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.

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-2K b /TRP2 180-188-Ig dimers, and anti-CD28. The resulting PEGylated and CD47-conjugated nanoellipsoidal aAPCs (EaAPC PEG/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 (EaAPC PEG). Results: EaAPC PEG/CD47 markedly reduced cellular uptake in vitro and in vivo, as compared with EaAPC PEG , EaAPC, SaAPC, and Blank-NPs and expanded naïve TRP2 180-188-specific CD8 + T cells in the co-cultures with spleen lymphocytes. After three infusions, the EaAPC PEG/CD47 showed much stronger effects on facilitating TRP2 180-188-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.

Biomimetic nanoparticles have been reported as immune modulators in autoimmune diseases and allograft rejections by numerous researchers. However, most of the therapeutics carrying antigens, toxins or cytokines underlay the mechanism of antigen presentation by cellular uptake of NPs through pinocytosis and phagocytosis. Few researches focus on the direct and antigen-specific modulation on T cells by NPs and combined use of multiple regulatory molecules. Here, polylactic-co-glycolic acid nanoparticles (PLGA-NPs) were fabricated as scaffold to cocoupling H-2Kb-Ig dimer, anti-Fas mAb, PD-L1-Fc, TGF-β and CD47-Fc for the generation of alloantigen-presenting and tolerance-inducing NPs, termed killer NPs and followed by i.v. injection into a single MHC-mismatched murine model of alloskin transplantation. Three infusions prolonged alloskin graft survival for 45 days; depleted most of H-2Kb alloreactive CD8+ T cells in peripheral blood, spleen and local graft, in an antigen-specific manner. The killer NPs circulated throughout vasculature into various organs and local allograft, with a retention time up to 30 h. They made contacts with CD8+ T cells to facilitate vigorous apoptosis, inhibit the activation and proliferation of alloreactive CD8+ T cells and induce regulatory T cells in secondary lymphoid organs, with the greatly minimized uptake by phagocytes. More importantly, the impairment of host overall immune function and visible organ toxicity were not found. Our results provide the first experimental evidence for the direct and on-target modulation on alloreactive T cells by the biodegradable 200-nm killer NPs via co-presentation of alloantigen and multiple regulatory molecules, thus suggest a novel antigen-specific immune modulator for allograft rejections.

PurposeNumerous 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 methodsPoly(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.ResultsFour 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.ConclusionOur 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.

emerging as a potential alternative to these problems. The tissue engineering triad involves the effective integration of cells, scaffolds, and signals for the development of biological substitutes to restore, maintain, and replace injured tissues and organs. [2] The scaffold plays a substantial role in the field of tissue engineering, primarily by providing physical support (structures and substrates) for cells to attach and grow, subsequently resulting in tissue formation. [3] A variety of natural and synthetic biomaterials have been explored to develop tissue-engineered scaffolds. By incorporating exogenous growthstimulating signals, such as growth factors or small molecules, scaffolds can provide specific bioactivities, that is, biochemical signals required for cellular behaviors and tissue regeneration. [4] As biomaterials are essentially comprised of chemical molecules, they are capable of supplying inherent biochemical signals to guide and influence cells. For example, cells residing in tissue-engineered scaffolds can establish cell-biomaterial communication networks partially through biochemical signaling. The degradation products of various biodegradable biomaterials after implantation in vivo also provide specific biochemical signals to the cells in and around the 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.

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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/β 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.Vaccines 2019, 7, 161 2 of 16 with microcephaly, fetal demise, and other congenital disorders [4][5][6]. Thus, ZIKV has emerged as a global health threat and there is an urgent requirement for an effective vaccine to combat ZIKV-associated diseases.The ZIKV genome encodes a single polyprotein, which is processed into three structural proteins (capsid (C), precursor of membrane/membrane (prM/M), and envelope (E)) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [7][8][9]. The E protein has important roles in infection and pathogenesis, is involved in virus binding to target cells and membrane fusion, and also serves as an important vaccine target [7,8]. The E protein is a transmembrane protein and consists of three domains termed I, II, and III (DI-DIII), a fusion loop (FL), and a stalk region (S) [8]. The DI-DII region of ZIKV E protein is a target for cross-reactive antibodies with other flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV), whereas the DIII domain induces ZIKV-specific antibodies [10][11][12], and therefore, is a key target for ZIKV vaccine development. ZIKV vaccine candidates under development include inactivated virus, live attenuated virus, DNA, mRNA, viral vectors, virus-like particles (VLPs), and viral proteins (or subunit vaccines) [13][14][15][16]. Although ma...