Molecular characterization of antigen-peptide pulsed dendritic cells: immature dendritic cells develop a distinct molecular profile when pulsed with antigen peptide.
ABSTRACT: As dendritic cells (DCs) are the most potent professional antigen-presenting cells, they are being tested as cancer vaccines for immunotherapy of established cancers. Although numerous studies have characterized DCs by their phenotype and function, few have identified potential molecular markers of antigen presentation prior to vaccination of host. In this study we generated pre-immature DC (piDC), immature DC (iDC), and mature DC (mDC) from human peripheral blood monocytes (PBMC) obtained from HLA-A2 healthy donors, and pulsed them with human papillomavirus E7 peptide (p11-20), a class I HLA-A2 binding antigen. We then characterized DCs for cell surface phenotype and gene expression profile by microarray technology. We identified a set of 59 genes that distinguished three differentiation stages of DCs (piDC, iDC and mDC). When piDC, iDC and mDC were pulsed with E7 peptide for 2 hrs, the surface phenotype did not change, however, iDCs rather than mDCs showed transcriptional response by up-regulation of a set of genes. A total of 52 genes were modulated in iDC upon antigen pulsing. Elongation of pulse time for iDCs to 10 and 24 hrs did not significantly bring further changes in gene expression. The E7 peptide up-modulated immune response (KPNA7, IGSF6, NCR3, TREM2, TUBAL3, IL8, NFKBIA), pro-apoptosis (BTG1, SEMA6A, IGFBP3 and SRGN), anti-apoptosis (NFKBIA), DNA repair (MRPS11, RAD21, TXNRD1), and cell adhesion and cell migration genes (EPHA1, PGF, IL8 and CYR61) in iDCs. We confirmed our results by Q-PCR analysis. The E7 peptide but not control peptide (PADRE) induced up-regulation of NFKB1A gene only in HLA-A2 positive iDCs and not in HLA-A2 negative iDCs. These results suggest that E7 up-regulation of genes is specific and HLA restricted and that these genes may represent markers of antigen presentation and help rapidly assess the quality of dendritic cells prior to administration to the host.
Project description:Induction of optimal HIV-1-specific T-cell responses, which can contribute to controlling viral infection in vivo, depends on antigen processing and presentation processes occurring in DCs. Opsonization can influence the routing of antigen processing and pathways used for presentation. We studied antigen proteolysis and the role of endocytic receptors in MHC class I (MHCI) and II (MHCII) presentation of antigens derived from HIV-1 in human monocyte-derived immature DCs (IDCs) and mature DCs, comparing free and complement opsonized HIV-1 particles. Opsonization of virions promoted MHCI presentation by DCs, indicating that complement opsonization routes more virions toward the MHCI presentation pathway. Blockade of macrophage mannose receptor (MMR) and ?7-integrin enhanced MHCI and MHCII presentation by IDCs and mature DCs, whereas the block of complement receptor 3 decreased MHCI and MHCII presentation. In addition, we found that IDC and MDC proteolytic activities were modulated by HIV-1 exposure; complement-opsonized HIV-1 induced an increased proteasome activity in IDCs. Taken together, these findings indicate that endocytic receptors such as MMR, complement receptor 3, and ?7-integrin can promote or disfavor antigen presentation probably by routing HIV-1 into different endosomal compartments with distinct efficiencies for degradation of viral antigens and MHCI and MHCII presentation, and that HIV-1 affects the antigen-processing machinery.
Project description:Mast cells (MCs) are tissue resident cells, rich in inflammatory mediators, involved in allergic reactions, and with an increasingly recognized role in immunomodulation. Dendritic cells (DCs), on the other hand, are central to the determination of immune response patterns, being highly efficient antigen-presenting cells that respond promptly to changes in their microenvironment. Here, we show that direct cell contact between immature monocyte-derived DCs (iDCs) and MC bends DCs toward tolerance induction. DCs that had direct contact with MC (MC-iDC) decreased HLA-DR but increased PD-L1 expression and stimulated regulatory T lymphocytes, which expresses FoxP3(+), secrete TGF-? and IL-10, and suppress the proliferation of mitogen-stimulated naïve T lymphocytes. Furthermore, MC-iDC expressed higher levels of indoleamine-2,3-deoxigenase (IDO), a phenomenon that was blocked by treatment of MC with anti-PD-1 or by the treatment of DCs with anti-PD-L1 or anti-PD-L2, but not by blocking of H1 and H2 histamine receptors on DCs. Contact with MC also increased phosphorylated STAT-3 levels in iDCs. When a STAT-3 inhibitor, JSI-124, was added to the DCs before contact with MC, the MC-iDC recovered their ability to induce allogeneic T cell proliferation and did not increase their IDO expression.
Project description:Persistent infection with high-risk human papilloma virus (HPV) is the primary cause of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. HPV58 is the third most common HPV genotype in China after HPV16 and HPV18. HPV E6 and E7 are oncoproteins and are constitutively expressed in HPV-associated cancer cells, therefore they are considered to be ideal target antigens for immunotherapy, including HPV therapeutic vaccine. In the present study, human leukocyte antigen (HLA)-A2-restricted cytotoxic T lymphocyte (CTL) epitope peptides were predicted and screened from HPV58 E7 antigen and their immunogenicity was subsequently determined. A total of 6 HLA-A2-binding peptides derived from HPV58 E7 were predicted and selected using 3 different prediction programs. A negative control peptide and PBS were used as two negative controls. Peripheral blood mononuclear cells (PBMCs) with HLA-A2(+) allele were used to detect the specific cellular immune response among the 6 predicted peptides by enzyme-linked immunospot assay (ELISOPT). Following preliminary screening for the predicted peptides, the antigenicity of the peptide HPV58 E772-80 was further assessed by an immunoassay to a vaccine contained HPV58 E7 antigen. Specific humoral and cellular immunity were detected using the peptide HPV58 E772-80 as the specific antigen. A total of 6 peptides from HPV58 E7 protein were predicted and subsequently named P1 (E77-15: TLREYILDL), P2 (E714-22: DLHPEPTDL), P3 (E769-77: CINSTTTDV), and P4 (E772-80: STTTDVRTL), P5 (E779-87: TLQQLLMGT) and P6 (E783-91: LLMGTCTIV). In the ELISPOT assay on HLA-A2 (+) human PBMCs, interferon (IFN)-?-production was evident in the P2 and P4 groups. The average numbers of IFN-? associated spots in the P2 and P4 groups was 50.61±5.37 spot-forming cells (SFC)/1×105 and 266±34.42 SFC/1×105, respectively. The numbers of spots in the two peptides were significantly increased compared with the other 4 peptides and the control groups (P<0.05). In the further antigenicity verification of P4 (HPV58 E772-80), the peptide only stimulated the humoral immune response of the AD-HPV16/18/58 mE6E7 vaccine containing HPV58 E7 antigen. Compared with the 2 negative control groups (1:400), the antibody titers of the vaccine group (1:25,600) were significantly increased (P<0.05). In cellular immunoassays the average number of IFN-? associated spots was 143.3±32.13 SFC/1×105 in the vaccine group, which was significantly enhanced compared with the PBS group (8±5.29 SFC/1×105; P<0.01) and the AD-NC group (28±5.13 SFC/1×105; P<0.01). The peptide HPV58 E772-80 (STTTDVRTL) displayed sufficient antigenicity to a vaccine contained HPV58 E7 antigen. Therefore, HPV58 E772-80 peptide may be considered as a candidate epitope peptide for the construction of HPV58 peptide vaccines.
Project description:The pro-inflammatory S100 calcium binding protein A8 (S100A8) is elevated in the serum of patients with Plasmodium falciparum malaria, but its function in Plasmodium vivax malaria is not yet clear. This function was investigated in P. vivax-infected patients in this study.The level of S100A8 in the serum was measured with ELISA. Full amino acids of S100A8 were synthesized to verify the functions for maturation of immature dendritic cell (iDC) and evaluation of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) generation by mature DC (mDC).A higher amount of S100A8 was detected in vivax-infected patients (141.2 ± 61.849 ng/ml, n = 40) compared with normal control group (48.1 ± 27.384 ng/ml, n = 40). The level of S100A8 did not coincide with that of anti-malarial antibody measured by indirect fluorescent antibody test (IFAT) using parasite-infected red blood cells as antigen. Programmed death-ligand 1 (PD-L1) was up-regulated on the surface of iDCs following treatment with synthetic S100A8, not with synthetic MSP-1, AMA-1 and CSP, as compared to the expression seen for non-treated iDCs. The addition of red blood cells of infected patients to iDCs also elevated their surface expression of CD86. However, the serum levels of S100A8 decreased with increase in parasitaemia. DCs matured by sera containing S100A8 generated Treg cells from naïve T cells. The ratio of Treg cells generated was inversely proportional to the concentration of S100A8 in sera.Treg cells suppress the activity of cytotoxic T cells, which kill malaria parasites; therefore, the up-regulation of S100A8 in malaria patients may contribute to pathogen immune escape or tolerance.
Project description:Intramuscular administration of DNA vaccines can lead to the generation of antigen-specific immune responses through cross-priming mechanisms. We propose a strategy that is capable of leading to local inflammation and enhancing cross-priming, thus resulting in improved antigen-specific immune responses. Therefore, in this study, we evaluated the immunological responses elicited through electroporation-mediated intramuscular administration of a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 (CRT-E7) in combination with DNA expressing HLA-A2 as compared with CRT-E7 DNA vaccination alone. We found that the co-administration of a DNA vaccine in conjunction with a DNA encoding a xenogenic major histocompatibility complex (MHC) molecule could significantly enhance the E7-specific CD8+ T-cell immune responses and antitumor effects against an E7-expressing tumor, TC-1, in C57BL/6 tumor-bearing mice. Furthermore, a similar enhancement in E7-specific immune responses was observed by the co-administration of CRT-E7 DNA with DNA encoding other types of xenogenic MHC class-I molecules. This strategy was also applicable to another antigenic system, ovalbumin. Further characterization of the injection site revealed that the co-administration of HLA-A2 DNA led to a significant increase in the number of infiltrating CD8+ T lymphocytes and CD11b/c+ antigen-presenting cells. Furthermore, the E7-specific immune responses generated by intramuscular co-administration of CRT-E7 with HLA-A2 DNA were reduced in HLA-A2 transgenic mice. Thus, our data suggest that intramuscular co-administration of DNA encoding xenogenic MHC class-I can further improve the antigen-specific immune responses, as well as antitumor effects generated by DNA vaccines through enhancement of cross-priming mechanisms.
Project description:In order to detect the gene expression profile of in vitro generated dendritic cells , purified monocytes from PBMCs were used as dendritic cell (DCs) precursors and were cultured in medium with cocktail for differentiation and maturation to immature dendritic cells (iDCs) and mature dendritic cells (mDCs). Total RNA samples were isolated from precursor, iDCs and mDCs and used for microarray-based gene expression profiles. Overall design: To generate enough amount of immature DC (iDCs) and mature DCs (mDCs), monocytes were differentiated with GM-CSF and rhIL-4 for 2 days and maturated in the presence of TNF-α, IL-1β, IL-6 and PGE2 for another 2 days. With the anticipation to insight developmental-stage-specific genes with potential functions related to monocyte derived DCs, global gene expression profiling was set using microarray technology.gene expression profiles were performed in triplicate independent experiments starting for 3 groups of precursor, iDC and mDC generated from different blood donors.
Project description:In order to detect the microRNA expression profile of in vitro generated dendritic cells , purified monocytes from PBMCs were used as dendritic cell (DCs) precursors and were cultured in medium with cocktail for differentiation and maturation to immature dendritic cells (iDCs) and mature dendritic cells (mDCs). microRNA samples were isolated from precursor, iDCs and mDCs and used for microarray-based microRNAs expression profiles. Overall design: To generate enough amount of immature DC (iDCs) and mature DCs (mDCs), monocytes were differentiated with GM-CSF and rhIL-4 for 2 days and maturated in the presence of TNF-α, IL-1β, IL-6 and PGE2 for another 2 days. With the anticipation to insight developmental-stage-specific microRNAs with potential functions related to monocyte derived DCs, global microRNAs expression profiling was set using microarray technology.microRNA expression profiles were performed in triplicate independent experiments starting for 3 groups of precursor, iDC and mDC generated from different blood donors.
Project description:EphA2 is a receptor tyrosine kinase and is frequently overexpressed in a wide array of advanced cancers. We demonstrate in the current study that the EphA2 protein is restrictedly expressed in primary glioblastoma multiforme and anaplastic astrocytoma tissues in comparison to normal brain tissues. To evaluate the possibility of targeting EphA2 in glioma vaccine strategies, we stimulated human leukocyte antigen (HLA) A2+ peripheral blood mononuclear cells (PBMCs) obtained from healthy donors and glioma patients with autologous dendritic cells (DCs) loaded with synthetic EphA2883-891 peptide (TLADFDPRV), which has previously been reported to induce interferon-gamma in HLA-A2+ PBMCs. Stimulated PBMCs demonstrated antigen-specific cytotoxic T lymphocyte (CTL) responses as detected by specific lysis of T2 cells loaded with the EphA2883 peptide as well as HLA-A2+ glioma cells, SNB19 and U251, that express EphA2. Furthermore, in vivo immunization of HLA-A2 transgenic HHD mice with the EphA2883-891 peptide resulted in the development of an epitope-specific CTL response in splenocytes, despite the fact that EphA2883-891 is an autoantigen in these mice. Taken together, these data suggest that EphA2883-891 may be an attractive antigen epitope for molecularly targeted glioma vaccines.
Project description:Human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are required for cellular transformation and represent candidate targets for HPV-specific and major histocompatibility complex class I-restricted CD8(+)-T-cell responses in patients with cervical cancer. Recent evidence suggests that cross-reactivity represents the inherent nature of the T-cell repertoire. We identified HLA-A2 binding HPV16 E7 variant peptides from human, bacterial, or viral origin which are able to drive CD8(+)-T-cell responses directed against wild-type HPV16 E7 amino acid 11 to 19/20 (E7(11-19/20)) epitope YMLDLQPET(T) in vitro. CD8(+) T cells reacting to the HLA-A2-presented peptide from HPV16 E7(11-19(20)) recognized also the HLA-A2 binding peptide TMLDIQPED (amino acids 52 to 60) from the human coronavirus OC43 NS2 gene product. Establishment of coronavirus NS2-specific, HLA-A2-restricted CD8(+)-T-cell clones and ex vivo analysis of HPV16 E7 specific T cells obtained by HLA-A2 tetramer-guided sorting from PBL or tumor-infiltrating lymphocytes obtained from patients with cervical cancer showed that cross-reactivity with HPV16 E7(11-19(20)) and coronavirus NS2(52-60) represents a common feature of this antiviral immune response defined by cytokine production. Zero of 10 patients with carcinoma in situ neoplasia and 3 of 18 patients with cervical cancer showed > or =0.1% HPV16 E7-reactive T cells in CD8(+) peripheral blood lymphocytes. In vivo priming with HPV16 was confirmed in patients with cervical cancer or preinvasive HPV16-positive lesions using HLA-A2 tetramer complexes loaded with the E6-derived epitope KLPQLCTEL. In contrast, we could not detect E6-reactive T cells in healthy individuals. These data imply that the measurement of the HPV16 E7(11-19(20)) CD8(+)-T-cell response may reflect cross-reactivity with a common pathogen and that variant peptides may be employed to drive an effective cellular immune response against HPV.
Project description:Monocytes cultured in media containing GM-CSF and IL4 for 5 days normally differentiate into immature dendritic cells which, upon further stimulation with LPS for an additional 2 days, acquire a mature phenotype. In the present study, we evaluated the effect of human cord-blood mesenchymal stem cells (MSC) on human monocyte differentiation into immature and mature DCs by examining the trancriptional profile of the cells that were obtained after both the differentiation and maturation stages. Overall design: This is a six condition experiment (iDC, iDC-MSC, mDC, mDC-MSCd, mDC-MSCm, mDC-MSCdm) where each condition is in biological triplicates, that is 3 donors (A, B, C) per condition. iDC are cells obtained after 5-day differentiation of monocytes, otherwise known as immature DCs. iDC-MSC are the cells obtained at the end of the 5-day differentiation phase of monocytes, during which MSC were also present. mDC are cells obtained after a 2-day maturation of iDC with LPS, also known as mature DCs. mDC-MSC are cells obtained after MSC have been present during only the differentiation (mDC-MSCd) phase, only the maturation (mDC-MSCm) phase or during both the differentiation and maturation phases (mDC-MSCdm).