PIBF (progesterone induced blocking factor) is overexpressed in highly proliferating cells and associated with the centrosome.

PIBF was previously identified as a 34 kDa immunomodulatory molecule secreted by pregnancy lymphocytes and is thought to play a crucial role in preventing rejection of the embryo by the maternal immune response. Recent data suggested that PIBF protein was also expressed by the progesterone receptor (PR) positive MCF-7 breast tumor cell line. Therefore our study was designed to analyze the expression of PIBF in malignant cell lines and primary tumors both at the mRNA and protein levels. RNA expression analyses of several human cell lines with different tissue origin and paired human tumor/normal tissues, as well as of several PR+ and PR- breast tumors revealed that PIBF mRNA was overexpressed in highly proliferating cells independent of the presence of PR. In addition to the full-length PIBF mRNA encoding for a 90 kDa protein, several alternatively spliced species were detected, all resulting from perfect exon skipping. The most frequently identified splice variant is predicted to encode for an approximately 35 kDa protein. Immunofluorescence microscopy revealed a centrosomal localization for the full-length PIBF, while the 35 kDa form showed a diffuse cytoplasmic staining. These data, together with the identification of the PIBF gene in the chromosomal region associated with breast cancer susceptibility, reveal a strong parallel with known tumor suppressor proteins, such as BRCA1 and p53 having the same centrosomal localization. Given the notion that a number of proteins shown to be involved in tumorigenesis are associated with the centrosome and disturbed centrosome function causes unequal segregation of chromosomes, studies to evaluate whether or not PIBF that is highly expressed in tumors is directly involved in tumorigenesis are thus warranted.

The dendritic cell-specific chemokine, dendritic cell-derived CC chemokine 1, enhances protective cell-mediated immunity to murine malaria.

Cell-mediated immunity plays a crucial role in the control of many infectious diseases, necessitating the need for adjuvants that can augment cellular immune responses elicited by vaccines. It is well established that protection against one such disease, malaria, requires strong CD8(+) T cell responses targeted against the liver stages of the causative agent, Plasmodium spp. In this report we show that the dendritic cell-specific chemokine, dendritic cell-derived CC chemokine 1 (DC-CK1), which is produced in humans and acts on naive lymphocytes, can enhance Ag-specific CD8(+) T cell responses when coadministered with either irradiated Plasmodium yoelii sporozoites or a recombinant adenovirus expressing the P. yoelii circumsporozoite protein in mice. We further show that these enhanced T cell responses result in increased protection to malaria in immunized mice challenged with live P. yoelii sporozoites, revealing an adjuvant activity for DC-CK1. DC-CK1 appears to act preferentially on naive mouse lymphocytes, and its adjuvant effect requires IL-12, but not IFN-gamma or CD40. Overall, our results show for the first time an in vivo role for DC-CK1 in the establishment of primary T cell responses and indicate the potential of this chemokine as an adjuvant for vaccines against malaria as well as other diseases in which cellular immune responses are important.

Retrovirus-mediated IL-7 expression in leukemic dendritic cells generated from primary acute myelogenous leukemias enhances their functional properties.

Myeloid lineage-derived dendritic cells (DCs) are considered the professional antigen-presenting cell type responsible for eliciting T-cell-mediated immune responses. Acute myelogenous leukemia (AML) is a disease in which tumor antigens are expressed by the malignant clone that also has the potential to differentiate into DC-like cells (leukemic DCs) with antigen-presenting capacity. This study investigated whether the constitutive expression of the cytokine interleukin-7 (IL-7) in primary AML cells during their differentiation toward leukemic DCs results in superior antigen-presenting cells. A bicistronic retroviral vector encoding the IL-7 cytokine and the surface immunoselectable low-affinity nerve growth factor receptor (LNGFr) gene was constructed and used for transduction experiments. A serum-free system was used to transduce and differentiate leukemic cells toward leukemic DCs. The study included 8 patients with AML. The transduction efficiency with the cytokine vector varied among patients, ranging from 5% to 30% as judged by LNGFr expression. The leukemic origin of the transduced cells was confirmed in a patient with a chromosomal translocation t(9:11) by fluorescence in situ hybridization analysis. Cytokine modified-cells consistently secreted IL-7 (mean, 415 pg +/- 190/10(6) cells/48 hours; n = 5). We demonstrate that IL-7-transduced cells are included in the differentiated leukemic DC subset, and, as shown in a particular case, that about half of the mature CD80(+) and CD83(+) populations coexpress the LNGFr transgene. In addition, IL-7-modified leukemic cells induce stronger allo-T-cell stimulation and higher amounts of IL-2 production in T cells compared with control groups. Finally, cytokine-transduced leukemic DCs can effectively prime and generate cytotoxic T lymphocytes against autologous leukemic blasts.

Induction of specific immune responses by polycation-based vaccines.

The s.c injection of tumor Ag-derived, MHC class I-binding peptides together with cationic poly-amino acids (e.g., poly-L-arginine; pR) has been shown to protect animals against a challenge with tumor cells expressing the respective peptide(s). Given our only restricted knowledge about immunogenic tumor-associated peptides, we sought to determine whether this pR-based vaccination protocol would also induce protective cancer immunity if large proteins were used instead of peptide epitopes. We found that the intracutaneous administration of the model Ag beta-galactosidase (beta-gal) together with pR (referred to as pR-based protein vaccine; pR-PV) was significantly more potent in protecting mice against the growth of beta-gal-expressing RENCA cells than the protein alone. Coadministration of pR enhanced both the beta-gal-induced specific humoral and CD8 response. The protective effect required CD8(+), but neither CD4(+) T lymphocytes nor beta-gal-specific Abs. beta-Gal priming of protective CD8(+) T lymphocytes was found to be CD4(+) T cell-independent, to take place within the draining lymph nodes, and to be accomplished by day 5 after vaccination. Ablation of the injection sites as early as 1.5 h after pR-PV administration still led to protection in a large proportion of the animals, indicating that certain protein Ags administered intradermally in the context of polycations are quickly transported to the draining nodes, where they induce molecular and cellular events resulting in the helper-independent priming and expansion of Tc1 cells. However, optimal protection required the prolonged presence of the injection site, suggesting that pR-PV injection facilitates the formation of a cutaneous depot of Ag-charged cells capable of migration and T cell activation.

Poly-L-arginine synergizes with oligodeoxynucleotides containing CpG-motifs (CpG-ODN) for enhanced and prolonged immune responses and prevents the CpG-ODN-induced systemic release of pro-inflammatory cytokines.

This study describes an entirely synthetic vaccine composed of antigenic peptides (T cell epitopes), oligodeoxynucleotides containing CpG-motifs (CpG-ODN) and poly-L-arginine (pR). CpG-ODN are known to be potent inducers of predominantly type 1-like immune responses, while polycationic amino acids, like pR, facilitate the uptake of antigens into antigen presenting cells (APCs). We demonstrate that the application of peptides and pR/CpG-ODN results in strongly enhanced peptide-specific immune responses as compared to the application of peptides with either of the immunomodulators alone. High numbers of antigen-specific T cells can be observed even after only one injection of the vaccine for a remarkably long period of time (at least 372 days). Furthermore, the potentially harmful systemic release of pro-inflammatory cytokines induced upon injection of CpG-ODN is inhibited. Thus, the combined application of CpG-ODN and pR may represent a novel vaccine strategy in humans.

Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus.

For the design of potent subunit vaccines, it is of paramount importance to identify all antigens immunologically recognized by a patient population infected with a pathogen. We have developed a rapid and efficient procedure to identify such commonly recognized antigens, and here we provide a comprehensive in vivo antigenic profile of Staphylococcus aureus, an important human pathogen. S. aureus peptides were displayed on the surface of Escherichia coli via fusion to one of two outer membrane proteins (LamB and FhuA) and probed with sera selected for high Ab titer and opsonic activity. A total of 60 antigenic proteins were identified, most of which are located or predicted to be located on the surface of the bacterium or secreted. The identification of these antigens and their reactivity with individual sera from patients and healthy individuals greatly facilitate the selection of promising vaccine candidates for further evaluation. This approach, which makes use of whole genome sequence information, has the potential to greatly accelerate and facilitate the formulation of novel vaccines and is applicable to any pathogen that induces Abs in humans and/or experimental animals.

Vaccination with poly-L-arginine as immunostimulant for peptide vaccines: induction of potent and long-lasting T-cell responses against cancer antigens.

Vaccines that induce high numbers of sustained T cell responses are urgently needed for the treatment of numerous diseases including cancer. Antigen-presenting cells (APCs), the most important of which are dendritic cells, orchestrate antigen-dependent T cell responses in that they present antigens to T cells in an appropriate environment. Here we present evidence that after vaccination with a simple mixture of the cationic poly-amino acid poly-L-arginine and tumor antigen-derived peptide antigens, large numbers of antigen-specific T cells are induced and APCs mediate the generation of T lymphocytes. We observe that after s.c. injection, MHC class II(+) cells infiltrate injection sites and are loaded with large amounts of antigen in vivo under the influence of poly-L-arginine. Consequently, numerous antigen-charged APCs can be detected in draining lymph nodes of vaccinated animals. Antigen-specific T cell responses induced are systemic and were readily detected more than 4 months after the last vaccination, the latest time point we measured. By contrast, even after repeat injections, we were consistently unable to detect antibody responses against poly-L-arginine, allowing this compound to be used for numerous booster injections. Clinical trials in cancer patients using poly-L-arginine as immunostimulant will be carried out in the near future.