Routine evaluation of HBV-specific T cell reactivity in chronic hepatitis B using a broad-spectrum T-cell epitope peptide library and ELISpot assay.

BACKGROUND: The clinical routine test of HBV-specific T cell reactivity is still limited due to the high polymorphisms of human leukocyte antigens (HLA) in patient cohort and the lack of universal detection kit, thus the clinical implication remains disputed. METHODS: A broad-spectrum peptide library, which consists of 103 functionally validated CD8+ T-cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and fits to the HLA polymorphisms of Chinese and Northeast Asian populations, was grouped into eight peptide pools and was used to establish an ELISpot assay for enumerating the reactive HBV-specific T cells in PBMCs. Totally 294 HBV-infected patients including 203 ones with chronic hepatitis B (CHB), 13 ones in acute resolved stage (R), 52 ones with liver cirrhosis (LC) and 26 ones with hepatocellular carcinoma (HCC) were detected, and 33 CHB patients were longitudinally monitored for 3 times with an interval of 3-5 months. RESULTS: The numbers of reactive HBV-specific T cells were significantly correlated with ALT level, HBsAg level, and disease stage (R, CHB, LC and HCC), and R patients displayed the strongest HBV-specific T cell reactivity while CHB patients showed the weakest one. For 203 CHB patients, the numbers of reactive HBV-specific T cells presented a significantly declined trend when the serum viral DNA load, HBsAg, HBeAg or ALT level gradually increased, but only a very low negative correlation coefficient was defined (r = - 0.21, - 0.21, - 0.27, - 0.079, respectively). Different Nucleotide Analogs (NUCs) did not bring difference on HBV-specific T cell reactivity in the same duration of treatment. NUCs/pegIFN-α combination led to much more reactive HBV-specific T cells than NUCs monotherapy. The dynamic numbers of reactive HBV-specific T cells were obviously increasing in most CHB patients undergoing routine treatment, and the longitudinal trend possess a high predictive power for the hepatitis progression 6 or 12 months later. CONCLUSION: The presented method could be developed into an efficient reference method for the clinical evaluation of cellular immunity. The CHB patients presenting low reactivity of HBV-specific T cells have a worse prognosis for hepatitis progression and should be treated using pegIFN-α to improve host T-cell immunity.

Dual FGFR and VEGFR inhibition synergistically restrain hexokinase 2-dependent lymphangiogenesis and immune escape in intrahepatic cholangiocarcinoma.

BACKGROUND: Therapies for cholangiocarcinoma are largely limited and ineffective. Herein, we examined the role of the FGF and VEGF pathways in regulating lymphangiogenesis and PD-L1 expression in intrahepatic cholangiocarcinoma (iCCA). METHODS: The lymphangiogenic functions of FGF and VEGF were evaluated in lymphatic endothelial cells (LECs) and iCCA xenograft mouse models. The relationship between VEGF and hexokinase 2 (HK2) was validated in LECs by western blot, immunofluorescence, ChIP and luciferase reporter assays. The efficacy of the combination therapy was assessed in LECs and xenograft models. Microarray analysis was used to evaluate the pathological relationships of FGFR1 and VEGFR3 with HK2 in human lymphatic vessels. RESULTS: FGF promoted lymphangiogenesis through c-MYC-dependent modulation of HK2 expression. VEGFC also upregulated HK2 expression. Mechanistically, VEGFC phosphorylated components of the PI3K/Akt/mTOR axis to upregulate HIF-1α expression at the translational level, and HIF-1α then bound to the HK2 promoter region to activate its transcription. More importantly, dual FGFR and VEGFR inhibition with infigratinib and SAR131675 almost completely inhibited lymphangiogenesis, and significantly suppressed iCCA tumor growth and progression by reducing PD-L1 expression in LECs. CONCLUSIONS: Dual FGFR and VEGFR inhibition inhibits lymphangiogenesis through suppression of c-MYC-dependent and HIF-1α-mediated HK2 expression, respectively. HK2 downregulation decreased glycolytic activity and further attenuated PD-L1 expression. Our findings suggest that dual FGFR and VEGFR blockade is an effective novel combination strategy to inhibit lymphangiogenesis and improve immunocompetence in iCCA.

Screening of Efficient Adjuvants for the RBD-Based Subunit Vaccine of SARS-CoV-2.

The variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more transmissible, with a reduced sensitivity to vaccines targeting the original virus strain. Therefore, developing an effective vaccine against both the original SARS-CoV-2 strain and its variants is an urgent need. It is known that the receptor-binding domain (RBD) in the S protein of SARS-CoV-2 is an important vaccine target, but subunit vaccines usually have lower immunogenicity and efficacy. Thus, selecting appropriate adjuvants to enhance the immunogenicity of protein-based subunit vaccine antigens is necessary. Here, an RBD-Fc subunit vaccine of SARS-CoV-2 has been generated, followed by vaccination in B6 mice, and four adjuvant regimens were investigated, including aluminum salts (Alum) + 3-O-desacyl-4'-monophosphoryl lipid A (MPL), AddaVax, QS21 + MPL, and Imiquimod. The adjuvant potency was evaluated by comparing the elicited polyclonal antibodies titers with measuring binding to RBD and S protein in ELISA and Western blot analysis, and also the cross-neutralizing antibodies titers using a pseudovirus infection assay of hACE2-expressing 293T cells, with pseudoviruses expressing the S protein of the SARS-CoV-2 original strain and Delta strain. The presence of QS21 + MPL adjuvant induced stronger polyclonal antibody response and neutralization potency blocking the original strain and Delta strain, as compared with the non-adjuvant RBD-Fc group and other adjuvant groups. Meanwhile, Imiquimod even had a negative effect in inducing specific antibodies and cross-neutralizing antibody production as an adjuvant.

Silk Fibroin and Sericin Differentially Potentiate the Paracrine and Regenerative Functions of Stem Cells Through Multiomics Analysis.

Silk fibroin (SF) and sericin (SS), the two major proteins of silk, are attractive biomaterials with great potential in tissue engineering and regenerative medicine. However, their biochemical interactions with stem cells remain unclear. In this study, multiomics are employed to obtain a global view of the cellular processes and pathways of mesenchymal stem cells (MSCs) triggered by SF and SS to discern cell-biomaterial interactions at an in-depth, high-throughput molecular level. Integrated RNA sequencing and proteomic analysis confirm that SF and SS initiate widespread but distinct cellular responses and potentiate the paracrine functions of MSCs that regulate extracellular matrix deposition, angiogenesis, and immunomodulation through differentially activating the integrin/PI3K/Akt and glycolysis signaling pathways. These paracrine signals of MSCs stimulated by SF and SS effectively improve skin regeneration by regulating the behavior of multiple resident cells (fibroblasts, endothelial cells, and macrophages) in the skin wound microenvironment. Compared to SS, SF exhibits better immunomodulatory effects in vitro and in vivo, indicating its greater potential as a carrier material of MSCs for skin regeneration. This study provides comprehensive and reliable insights into the cellular interactions with SF and SS, enabling the future development of silk-based therapeutics for tissue engineering and stem cell therapy.

A systemic review of T-cell epitopes defined from the proteome of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection remains in a global pandemic, and no eradicative therapy is currently available. Host T cells have been shown to play a crucial role in the antiviral immune protection and pathology in Coronavirus disease 2019 (COVID-19) patients; thus, identifying sufficient T-cell epitopes from the SARS-CoV-2 proteome can contribute greatly to the development of T-cell epitope vaccines and the precise evaluation of host SARS-CoV-2-specific cellular immunity. This review presents a comprehensive map of T-cell epitopes functionally validated from SARS-CoV-2 antigens, the human leukocyte antigen (HLA) supertypes to present these epitopes, and the strategies to screen and identify T-cell epitopes. To the best of our knowledge, a total of 1349 CD8+ T-cell epitopes and 790 CD4+ T-cell epitopes have been defined by functional experiments thus far, but most are presented by approximately twenty common HLA supertypes, such as HLA-A0201, A2402, B0702, DR15, DR7 and DR11 molecules, and 74-80% of the T-cell epitopes are derived from S protein and nonstructural protein. These data provide useful insight into the development of vaccines and specific T-cell detection systems. However, the currently defined T-cell epitope repertoire cannot cover the HLA polymorphism of major populations in an indicated geographic region. More research is needed to depict an overall landscape of T-cell epitopes, which covers the overall SARS-CoV-2 proteome and global patients.

Exosomal Vaccine Loading T Cell Epitope Peptides of SARS-CoV-2 Induces Robust CD8+ T Cell Response in HLA-A Transgenic Mice.

Purpose: Current vaccines for the SARS-CoV-2 virus mainly induce neutralizing antibodies but overlook the T cell responses. This study aims to generate an exosomal vaccine carrying T cell epitope peptides of SARS-CoV-2 for the induction of CD8+ T cell response. Methods: Thirty-one peptides presented by HLA-A0201 molecule were conjugated to the DMPE-PEG-NHS molecules, and mixed with DSPE-PEG to form the peptide-PEG-lipid micelles, then fused with exosomes to generate the exosomal vaccine, followed by purification using size-exclusion chromatography and validation by Western blotting, liquid nuclear magnetic resonance (NMR) test and transmission electron microscopy. Furthermore, the exosomal vaccine was mixed with Poly (I:C) adjuvant and subcutaneously administered for three times into the hybrid mice of HLA-A0201/DR1 transgenic mice with wild-type mice. Then, the epitope-specific T cell responses were detected by ex vivo ELISPOT assay and intracellular cytokine staining. Results: The exosomal vaccine was purified from the Peak 2 fraction of FPLC and injected into the hybrid mice for three times. The IFN-γ spot forming units and the frequencies of IFN-γ+/CD8+ T cells were 10-82-fold and 13-65-fold, respectively, higher in the exosomal vaccine group compared to the Poly (I:C) control group, without visible organ toxicity. In comparison with the peptides cocktail vaccine generated in our recent work, the exosomal vaccine induced significantly stronger T cell response. Conclusion: Exosomal vaccine loading T cell epitope peptides of SARS-CoV-2 virus was initially generated without pre-modification for both peptides and exosomes, and elicited robust CD8+ T cell response in HLA-A transgenic mice.

The Use of Molecular Dynamics Simulation Method to Quantitatively Evaluate the Affinity between HBV Antigen T Cell Epitope Peptides and HLA-A Molecules.

Chronic hepatitis B virus (HBV), a potentially life-threatening liver disease, makes people vulnerable to serious diseases such as cancer. T lymphocytes play a crucial role in clearing HBV virus, while the pathway depends on the strong binding of T cell epitope peptide and HLA. However, the experimental identification of HLA-restricted HBV antigenic peptides is extremely time-consuming. In this study, we provide a novel prediction strategy based on structure to assess the affinity between the HBV antigenic peptide and HLA molecule. We used residue scanning, peptide docking and molecular dynamics methods to obtain the molecular docking model of HBV peptide and HLA, and then adopted the MM-GBSA method to calculate the binding affinity of the HBV peptide-HLA complex. Overall, we collected 59 structures of HLA-A from Protein Data Bank, and finally obtained 352 numerical affinity results to figure out the optimal bind choice between the HLA-A molecules and 45 HBV T cell epitope peptides. The results were highly consistent with the qualitative affinity level determined by the competitive peptide binding assay, which confirmed that our affinity prediction process based on an HLA structure is accurate and also proved that the homologous modeling strategy for HLA-A molecules in this study was reliable. Hence, our work highlights an effective way by which to predict and screen for HLA-peptide binding that would improve the treatment of HBV infection.

Computer-Based Immunoinformatic Analysis to Predict Candidate T-Cell Epitopes for SARS-CoV-2 Vaccine Design.

Since the first outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, its high infectivity led to its prevalence around the world in an exceptionally short time. Efforts have been made to control the ongoing outbreak, and among them, vaccine developments are going on high priority. New clinical trials add to growing evidence that vaccines from many countries were highly effective at preventing SARS-CoV-2 virus infection. One of them is B cell-based vaccines, which were common during a pandemic. However, neutralizing antibody therapy becomes less effective when viruses mutate. In order to tackle the problem, we focused on T-cell immune mechanism. In this study, the mutated strains of the virus were selected globally from India (B.1.617.1 and B.1.617.2), United Kingdom (B.1.1.7), South Africa (B.1.351), and Brazil (P.1), and the overlapping peptides were collected based on mutation sites of S-protein. After that, residue scanning was used to predict the affinity between overlapping peptide and HLA-A*11:01, the most frequent human leukocyte antigen (HLA) allele among the Chinese population. Then, the binding free energy was evaluated with molecular docking to further verify the affinity changes after the mutations happen in the virus genomes. The affinity test results of three epitopes on spike protein from experimental validation were consistent with our predicted results, thereby supporting the inclusion of the epitope 374FSTFKCYGL382 in future vaccine design and providing a useful reference route to improve vaccine development.

Screening and Identification of HBV Epitopes Restricted by Multiple Prevalent HLA-A Allotypes.

Although host T cell immune responses to hepatitis B virus (HBV) have been demonstrated to have important influences on the outcome of HBV infection, the development of T cell epitope-based vaccine and T cell therapy and the clinical evaluation of specific T cell function are currently hampered markedly by the lack of validated HBV T cell epitopes covering broad patients. This study aimed to screen T cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and presenting by thirteen prevalent human leukocyte antigen (HLA)-A allotypes which gather a total gene frequency of around 95% in China and Northeast Asia populations. 187 epitopes were in silico predicted. Of which, 62 epitopes were then functionally validated as real-world HBV T cell epitopes by ex vivo IFN-γ ELISPOT assay and in vitro co-cultures using peripheral blood mononuclear cells (PBMCs) from HBV infected patients. Furthermore, the HLA-A cross-restrictions of each epitope were identified by peptide competitive binding assay using transfected HMy2.CIR cell lines, and by HLA-A/peptide docking as well as molecular dynamic simulation. Finally, a peptide library containing 105 validated epitopes which cross-binding by 13 prevalent HLA-A allotypes were used in ELISPOT assay to enumerate HBV-specific T cells for 116 patients with HBV infection. The spot forming units (SFUs) was significantly correlated with serum HBsAg level as confirmed by multivariate linear regression analysis. This study functionally validated 62 T cell epitopes from HBV main proteins and elucidated their HLA-A restrictions and provided an alternative ELISPOT assay using validated epitope peptides rather than conventional overlapping peptides for the clinical evaluation of HBV-specific T cell responses.

A Systematic Review of T Cell Epitopes Defined from the Proteome of Hepatitis B Virus.

Hepatitis B virus (HBV) infection remains a worldwide health problem and no eradicative therapy is currently available. Host T cell immune responses have crucial influences on the outcome of HBV infection, however the development of therapeutic vaccines, T cell therapies and the clinical evaluation of HBV-specific T cell responses are hampered markedly by the lack of validated T cell epitopes. This review presented a map of T cell epitopes functionally validated from HBV antigens during the past 33 years; the human leukocyte antigen (HLA) supertypes to present these epitopes, and the methods to screen and identify T cell epitopes. To the best of our knowledge, a total of 205 CD8+ T cell epitopes and 79 CD4+ T cell epitopes have been defined from HBV antigens by cellular functional experiments thus far, but most are restricted to several common HLA supertypes, such as HLA-A0201, A2402, B0702, DR04, and DR12 molecules. Therefore, the currently defined T cell epitope repertoire cannot cover the major populations with HLA diversity in an indicated geographic region. More researches are needed to dissect a more comprehensive map of T cell epitopes, which covers overall HBV proteome and global patients.

Identification of HLA-A2 restricted epitopes of glypican-3 and induction of CTL responses in HLA-A2 transgenic mice.

Hepatocellular carcinoma (HCC) is a malignant tumor with high mortality, but lacks effective treatments. Carcinoembryonic antigen glypican-3 (GPC3) is a tumor-associated antigen overexpressed in HCC but rarely expressed in healthy individuals and thus is one of the most promising therapeutic targets. T cell epitope-based vaccines may bring light to HCC patients, especially to the patients at a late stage. However, few epitopes from GPC3 were identified to date, which limited the application of GPC3-derived epitopes in immunotherapy and T cell function detection. In this study, a total of 25 HLA-A0201 restricted GPC3 epitopes were in silico predicted and selected as candidate epitopes. Then, HLA-A0201+/GPC3+ HCC patients' PBMCs were collected and co-stimulated with the candidate epitope peptides in ex vivo IFN-γ Elispot assay, by which five epitopes were identified as real-world epitopes. Their capacity to elicit specific CD8+ T cells activation and proliferation was further confirmed by in vitro co-cultures of patients' PBMCs with peptide, in vitro co-cultures of healthy donors' PBLs with DCs and peptide, T2 cell binding assay as well as HLA-A2 molecule stability assay. Moreover, the in vivo immunogenicity of the five validated epitopes was confirmed by peptides cocktail/poly(I:C) vaccination in HLA-A0201/DR1 transgenic mice. Robust epitope-specific CD8+ T cell responses and cytotoxicity targeting HepG2 cells were observed as detected by IFN-γ Elispot, intracellular IFN-γ staining and cytolysis assay. This study provided novel GPC3 CTL epitopes for the development of T cell epitope vaccines and evaluation of GPC3 specific T cell responses.

Receptor-Binding Domain Proteins of SARS-CoV-2 Variants Elicited Robust Antibody Responses Cross-Reacting with Wild-Type and Mutant Viruses in Mice.

Multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have spread around the world, but the neutralizing effects of antibodies induced by the existing vaccines have declined, which highlights the importance of developing vaccines against mutant virus strains. In this study, nine receptor-binding domain (RBD) proteins of the SARS-CoV-2 variants (B.1.1.7, B.1.351 and P.1 lineages) were constructed and fused with the Fc fragment of human IgG (RBD-Fc). These RBD-Fc proteins contained single or multiple amino acid substitutions at prevalent mutation points of spike protein, which enabled them to bind strongly to the polyclonal antibodies specific for wild-type RBD and to the recombinant human ACE2 protein. In the BALB/c, mice were immunized with the wild-type RBD-Fc protein first and boosted twice with the indicated mutant RBD-Fc proteins later. All mutant RBD-Fc proteins elicited high-level IgG antibodies and cross-neutralizing antibodies. The RBD-Fc proteins with multiple substitutions tended to induce higher antibody titers and neutralizing-antibody titers than the single-mutant RBD-Fc proteins. Meanwhile, both wild-type RBD-Fc protein and mutant RBD-Fc proteins induced significantly decreased neutralization capacity to the pseudovirus of B.1.351 and P.1 lineages than to the wild-type one. These data will facilitate the design and development of RBD-based subunit vaccines against SARS-COV-2 and its variants.

Screening HLA-A-restricted T cell epitopes of SARS-CoV-2 and the induction of CD8+ T cell responses in HLA-A transgenic mice.

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific T cells have been found to play essential roles in host immune protection and pathology in patients with coronavirus disease 2019 (COVID-19), this study focused on the functional validation of T cell epitopes and the development of vaccines that induce specific T cell responses. A total of 120 CD8+ T cell epitopes from the E, M, N, S, and RdRp proteins were functionally validated. Among these, 110, 15, 6, 14, and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; in addition, four epitopes from the S protein displayed one amino acid that was distinct from the current SARS-CoV-2 variants. Then, 31 epitopes restricted by the HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or poly (lactic-co-glycolic acid) nanoparticles, and these vaccines elicited robust and specific CD8+ T cell responses in HLA-A2/DR1 transgenic mice as well as wild-type mice. In contrast to previous research, this study established a modified DC-peptide-PBL cell coculture system using healthy donor PBMCs to validate the in silico predicted epitopes, provided an epitope library restricted by nine of the most prevalent HLA-A allotypes covering broad Asian populations, and identified the HLA-A restrictions of these validated epitopes using competitive peptide binding experiments with HMy2.CIR cell lines expressing the indicated HLA-A allotype, which initially confirmed the in vivo feasibility of 9- or 10-mer peptide cocktail vaccines against SARS-CoV-2. These data will facilitate the design and development of vaccines that induce antiviral CD8+ T cell responses in COVID-19 patients.

Design and Optimization of PLGA Particles to Deliver Immunomodulatory Drugs for the Prevention of Skin Allograft Rejection.

Background: Recent advancements in therapeutic strategies have attracted considerable attention to control the acute organs and tissues rejection, which is the main cause of mortality in transplant recipients. The long-term usage of immunosuppressive drugs compromises the body immunity against simple infections and decrease the patients' quality of life. Tolerance of allograft in recipients without harming the rest of host immune system is the basic idea to develop the therapeutic approaches after induction of donor-specific transplant. Methods: Controlled and targeted delivery system by using biomimetic micro and nanoparticles as carriers is an effective strategy to deplete the immune cells in response to allograft in an antigen-specific manner. Polylactic-co-glycolic acid (PLGA) is a biocompatible and biodegradable polymer, which has frequently being used as drug delivery vehicle. Results: This review focuses on the biomedical applications of PLGA based biomimetic micro and nano-sized particles in drug delivery systems to prolong the survival of alloskin graft. Conclusion: We will discuss the mediating factors for rejection of alloskin graft, selective depletion of immune cells, controlled release mechanism, physiochemical properties, size-based body distribution of PLGA particles and their effect on overall host immune system.

Effects of Adjuvants on the Immunogenicity and Efficacy of a Zika Virus Envelope Domain III Subunit Vaccine.

Zika virus (ZIKV), a mosquito-borne flavivirus, has attracted global attention due to its close association with congenital Zika syndrome and neurological diseases, and transmission through additional routes, such as sexual contact. Currently there are no vaccines approved for ZIKV, and thus, there is an urgent need to develop an effective and safe ZIKV vaccine. Domain III (DIII) of the ZIKV envelope (E) protein is an important vaccine target, and a vaccine developed using a mutant DIII of E (EDIII) protein protects adult and pregnant mice, and unborn offspring, against ZIKV infection. Here, we have used immunocompetent BALB/c mice treated with anti-interferon-α/ß receptor 1 (Ifnar1) antibodies to investigate whether three adjuvants (aluminum (Alum), monophosphoryl lipid A (MPL), and MF59), either alone or in combination, could improve the efficacy of this EDIII subunit vaccine. Our data show that, although vaccine formulated with a single adjuvant induced a specific antibody and cellular immune response, and reduced viral load in mice challenged with ZIKV, the combination of Alum and MPL adjuvants led to a more robust and balanced immune response, stronger neutralizing activity against three recent ZIKV human strains, and greater protection against a high-dose ZIKV challenge. Particularly, the combination of Alum with MPL significantly reduced viral titers and viral RNA copy numbers in sera and tissues, including the male reproductive organs. Overall, this study has identified the combination of Alum and MPL as the most effective adjuvant for ZIKV EDIII subunit vaccines, and it has important implications for subunit vaccines against other enveloped viruses, including non-ZIKV flaviviruses.

Selective downregulation of distinct circRNAs in the tissues and plasma of patients with primary hepatic carcinoma.

Multiple studies have indicated that circular RNAs (circRNAs) are closely associated with malignant tumor development and metastasis. However, the significance of circRNAs in primary hepatic carcinoma (PHC), particularly in the plasma, remains largely undetermined. In the current study, circRNA expression profiles in three pairs of tumor and adjacent normal samples from patients with PHC, were examined using circRNA chip screening. A total of 80 circRNAs were upregulated, while 75 circRNAs were downregulated in PHC tissues, relative to para-tumor tissues (fold change, ≥1.5). A total of two upregulated circRNAs and three downregulated circRNAs were selected as candidates for further validation of their differential expression. This was performed using reverse transcription-quantitative PCR with 11 pairs of PHC tissues and para-tumor tissues. The results indicated that hsa_circ_0003056 exhibited reduced expression in PHC tissues. Moreover, hsa_circ_0003056 and hsa_circ_0067127 were quantified in the plasma samples of 35 PHC patients and 32 healthy donors. The results revealed that hsa_circ_0067127 was significantly downregulated in the patients' plasma. Finally, a competing endogenous RNA network was constructed, which consisted of one circRNA (hsa_circ_0003056 or has_circ_0067127), five miRNAs and miRNA-targeted genes (mRNAs). Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that differentially expressed (DE) genes were significantly enriched in the pathway associated with 'regulation of the pluripotency of stem cells' for hsa_circ_0003056, and 'ubiquitin-mediated proteolysis' and 'prostate cancer' for hsa_circ_0067127. Gene ontology analysis revealed that DE genes were primarily associated with the 'modulation of kinase activity' and 'intracellular and transmembrane-ephrin receptor activity' for hsa_circ_0003056, 'artery morphogenesis activity', 'HOPS complex and transferase activity' and in 'transferring acyl groups' for hsa_circ_0067127. This approach indicated that hsa_circ_0003056 in PHC tissue, and hsa_circ_0067127 in PHC plasma, are downregulated and may be implicated in the tumorigenesis of PHC.

PEGylated and CD47-conjugated nanoellipsoidal artificial antigen-presenting cells minimize phagocytosis and augment anti-tumor T-cell responses.

PURPOSE: Antigen-presenting cells (APCs) are powerful tools to expand antigen-specific T cells ex vivo and in vivo for tumor immunotherapy, but suffer from time-consuming generation and biosafety concerns raised by live cells. Alternatively, the cell-free artificial antigen-presenting cells (aAPCs) have been rapidly developed. Nanoscale aAPCs are recently proposed owing to their superior biodistribution and reduced embolism than conventional cell-sized aAPCs, but pose the challenges: easier cellular uptake and smaller contact surface area with T cells than the cell-sized counterparts. This study aimed to fabricate a new "stealth" nano-aAPCs with microscale contact surface area to minimize cellular uptake and activate antigen-specific T cells by combination uses of ellipsoidal stretch, PEGylation, and self-marker CD47-Fc conjugation. METHODS: The spherical polylactic-co-glycolic acid nanoparticles were fabricated using a double-emulsion method, and then stretched twofold using film-stretching procedure followed by PEGylation and co-coupling with CD47-Fc, H-2Kb/TRP2180-188-Ig dimers, and anti-CD28. The resulting PEGylated and CD47-conjugated nanoellipsoidal aAPCs (EaAPCPEG/CD47) were co-cultured with macrophages or spleen lymphocytes and also infused into melanoma-bearing mice. The in vitro and in vivo effects were evaluated and compared with the nanospherical aAPCs (SaAPC), nanoellipsoidal aAPCs (EaAPC), or PEGylated nanoellipsoidal aAPC (EaAPCPEG). RESULTS: EaAPCPEG/CD47 markedly reduced cellular uptake in vitro and in vivo, as compared with EaAPCPEG, EaAPC, SaAPC, and Blank-NPs and expanded naïve TRP2180-188-specific CD8+ T cells in the co-cultures with spleen lymphocytes. After three infusions, the EaAPCPEG/CD47 showed much stronger effects on facilitating TRP2180-188-specific CD8+ T-cell proliferation, local infiltration, and tumor necrosis in the melanoma-bearing mice and on inhibiting tumor growth than the control aAPCs. CONCLUSION: The superimposed or synergistic effects of ellipsoidal stretch, PEGylation, and CD47-Fc conjugation minimized cellular uptake of nano-aAPCs and enhanced their functionality to expand antigen-specific T cells and inhibit tumor growth, thus suggesting a more valuable strategy to design "stealth" nanoscale aAPCs suitable for tumor active immunotherapy.

An Artificial Antigen-Presenting Cell Delivering 11 Immune Molecules Expands Tumor Antigen-Specific CTLs in Ex Vivo and In Vivo Murine Melanoma Models.

Antigen-presenting cells expand antigen-specific T cells ex vivo and in vivo for tumor immunotherapy, but are time-consuming to generate and, as live cells, raise biosafety concerns. An alternative is found in cell-free artificial antigen-presenting cells (aAPC), but these only present two or three kinds of immune molecules. Here, we describe a multipotent artificial antigen-presenting cell (MaAPC) that delivered 11 kinds of immune moleclues. This MaAPC simulated natural APCs through the concurent coupling of target antigens (H-2Kb/TRP2180-188-Ig dimers and H-2Db/gp10025-33-Ig dimers), costimulatory molecules (anti-CD28, anti-4-1BB, and anti-CD2), and "self-marker" CD47-Fc onto surface-modified polylactic-co-glycolic acid microparticles (PLGA-MP). These PLGA-MPs also encapsulated cytokines (IL2 and IL15), a chemokine (CCL21), and checkpoint inhibitors (anti-CTLA-4 and anti-PD-1). Culture of MaAPCs with naïve T cells for 1 week elevated the frequencies of TRP2180-188-specific and gp10025-33-specific CTLs to 51.0% and 43.3%, respectively, with enhanced cytotoxicity. Three infusions of MaAPCs inhibited subcutaneous melanoma growth in a mouse model and expanded TRP2180-188 and gp10025-33-specific CTLs 59-86-fold in peripheral blood, 76-77-fold in spleen, and 205-212-fold in tumor tissue, in an antigen-specific manner. Compared with conventional aAPCs carrying two or three immune molecules, the 11-signal MaAPCs exerted greater impact on T cells, including activation, proliferation, cytotoxicity, differentiation to memory CTLs or regulatory T cells and cytokines profiles, without detected side effects. Such MaAPCs could be used to individualize tumor immunotherapy.