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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.

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

Background Intrahepatic cholangiocarcinoma (iCCA) patients often lose the chance of radical resection due to early lymphatic metastasis. And most of the therapy in cholangiocarcinoma has been limited and ineffective. Herein, we examined the role of FGF and VEGF pathways in regulating lymphangiogenesis and PD-L1 expression. We also validated the possibility to achieve a powerful anti-lymphangiogenesis effect and improve the immunocompetence with combination therapy in iCCA. Methods The expression of FGFR1 and VEGFR3 in primary lymphatic endothelial cells (LECs) were detected by Flow cytometry. The lymphngiogenic function of FGF and VEGF were evaluated in LECs and iCCA xenograft mice model. The relationship between VEGF and hexokinase2 (HK2) was validated in LECs by western blot, immunofluorescence and luciferase reporter assays. The therapeutic efficacy of infigratinib in combination with SAR131675 were assessed in LECs and xenograft models. Microarray analysis was used to evaluate the pathological relevance between FGFR1 and VEGFR3 with HK2 in human lymphatic vessels. PD-L1 expression in LECs affected by combined treatment were analysis by flow cytometry and western blot. Results FGF promoted lymphangiogenesis through c-MYC-dependent modulation of HK2. In addition, VEGFC also upregulated HK2 expression. Mechanically, VEGFC phosphorylated PI3K/Akt/mTOR axis to upregulate HIF-1α expression at the translational level, HIF-1α then bind to the HK2 promoter region for transcriptional activation. More importantly, dual FGFR and VEGFR inhibition with infigratinib and SAR131675 almost completely inhibited lymphangiogenesis in LECs and iCCA xenograft mice model. Histologically, high HK2 expression in lymphatic vessels were significantly associated with poor iCCA prognosis and the expression of HK2, FGFR1 and VEGFR3 in lymphatic vessels were related to lymph node metastasis in intrahepatic cholangiocarcinoma. Meanwhile, FGFR1 and VEGFR3 were significantly correlated with HK2 in lymphatic vessels. Furthermore, the combination of infigratinib and SAR131675 significantly reduced PD-L1 expression in LECs through inhibiting lactic acid production. Conclusions Dual FGFR and VEGFR inhibition restrain lymphangiogenesis through suppression c-MYC-dependent and HIF-1α-mediated HK2 expression respectively. Decreased HK2 down-regulated glycolytic activity and further attenuated PD-L1 expression. Our findings suggest that dual FGFR and VEGFR blocking is an effective novel combination strategy to inhibit lymphangiogenesis and improve the immunocompetence in iCCA.

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.

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.

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.

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.

While SARS-CoV-2-specific T cells have been characterized to play essential roles in host immune protection in COVID-19 patients, few researches focus on the functional validation of T cell epitopes and development of vaccines inducing specific T cell responses. In this study, 120 CD8+ T cell epitopes from E, M, N, S and RdRp proteins were validated. Among them, 110 epitopes have not been reported previously; 110, 15, 6, 14 and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; 4 epitopes from S protein displayed one amino acid distinct from the current variants of SARS-CoV-2. Thirty-one epitopes restricted by HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or polylactic-co-glycolic acid nanoparticles, which elicited robust specific CD8+ T cell responses in wild-type and HLA-A2/DR1 transgenic mice. Seven of the 31 epitopes were found to be cross-presented by HLA-A2 and H-2K/Db molecules. Unlike previous researches, this study established a modified cell co-culture system of DC-peptide-PBL using healthy donor’s PBMCs to validate the CD8+ T cell epitope on-silicon predicted; provided a library of CD8+ T cell epitopes restricted by a series of high-frequency HLA-A allotypes which covering broad Asian populations; identified the HLA-A cross-restrictions of these CD8+ T cell epitopes using competitive binding experiments with HMy2.CIR cell lines expressing indicated HLA-A molecules; and initially confirmed the in vivo feasibility of 9 or 10-mer peptide cocktail vaccines of SARS-CoV2. These data will facilitate the development of vaccines inducing antiviral CD8+ T cell responses.

In this study, a tolerogenic artificial APC (TaAPC) was developed to directly and selectively modulate myelin-autoreactive CD4+ and CD8+ T cells in the myelin oligodendrocyte glycoprotein (MOG)35–55 peptide–induced experimental autoimmune encephalomyelitis in C57BL/6J mice. Cell-sized polylactic-coglycolic acid microparticles were generated to cocouple target Ags (MOG40–54/H-2Db-Ig dimer, MOG35–55/I-Ab multimer), regulatory molecules (anti-Fas and PD-L1-Fc), and “self-marker” CD47-Fc and encapsulate inhibitory cytokine (TGF-β1). Four infusions of the TaAPCs markedly and durably inhibited the experimental autoimmune encephalomyelitis progression and reduced the local inflammation in CNS tissue. They circulated throughout vasculature into peripheral lymphoid tissues and various organs, but not into brain, with retention of 36 h and exerted direct effects on T cells in vivo and in vitro. Two infusions of the TaAPCs depleted 65–79% of MOG35–55-specific CD4+ and 46–62% of MOG40–54-specific CD8+ T cells in peripheral blood, spleen, and CNS tissues in an Ag-specific manner and regulatory molecule–dependent fashion; induced robust T cell apoptosis; inhibited the activation and proliferation of MOG peptide–reactive T cells; reduced MOG peptide–reactive Th1, Th17, and Tc17 cells; and expanded regulatory T cells. They also inhibited IFN-γ/IL-17A secretion and elevated IL-10/TGF-β1 production in splenocytes but not in CNS tissue. More importantly, the TaAPCs treatment did not obviously suppress the overall immune function of host. To our knowledge, this study provides the first experimental evidence for the capability of TaAPCs to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine, thus suggesting a novel Ag-specific immunotherapy for the T cell–mediated autoimmune diseases.

Purpose Numerous nanomaterials have been reported in the treatment of multiple sclerosis or experimental autoimmune encephalomyelitis (EAE). But most of these nanoscale therapeutics deliver myelin antigens together with toxins or cytokines and underlay the cellular uptake and induction of tolerogenic antigen-presenting cells by which they indirectly induce T cell tolerance. This study focuses on the on-target and direct modulation of myelin-autoreactive T cells and combined use of multiple regulatory molecules by generating a tolerogenic nanoparticle. Materials and methods Poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) were fabricated by co-coupling MOG40-54/H-2Db-Ig dimer, MOG35-55/I-Ab multimer, anti-Fas, PD-L1-Fc and CD47-Fc and encapsulating transforming growth factor-β1. The resulting 217 nm tolerogenic nanoparticles (tNPs) were administered intravenously into MOG35-55 peptide-induced EAE mice, which was followed by the investigation of therapeutic outcomes and the in vivo mechanism. Results Four infusions of the tNPs durably ameliorated EAE with a marked reduction of clinical score, neuroinflammation and demyelination. They were distributed in secondary lymphoid tissues, various organs and brain after intravenous injection, with retention over 36 h, and made contacts with CD4+ and CD8+ T cells. Two injections of the tNPs markedly decreased the MOG35-55-reactive Th1 and Th17 cells and MOG40-55-reactive Tc1 and Tc17 cells, increased regulatory T cells, inhibited T cell proliferation and elevated T cell apoptosis in spleen. Transforming growth factor-β1 and interleukin-10 were upregulated in the homogenates of central nervous system and supernatant of spleen cells. Conclusion Our data suggest a novel therapeutic nanoparticle to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine in the antigen-specific combination immunotherapy for T cell-mediated autoimmune diseases.

Background Mouse experimental autoimmune encephalomyelitis (EAE) is widely used model of multiple sclerosis (MS). The role of autoreactive CD4+ and CD8+ T cells in the development of mouse EAE has been demonstrated. However, little information is available about the relation between the frequency and reactivity of myelin antigen-reactive CD4+ and CD8+ T cells in secondary lymphoid organs and their relevance with the inflammation and pathological lesion of CNS during the course of EAE mouse model. Methods In this study, an EAE model with a clinical course containing acute onset, peak and chronic remission stages was established in C57BL/6J mice by myelin oligodendrocyte protein (MOG)35-55 peptide immunization, and followed by the monitoring of clinical and pathological parameters and autoreactive T cells at different stages during the course. Results The dynamic changes of inflammatory infiltration, myelin loss, and astrocyte proliferation in brain and spinal cord were highly consistent with clinical severity observed in EAE course. However, the frequencies of both MOG-specific CD4+ and CD8+ T cells in secondary lymphoid organs presented different dynamic trends from the IFN-γ production by MOG-reactive T cells. Meanwhile, the IL-17 production by MOG-reactive CD4+ T cells was consistent with the proliferation of MOG-specific CD4+ T cells. Conclusions Both CD4+ and CD8+ T cells were most sensitive to MOG antigen stimulation for IFN-γ production during the early stage of EAE, but then rapidly lost the function despite their vigorous proliferation at the peak stage and later.

Shilong Song, Xiaoxiao Jin, Lei Zhang, Chen Zhao, Yan Ding, Qianqian Ang, Odontuya Khaidav, Chuanlai Shen Department of Microbiology and Immunology, Medical School, Southeast University, Nanjing, Jiangsu Province 210009, People’s Republic of China 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. Keywords: PLGA nanoparticles, artificial antigen-presenting cells, phagocytosis, cancer active immunotherapy

The specific eradication of pathogenic T cells for the treatment of allograft rejections and autoimmune disorders without impairment of overall immune function is a fundamental goal. Here, cell-sized poly(lactic-co-glycolic acid) microparticles (PLGA MPs) were prepared as a scaffold to co-display the peptide/major histocompatibility complex (pMHC, target antigen) and anti-Fas monoclonal antibody (apoptosis-inducing molecule) for the generation of biodegradable killer MPs. Ovalbumin (OVA) antigen-targeted killer MPs significantly depleted OVA-specific CD8+ T cells in an antigen-specific manner, both in vitro and in OT-1 mice. After intravenous administration, the killer MPs predominantly accumulated in the liver, lungs, and gut of OT-1 mice with a retention time of up to 48 hours. The killing effects exerted by killer MPs persisted for 4 days after two injections. Moreover, the H-2Kb alloantigen-targeted killer MPs were able to eliminate low-frequency alloreactive T cells and prolong alloskin graft survival for 41.5 days in bm1 mice. Our data indicate that PLGA-based killer MPs are capable of specifically depleting pathogenic T cells, which highlights their therapeutic potential for treating allograft rejection and autoimmune disorders.

In this study, a tolerogenic artificial APC (TaAPC) was developed to directly and selectively modulate myelin-autoreactive CD4

The strategy of specifically depleting antigen-specific T cells can potentially be used for the treatment of allograft rejection and autoimmunity because it does not suppress the overall immune systems.In this study, we generated killer polylactic-co-glycolic acid (PLGA) microspheres by covalently coupling major histocompatibility complex (MHC) class I antigens and apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto PLGA microspheres. A modified double-emulsion method was used for the preparation of cell-sized PLGA microspheres. H-2K(b)/peptide monomers were generated in-house and analyzed through flow cytometry. The killer PLGA microspheres were administered intravenously into BALB/c mice (H-2K(d)) that had previously been grafted with skin squares from C57BL/6 mice (H-2K(b)). Tumor cell challenge and third-party mixed lymphocyte culture were used to assess the general immune functions of host.The alloskin graft survival was prolonged by 4 days. The killer PLGA microspheres could specifically deplete the H-2K(b) alloantigen-reactive CD8(+) T cells that infiltrated into the alloskin graft but not CD4(+) T cells, without impairment of host overall immune function.Here, we initially report that PLGA microspheres, which have been widely used as medicine-delivering carriers, were used to prepare antigen-specific killer complexes and treat allograft rejection. Our data highlight the therapeutic potential of this biocompatible and biodegradable antigen-specific killer effector for the treatment of allograft rejection and autoimmune disease.

Conventional peptide-major histocompatibility complex (pMHC) multimer staining, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay cannot concurrently determine the frequency and reactivity of antigen-specific T cells (AST) in a single assay. In this report, pMHC multimer, magnetic-activated cell sorting (MACS), and ELISPOT techniques have been integrated into a micro well by coupling pMHC multimers onto cell-sized magnetic beads to characterize AST cell populations in a 96-well microplate which pre-coated with cytokine-capture antibodies. This method, termed AAPC-microplate, allows the enumeration and local cytokine production of AST cells in a single assay without using flow cytometry or fluorescence intensity scanning, thus will be widely applicable. Here, ovalbumin257–264-specific CD8+ T cells from OT-1 T cell receptor (TCR) transgenic mice were measured. The methodological accuracy, specificity, reproducibility, and sensitivity in enumerating AST cells compared well with conventional pMHC multimer staining. Furthermore, the AAPC-microplate was applied to detect the frequency and reactivity of Hepatitis B virus (HBV) core antigen18–27- and surface antigen183–191-specific CD8+ T cells for the patients, and was compared with conventional method. This method without the need of high-end instruments may facilitate the routine analysis of patient-specific cellular immune response pattern to a given antigen in translational studies.

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-&alpha, /&beta, 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.

Accumulating evidence indicates that bead-based artificial antigen-presenting cells (aAPCs) are a powerful tool to induce antigen-specific T cell responses in vitro and in vivo. To date, most conventional aAPCs have been generated by coupling an antigen signal (signal 1) and one or two costimulatory signals, such as anti-CD28 with anti-LFA1 or anti-4-1BB (signal 2), onto the surfaces of cell-sized or nanoscale magnetic beads or polyester latex beads. The development of a biodegradable scaffold and the combined use of multiple costimulatory signals as well as third signals for putative clinical applications is the next step in the development of this technology. Here, a novel biodegradable aAPC platform for active immunotherapy was developed by co-encapsulating IL-2 and anti-CTLA-4 inside cell-sized polylactic-co-glycolic acid microparticles (PLGA-MPs) while co-coupling an H-2Kb/TRP2-Ig dimer and anti-CD28 onto the surface. Cytokines (activating signal) and antibodies (anti-inhibition signal) were efficiently co-encapsulated in PLGA-MP-based aAPCs and co-released without interfering with each other. The targeted, sustained co-release of IL-2 and anti-CTLA-4 achieved markedly enhanced, synergistic effects in activating and expanding tumor antigen-specific T cells both in vitro and in vivo, as well as in inhibiting tumor growth in a mouse melanoma model, as compared with conventional two-signal aAPCs and IL-2 or anti-CTLA-4 single-released aAPCs. These data revealed the feasibility and importance of the paracrine release of multiple costimulatory molecules and cytokines from biodegradable aAPCs and thus provide a proof of principle for the future use of polymeric aAPCs for active immunotherapy of tumors and infectious diseases.

The time-consuming and high-cost preparation of soluble peptide-major histocompatibility complexes (pMHC) currently limits their wide uses in monitoring antigen-specific T cells. The single-chain trimer (SCT) of peptide-β2m-MHC class I heavy chain was developed as an alternative strategy, but its gene fusion is hindered in many cases owing to the incompatibility between the multiple restriction enzymes and the restriction endonuclease sites of plasmid vectors. In this study, overlap extension PCR and one-step cloning were adopted to overcome this restriction. The SCT gene of the OVA₂₅₇₋₂₆₄ peptide-(GS₄)₃-β2m-(GS₄)₄-H-2K

Background and Aims Loss or downregulation of human leukocyte antigen (HLA) class I expression has been reported in a variety of human tumors including oral squamous cell carcinoma (OSCC). Methods Expression of HLA class I molecules were evaluated by immunohistochemistry, flow cytometry, semi-quantitative Western blot and RT-PCR in 43 tissue samples of primary oral squamous cell carcinomas (pOSCC) from Chinese patients and two OSCC cell lines. Results HLA class I heavy chain of B/C locus and A locus and β 2- microglobulin were obviously lost or downregulated in pOSCC with the percentage of 31, 55 and 35% respectively. The expression of HLA B/C, LMP2, LMP7, LMP10 and PA28β in OSCC cell lines was also presumably reduced in comparison with normal epithelial cell line. Conclusions These data suggested that the downregulation of HLA class I molecules in OSCC was closely associated with the low–efficient transcription and abnormality of post-transcription regulation of HLA class I genes and antigen presentation-related genes. These results can add more light to the mechanism by which OSCC escape from immunological surveillance.

The application of the peptide-linked beta2-microglobulin (beta2m) strategy is limited in some cases due to the incompatibility between the sequences of the peptides and the restriction sites of the plasmid vectors. An isocaudamer technique was adapted to overcome this restriction. Three peptide-linked beta2m genes, HBc(18-27)-hbeta2m gene, OVA(257-264)-mbeta2m gene and HER2/neu(369-377)-mbeta2m gene, were inserted into the pET28a vectors with this technique. The corresponding proteins were expressed in Escherichia coli with yields of over 50 mg/l culture and purities of over 80%. This strategy facilitates the construction of peptide-linked beta2m molecules and will simplify the preparation of major histocompatibility complex-peptide complexes.

Latex microspheres-based artificial antigen-presenting cell constructs (aAPCs) are proved to be valuable tools to expand T cells ex vivo for adoptive cell therapy, but little is known about their potential for active immunization. In this report, HLA-A2/peptide tetramers were generated and co-coated with anti-mouse CD28 monoclonal antibody onto surface of cell-sized latex microspheres followed by immunization of naive HLA-A2/K b transgenic mice. Five- to six-fold expansion of tumor antigen-specific CTLs was observed in the spleen after three rounds of immunization. The consequent splenocytes can efficiently recognize endogenously expressed tumor antigen on the surface of human target cells and cytolyze the tumor cells in an antigen-specific manner. This report provides initially the experimental evidence that latex microspheres-based aAPCs can effectively prime antigen-specific CTL proliferation and cytolysis in naive mice. This may contribute to a better insight into the potential of microspheres-based aAPCs for active immunization.

The uses of soluble HLA class I/peptide complexes to monitor antigen reactive T cells are often hampered by their low-yield and high-cost production. As an alternative strategy, the peptide-beta(2)m fused, 2-component (2C) HLA class I/peptide complex has been developed, but its application is limited due to the lack of the comparison of its structural and functional characteristics with those of its conventional 3-component (3C) counterpart. In this study, we have demonstrated that the 2C and 3C HLA-A2/MART1(27-35) complexes have a similar chromatographical profile and comparable stability, but the former has 2.5 times higher yield and significantly higher binding ability with HLA-A2/MART1(27-35) complex-specific receptors than the latter. Furthermore, the 2C complex has a comparable ability to stimulate specific CTL proliferation, but appears to be more effective in eliciting the cytotoxicity of antigen-specific CTL, as compared to its 3C counterpart.

FasL-expressing killer antigen-presenting cells (KAPCs) have the ability to delete antigen-specific T cells and, therefore, could potentially be used for the treatment of allograft rejection and autoimmunity; however, their cellular nature markedly limits their clinical use. Novel bead-based killer artificial antigen-presenting cells (KaAPCs), which are generated by coupling major histocompatibility complex (MHC) class I antigens together with the apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto magnetic beads, have recently attracted more attention. KaAPCs have a number of advantages over KAPCs and are able to deplete specific T cells in cocultures. However, it remains unknown whether bead-based KaAPCs can also induce apoptosis of alloreactive or autoreactive T cells and, consequently, generate hyporesponsiveness in vivo. In this study, H-2K b /peptide monomers and anti-Fas mAb have been covalently coupled to latex beads and administered intravenously into BALB/c mice (H-2K d ) that had previously been grafted with skin squares from C57BL/6 mice (H-2K b ). Alloskin graft survival was prolonged for 6 days. A 60% decrease of H-2K b antigen-alloreactive T cells was demonstrated by several measures 2 days after each injection of KaAPCs, but intact immune function, including antitumor activity, was maintained. These data provide the first in vivo evidence that bead-based KaAPCs can selectively deplete antigen-specific T cells without the loss of overall immune responsiveness and, therefore, highlight the therapeutic potential of this novel strategy for the treatment of allograft rejection and autoimmune disorders.

The current refolding process of MHC/peptide complexes is low-yield and time-consuming, thereby limiting the wide uses of MHC/peptide multimers. Here the heavy chain protein of MHC/peptide complex (H-2K(b)/TRP2(180-188)) was immobilized onto an ion-exchange chromatography column, and the β2m or TRP2(180-188)-fused β2m protein, which renatured previously in refolding buffer, was able to pass through the column for the gradient refolding. This strategy refolds, concentrates and purifies MHC/peptide complexes in a single integrated step, achieving a high level of process simplification and automation. Using this on-column refolding method, MHC/peptide complexes could be prepared within 24h with a refolding yield of over 20%. Anti-H-2K(b) mAb staining and flow cytometric analyses revealed that the on-column refolded H-2K(b)/TRP2(180-188) complexes had conformational characteristics similar to the dilution refolded H-2K(b)/TRP2(180-188) complexes and the commercial ones. Furthermore, H-2K(b)/TRP2(180-188) tetramer staining and the enumeration of TRP2(180-188)-specific T cells and H-2K(b)-alloreactive T cells confirmed that the H-2K(b)/TRP2(180-188) complexes prepared by on-column refolding or dilution refolding had comparable TCR-binding ability. These data demonstrate a novel, simple and efficient refolding strategy for the generation of MHC class I/peptide complexes.

Chronic hepatitis B virus infection is associated with a high risk of developing into hepatocellular carcinoma, while tumor recognition is important during the immune surveillance process that prevents cancer development in humans. The mechanisms of immune evasion and the role of the early immune response in chronic infection caused by hepatitis B virus (HBV) are still unclear. In the present study, 1 copy or 1.2 copies of HBV genome was transfected into a hepatocellular carcinoma cell line BEL7405. RT-PCR, Western blot and flow cytometry analysis were used to evaluate the expression of HLA class I molecules and transporter associated with antigen processing 1 (TAP1). Finally, the cytotoxic activity of natural killer (NK) cells against HBV transfected liver cells was detected by MTT colorimetry method. Following transfection of 1 copy or 1.2 copies of HBV genome, HLA class I expression was up-regulated in BEL7405 cell line in a dose-dependent manner. Furthermore, increased the surface HLA class I expression were caused by enhanced expression of TAP1 at mRNA and protein levels in those transfected cells. Consequently, a significantly down-regulated cytotoxic activity of NK cells against HBV transfected liver cells was observed. These results may demonstrate a way by which HBV avoids recognition by NK cells that might be associated with the establishment of chronic infection and tumor formation.

The heavy chain protein of HLA-peptide complexes (HLA/HBc(18-27) and HLA/CEA(694-702)) immobilized onto an ion exchange chromatography column and then the dilution-refolded HBc(18-27)-fused or CEA(694-702)-fused β2m protein was able to pass through the column. Using this method, HLA/peptide complexes were prepared within 30 h with a refolding yield of at least 20% (w/w) and purity of over 80% (w/w). This strategy refolds, concentrates, and purifies HLA/peptide complexes in a single integrated step and offers a potential tool to refold multiple-subunit proteins other than the major histocompatibility complex (MHC)/peptide complexes.

Down-regulated expression of human leukocyte antigen (HLA) class I molecules in many human cancers facilitate tumor cells to escape from immune attack. Promoter hypermethylation, one of the major epigenetic changes responsible for gene inactivation, plays an important role in gastric carcinogenesis. This study evaluated the expression and alteration of HLA class I molecules in a panel of 47 pairs of gastric cancer specimens with their noncancerous parts from Chinese patients by using immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific PCR (MSP) analysis. The expression of HLA-A, HLA-B/C and HLA class I complex was lost or down-regulated in human gastric cancer. The percentage of promoter methylation was 59.57% for HLA-A gene, 55.32% for HLA-B gene and 48.94% for HLA-C gene in gastric cancer, while it was decreased to 19.15%, 12.77% and 6.38% in the adjacent nontumor tissues, respectively. Seven of 10 (70%), 4 of 6 (66.7%) and 3 of 4 (75%) gastric cancer specimens with promoter hypermethylation at HLA-A, -B and -C loci showed transcriptional inactivation of HLA-A,-B and -C genes, suggesting an association between promoter hypermethylation and down-regulated expression of HLA class I molecules. Human gastric cancer cell line BGC-823 showed HLA-A down-regulation with promoter methylation of HLA-A locus. Treatment with DNA methyltransferase inhibitor restored the expression of HLA-A mRNA and surface HLA-A complex. Thus, our results showed that promoter hypermethylation might be one of the mechanisms that lead to HLA class I antigen down-regulation in gastric cancer.

Loss or downregulation of human leukocyte antigen (HLA) class I molecules is a widespread mechanism used by tumor cells to avoid tumor recognition by cytotoxic T lymphocytes favoring tumor immune escape. Multiple molecular mechanisms are responsible for these altered HLA class I tumor phenotypes, such as the loss of heterozygosity (LOH) at chromosome region 6p21.3. In this study, we used immunohistological techniques with a highly selective panel of anti-HLA monoclonal antibodies to analyze the expression of HLA class I molecules in 84 formalin-fixed, paraffin-embedded section and 49 frozen-fresh tissues of primary esophageal squamous cell carcinomas (pESCC) from Chinese patients. To elucidate the underlying mechanism of HLA class I loss or downregulation, we also analyzed LOH of previously selected microsatellite markers located in chromosomes 6 and 15 by polymerase chain reaction. DNA was obtained from frozen-fresh tumor tissues and surrounding stroma to define the LOH associated with chromosomes 6p21 and 15q21. Our results showed that HLA-A, HLA-B/C, HLA class I heavy chain, beta2-microglobuline, and HLA class I complex were lost or downregulated in pESCC (P

Background and Aim: The interferon sensitivity determining region (ISDR) in the non-structural 5A protein (NS5A) gene of hepatitis C virus genotype 1b (HCV-1b) has been demonstrated in Japanese patients; however, contradictory data have been reported from other parts of the world, particularly from Europe and the USA. This study investigated whether mutations in the ISDR of NS5A gene influence interferon efficacy in Chinese patients with chronic HCV-1b infection Methods: Forty-five Chinese patients with chronic HCV infection were enrolled in a retrospective study. Of these patients, 20 with HCV-1b infection completed an interferon monotherapy course and 6-month follow-up. The pretreatment mutations in the nucleic acid sequence of NS5A 2209–2248 (ISDR) of HCV-1b were identified by sequencing RT-PCR products followed by comparison with the prototype sequence (HCV-J). Correlation with the clinical efficacy of interferon alpha therapy was then analyzed retrospectively. Results: The statistical analysis indicated a significant correlation between the mutations in ISDR of HCV-1b and sustained virological response to interferon alpha treatment (P

The strategy of specifically depleting antigen-specific T cells can potentially be used to treat allograft rejection and autoimmunity because it does not suppress the overall immune systems. In this study, we generated killer polylactic-co-glycolic acid (PLGA) microspheres by covalently coupling H-2Kb-Ig and apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto cell-sized PLGA microspheres. The Kb/OVA257-264-Killer PLGAs resulted in a dramatic reduction in the number of OVA257-264-specific CD8+ T cells in an antigen-specific manner in vitro and in vivo, by using a transgenic OT-1 mice model. The bioluminescence imaging revealed that Killer PLGAs dominantly distributed in lung, liver and gut, and was maintained for up to 48 hours in OT-1 mice and a longer time in OT-1 mice than in C57BL/6 mice. Moreover, the Kb-Killer PLGAs were administered intravenously into bm1 mice (H-2Kbm1) that had previously been grafted with ear skin from C57BL/6 mice (H-2Kb). The killer PLGAs selectively decreased the H-2Kb alloantigen-reactive CD8+ T cells infiltrating into the alloskin graft but not CD4+ T cells, and significantly prolonged allograft survival for 41.5 days without the impairment of host overall immune function. Thus, our data highlight the therapeutic potential of this biocompatible and biodegradable killer effectors for the treatment of allograft rejection and autoimmune disease. Keywords: Allograft rejection, PLGA, H-2Kb-Ig

To observe the immune reaction in the mixed culture of host lymphocytes with allogenic and host endothelial cells.The host epithelial cells and lymphocytes from burn patients and allogenic epithelial cells were mix-cultured in different ratios, so as to simulate the local immune micro-environment of host skin island in intermingled skin grafting. In addition, the cells from normal human subjects were also mix-cultured as control. The lymphocyte cpm values were detected by (3)H-TdR and HLA molecules and T cell subgroup were determined by immunohistological technique.(1) The lymphocyte proliferation reaction could be effectively inhibited by the epithelial cells from burn patients but not from normal control. (2) The inhibition of host lymphocyte proliferation could not be mediated by the HLA-DQ molecules of epithelium from burn patients. (3) The positive expression rate of HLA-DR of epithelia from burn patients was evidently higher that that from normal control (P0.05), (4) The CD8 expression of lymphocyte in burn patients was significantly higher than that in normal control (P0.01), while the CD4 expression in burn patients was lower than that in normal control (P0.01). But there was no obvious difference of the CD3 expression between patients and normal subjects (P0.05).The lymphocyte proliferation reaction could be obviously inhibited by the host epithelium, which might be related to the specific immune state of the host lymphocytes and epithelium of burn patients.