The serine repeat antigen (SERA) gene family phylogeny in Plasmodium: the impact of GC content and reconciliation of gene and species trees.

Plasmodium falciparum is the parasite responsible for the most acute form of malaria in humans. Recently, the serine repeat antigen (SERA) in P. falciparum has attracted attention as a potential vaccine and drug target, and it has been shown to be a member of a large gene family. To clarify the relationships among the numerous P. falciparum SERAs and to identify orthologs to SERA5 and SERA6 in Plasmodium species affecting rodents, gene trees were inferred from nucleotide and amino acid sequence data for 33 putative SERA homologs in seven different species. (A distance method for nucleotide sequences that is specifically designed to accommodate differing GC content yielded results that were largely compatible with the amino acid tree. Standard-distance and maximum-likelihood methods for nucleotide sequences, on the other hand, yielded gene trees that differed in important respects.) To infer the pattern of duplication, speciation, and gene loss events in the SERA gene family history, the resulting gene trees were then "reconciled" with two competing Plasmodium species tree topologies that have been identified by previous phylogenetic studies. Parsimony of reconciliation was used as a criterion for selecting a gene tree/species tree pair and provided (1) support for one of the two species trees and for the core topology of the amino acid-derived gene tree, (2) a basis for critiquing fine detail in a poorly resolved region of the gene tree, (3) a set of predicted "missing genes" in some species, (4) clarification of the relationship among the P. falciparum SERA, and (5) some information about SERA5 and SERA6 orthologs in the rodent malaria parasites. Parsimony of reconciliation and a second criterion--implied mutational pattern at two key active sites in the SERA proteins-were also seen to be useful supplements to standard "bootstrap" analysis for inferred topologies.

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity.

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.

IL-12 controls cytotoxicity of a novel subset of self-antigen-specific human CD28+ cytolytic T cells.

Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28(-)) T cells strongly expressing granzyme/perforin, and two EM28(+) subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28(-)-derived clones lysed target cells with high efficacy. In contrast, EM28(+)-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28(+) conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.

From the Cover: Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination.

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

Biomedical nanoparticles modulate specific CD4(+) T cell stimulation by inhibition of antigen processing in dendritic cells

Understanding how nanoparticles may affect immune responses is an essential prerequisite to developing novel clinical applications. To investigate nanoparticle-dependent outcomes on immune responses, dendritic cells (DCs) were treated with model biomedical poly(vinylalcohol)-coated super-paramagnetic iron oxide nanoparticles (PVA-SPIONs). PVA-SPIONs uptake by human monocyte-derived DCs (MDDCs) was analyzed by flow cytometry (FACS) and advanced imaging techniques. Viability, activation, function, and stimulatory capacity of MDDCs were assessed by FACS and an in vitro CD4(+) T cell assay. PVA-SPION uptake was dose-dependent, decreased by lipopolysaccharide (LPS)-induced MDDC maturation at higher particle concentrations, and was inhibited by cytochalasin D pre-treatment. PVA-SPIONs did not alter surface marker expression (CD80, CD83, CD86, myeloid/plasmacytoid DC markers) or antigen-uptake, but decreased the capacity of MDDCs to process antigen, stimulate CD4(+) T cells, and induce cytokines. The decreased antigen processing and CD4(+) T cell stimulation capability of MDDCs following PVA-SPION treatment suggests that MDDCs may revert to a more functionally immature state following particle exposure.

Lymphoid environment limits superantigen and antigen-induced T cell proliferation at high precursor frequency.

After superantigen challenge a significant proportion of superantigen-reactive T cells remain undivided. We provide evidence that the lymphoid environment limits T cell proliferation in the secondary lymphoid organs when the frequency of superantigen reactive T cells is unusually high. We monitored T cell proliferation and the percentage of undivided cells when the frequency of superantigen-reactive T cells was low (1%), intermediate (15%) or high (30-100%) by transferring fluorescently labeled cells into different recipients. When the frequency was low, practically all the reactive T cells entered cell cycle and proliferated maximally. At intermediate frequencies a large proportion of reactive T cells did not enter cell cycle and the whole population divided less. A further increase in reactive T cells did not alter the percentage of undivided cells but induced a further decrease in the number of cell divisions. Interestingly, the observations made with superantigens were confirmed with peptide antigen and TCR-transgenic mice. Moreover, in vivo and in vitro data suggest that dendritic cells are the most likely candidates in limiting T cell proliferation in the lymphoid environment. In conclusion, we show that the availability of APC in the lymphoid environment can quantitatively limit T cell priming.

In vitro attachment of glycosyl-inositolphospholipid anchor structures to mouse Thy-1 antigen and human decay-accelerating factor.

Glycosyl-inositolphospholipid (GPL) anchoring structures are incorporated into GPL-anchored proteins immediately posttranslationally in the rough endoplasmic reticulum, but the biochemical and cellular constituents involved in this "glypiation" process are unknown. To establish whether glypiation could be achieved in vitro, mRNAs generated by transcription of cDNAs encoding two GPL-anchored proteins, murine Thy-1 antigen and human decay-accelerating factor (DAF), and a conventionally anchored control protein, polymeric-immunoglobulin receptor (IgR), were translated in a rabbit reticulocyte lysate. Upon addition of dog pancreatic rough microsomes, nascent polypeptides generated from the three mRNAs translocated into vesicles. Dispersal of the vesicles with Triton X-114 detergent and incubation of the hydrophobic phase with phosphatidylinositol-specific phospholipases C and D, enzymes specific for GPL-anchor structures, released Thy-1 and DAF but not IgR protein into the aqueous phase. The selective incorporation of phospholipase-sensitive anchoring moieties into Thy-1 and DAF but not IgR translation products during in vitro translocation indicates that rough microsomes are able to support and regulate glypiation.

Circulating estrogens and progesterone during primiparous pregnancies and risk of maternal breast cancer.

Pregnancy reduces maternal risk of breast cancer in the long term, but the biological determinants of the protection are unknown. Animal experiments suggest that estrogens and progesterone could be involved, but direct human evidence is scant. A case-control study (536 cases and 1,049 controls) was nested within the Finnish Maternity Cohort. Eligible were primiparous women who delivered at term a singleton offspring before age 40. For each case, two individually matched controls by age (±6 months) and date of sampling (±3 months) were selected. Estradiol, estrone and progesterone in first-trimester serum were measured by high-performance liquid chromatography tandem mass spectrometry and sex-hormone binding globulin (SHBG) by immunoassay. Odds ratios (OR) and 95% confidence intervals (CI) were estimated through conditional logistic regression. In the whole study population there was no association of breast cancer with any of the studied hormones. In analyses stratified by age at diagnosis, however, estradiol concentrations were positively associated with risk of breast cancer before age 40 (upper quartile OR, 1.81; CI, 1.08-3.06), but inversely associated with risk in women who were diagnosed ≥age 40 (upper quartile OR, 0.64; CI, 0.40-1.04), p(interaction) 0.004. Risk estimates for estrone mirrored those for estradiol but were less pronounced. Progesterone was not associated with risk of subsequent breast cancer. Our results provide initial evidence that concentrations of estrogens during the early parts of a primiparous pregnancy are associated with maternal risk of breast cancer and suggest that the effect may differ for tumors diagnosed before and after age 40.

Trachea, lung, and tracheobronchial lymph nodes are the major sites where antigen-presenting cells are detected after nasal vaccination of mice with human papillomavirus type 16 virus-like particles.

Vaccination by the nasal route has been successfully used for the induction of immune responses. Either the nasal-associated lymphoid tissue (NALT), the bronchus-associated lymphoid tissue, or lung dendritic cells have been mainly involved. Following nasal vaccination of mice with human papillomavirus type 16 (HPV16) virus-like-particles (VLPs), we have previously shown that interaction of the antigen with the lower respiratory tract was necessary to induce high titers of neutralizing antibodies in genital secretions. However, following a parenteral priming, nasal vaccination with HPV16 VLPs did not require interaction with the lung to induce a mucosal immune response. To evaluate the contribution of the upper and lower respiratory tissues and associated lymph nodes (LN) in the induction of humoral responses against HPV16 VLPs after nasal vaccination, we localized the immune inductive sites and identified the antigen-presenting cells involved using a specific CD4(+) T-cell hybridoma. Our results show that the trachea, the lung, and the tracheobronchial LN were the major sites responsible for the induction of the immune response against HPV16 VLP, while the NALT only played a minor role. Altogether, our data suggest that vaccination strategies aiming to induce efficient immune responses against HPV16 VLP in the female genital tract should target the lower respiratory tract.

MicroRNA Profile of Circulating CD4-positive Regulatory T Cells in Human Adults and Impact of Differentially Expressed MicroRNAs on Expression of Two Genes Essential to Their Function.

Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4(+) Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3'-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3'-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4(+) adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3'-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4(+) Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.

Long circulating half-life and high tumor selectivity of the photosensitizer meta-tetrahydroxyphenylchlorin conjugated to polyethylene glycol in nude mice grafted with a human colon carcinoma.

In a mode of nude mice bearing a human colon carcinoma xenograft, the biodistribution and tumor localization of metatetrahydroxyphenylchlorin (m-THPC) coupled to polyethylene glycol (PEG) were compared with those of the free form of this photosensitizer used in photodynamic therapy (PDT). At different times after i.v. injection of both forms of 125I-labeled photosensitizer, m-THPC-PEG gave on average a 2-fold higher tumor uptake than free m-THPC. In addition, at early times after injection, m-THPC-PEG showed a 2-fold longer blood circulating half-life and a 4-fold lower liver uptake than free m-THPC. The tumor to normal tissue ratios of radioactivity concentrations were always higher for m-THPC-PEG than for free m-THPC at any time point studied from 2 to 96 hr post-injection. Significant coefficients of correlation between direct fluorescence measurements and radioactivity counting were obtained within each organ tested. Fluorescence microscopy studies showed that m-THPC-PEG was preferentially localized near the tumor vessels, whereas m-THPC was more diffusely distributed inside the tumor tissue. To verify whether m-THPC-PEG conjugate remained phototoxic in vivo, PDT experiments were performed 72 hr after injection and showed that m-THPC-PEG was as potent as free m-THPC in the induction of tumor regression provided that the irradiation does for m-THPC-PEG conjugate was adapted to a well-tolerated 2-fold higher level. The overall results demonstrate first the possibility of improving the in vivo tumor localization of a hydrophobic dye used for PDT by coupling it to PEG and second that a photosensitizer conjugated to a macromolecule can remain phototoxic in vivo.

Marked increase in the secretion of a fully antigenic recombinant carcinoembryonic antigen obtained by deletion of its hydrophobic tail.

Carcinoembryonic antigen (CEA) is a well-known tumor marker, consisting of a single heavily glycosylated polypeptide chain (mol. wt 200 kD), bound to the cell surface by a phosphatidylinositol-glycan anchor. The hydrophobic domain, encoded by the 3' end of the open reading frame of the CEA gene is not present in the mature protein. This domain is assumed to play an important role in the targeting and attachment of CEA to the cell surface. To verify this hypothesis, a recombinant CEA cDNA lacking the 78 b.p. of the 3' region, encoding the 26 a.a. hydrophobic domain, was prepared in a Rc/CMV expression vector containing a neomycin resistance gene. The construct was transfected by the calcium phosphate technique into CEA-negative human and rat colon carcinoma cell lines. Geneticin-resistant transfectants were screened for the presence of CEA in the supernatant and positive clones were isolated. As determined by ELISA, up to 13 micrograms of recombinant CEA per 10(6) cells was secreted within 72 hr by the human transfected cells and about 1 microgram by the rat cells. For comparison, two human carcinoma cell lines, CO112 and LS174T, selected for high CEA expression, shed about 45 and 128 ng per 10(6) cells within 72 hr, respectively. Western blot analysis showed that the size of the recombinant CEA secreted by the transfected human cells is identical to that of reference CEA purified from human colon carcinomas metastases (about 200 kD). The recombinant CEA synthesized by the transfected rat carcinoma cells has a smaller size (about 144 kD, possibly due to incomplete glycosylation), as has already been observed for CEA produced by rat colon carcinoma cells transfected with full-length CEA cDNA. The 100-fold increase in secretion of rCEA encoded by truncated CEA cDNA transfected in human cells confirms the essential role of this domain in the targeting and anchoring of the glycoprotein. These results suggest a new approach for the in vitro production of large amounts of CEA needed in research laboratories and for immunoassay kits.

Extended co-expression of inhibitory receptors by human CD8 T-cells depending on differentiation, antigen-specificity and anatomical localization.

Inhibitory receptors mediate CD8 T-cell hyporesponsiveness against cancer and infectious diseases. PD-1 and CTLA-4 have been extensively studied, and blocking antibodies have already shown clinical benefit for cancer patients. Only little is known on extended co-expression of inhibitory receptors and their ligands. Here we analyzed the expression of eight inhibitory receptors by tumor-antigen specific CD8 T-cells. We found that the majority of effector T-cells simultaneously expressed four or more of the inhibitory receptors BTLA, TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1 and CTLA-4. There were major differences depending on antigen-specificity, differentiation and anatomical localization of T-cells. On the other hand, naive T-cells were only single or double positive for BTLA and TIM-3. Extended co-expression is likely relevant for effector T-cells, as we found expression of multiple ligands in metastatic lesions of melanoma patients. Together, our data suggest that naive T-cells are primarily regulated by BTLA and TIM-3, whereas effector cells interact via larger numbers of inhibitory receptors. Blocking multiple inhibitory receptors simultaneously or sequentially may improve T-cell based therapies, but further studies are necessary to clarify the role of each receptor-ligand pair.

Utilité et limites du dosage de l'antigène carcinoembryonnaire (CEA) dans la surveillance postopératoire de malades atteints de carcinomes du côlon et du rectu

An attempt is made to define the usefulness and limitations of carcinoembryonic antigen (CEA) radioimmunoassay for evaluation of tumor resection and detection of tumor relapse in patients with large-bowel carcinoma. In 45 patients for whom complete tumor resection was reported, all but 5 showed a drop in CEA to normal values after surgery. The 5 patients whose CEA did not fall to below 5 ng/ml showed a subsequent rise in CEA level and later were all found to have a tumor relapse. The results indicate that an incomplete drop in circulating CEA level one month after surgery is a bad prognostic sign. Twenty-two of these patients were followed up by repeated CEA radioimmunoassay for several months after surgery; 8 showed a progressive increase in CEA levels preceding clinical diagnosis of tumor relapse by 2-10 months. The clinical history of these 8 patients is briefly described. The results demonstrate that relapses of colon and rectum carcinoma can be detected by increased CEA levels months before the appearance of any clinical evidence.