor function both in vitro and in vivo, and this suppression was cell contact independent. Using neutralizing anti-IL-4 Ab or IL-4(-/-) mice, we determined the suppressive factor derived from Vγ1 γδ T cells was IL-4. Indeed, treatment of Vγ4 γδ T cells with rIL-4 significantly reduced expression levels of NKG2D, perforin, and IFN-γ. Finally, Vγ1 γδ T cells produced more IL-4 and expressed significantly higher level of GATA-3 upon Th2 priming in comparison with Vγ4 γδ T cells. Therefore, to our knowledge, our results established for the first time a negative regulatory role of Vγ1 γδ T cells in Vγ4 γδ T cell-mediated antitumor immunity through cell contact-independent and IL-4-mediated mechanisms. Selective depletion of this suppressive subset of γδ T cells may be beneficial for tumor immune therapy.

Vγ4 γδ T cell-derived IL-17A negatively regulates NKT cell function in Con A-induced fulminant hepatitis.

Con A-induced fulminant hepatitis is a well-known animal model for acute liver failure. However, the role of γδ T cells in this model is undefined. In this report, using TCR δ(-/-) mice, we demonstrated a protective role of γδ T cells in Con A-induced hepatitis model. TCR δ(-/-) mice showed significantly decreased levels of IL-17A and IL-17F in the Con A-treated liver tissue, and reconstitution of TCR δ(-/-) mice with wild-type (Wt), but not IL-17A(-/-), γδ T cells significantly reduced hepatitis, strongly suggesting a critical role of IL-17A in mediating the protective effect of γδ T cells. Interestingly, only Vγ4, but not Vγ1, γδ T cells exerted such a protective effect. Furthermore, depletion of NKT cells in TCR δ(-/-) mice completely abolished hepatitis, and NKT cells from Con A-challenged liver tissues of TCR δ(-/-) mice expressed significantly higher amounts of proinflammatory cytokine IFN-γ than those from Wt mice, indicating that γδ T cells protected hepatitis through targeting NKT cells. Finally, abnormal capacity of IFN-γ production by NKT cells of TCR δ(-/-) mice could only be downregulated by transferring Wt, but not IL-17(-/-), Vγ4 γδ T cells, confirming an essential role of Vγ4-derived IL-17A in regulating the function of NKT cells. In summary, our report thus demonstrated a novel function of Vγ4 γδ T cells in mediating a protective effect against Con A-induced fulminant hepatitis through negatively regulating function of NKT cells in an IL-17A-dependent manner, and transferring Vγ4 γδ T cells may provide a novel therapeutic approach for this devastating liver disease.

The mouse Mageb18 gene encodes a ubiquitously expressed type I MAGE protein and regulates cell proliferation and apoptosis in melanoma B16-F0 cells.

Although many cancer vaccines have been developed against type I MAGE (melanoma antigen) genes owing to their shared tumour-specific expression properties, studies about their expression and functions are relatively limited. In the present study, we first identify a non-testis-specific type I MAGE gene, Mageb18 (melanoma antigen family B 18). Mouse Mageb18 is also expressed in digestion- and immune-related tissues as well as testis, and its expression in testis is age-dependent. Mageb18 is expressed in many mouse-derived cell lines, and DNA demethylation and histone acetylation mediate the reactivation of Mageb18 in Mageb18-negtive H22 and C6 cells. We also show that mouse Mageb18 encodes a 46 kDa protein which is predominantly localized in the cytoplasm. In testis, the endogenous MAGEB18 protein is mainly expressed in proliferative spermatogonia and primary and secondary spermatocytes, but less so in spermatids. Finally, we demonstrate that knockdown of MAGEB18 inhibits the growth of B16-F0 cells and induces apoptosis, which correlates with increased levels of TP53 (tumour protein 53), p21, Bax and caspase 3. The results of the present study thus uncover an important phenomenon that the expression of certain type I MAGE genes, at least for Mageb18, is non-testis-specific. Although they can regulate various malignant phenotypes of cancer cells, it is necessary to study further their expression pattern in normal tissues before using them to develop more effective and safer cancer vaccines.