Identification of secretory IgA, free secretory piece and serum IgA in the ovine and caprine species.

Caprine and ovine IgA were identified by cross-reaction with anti-human and anti-bovine IgA sera in colostrum, mature milk, saliva, urine and serum. Secretory component (SC) was shown in the free form and associated with polymeric serum IgA in secretions. Mean molecular weights were determined for the IgA and the free secretory components. The high IgA content of saliva suggested that it was a major secretory immunoglobulin in these species. Traces of secretory IgA were also found in normal sera but most of the serum IgA had no secretory determinant. Secretory IgA, serum IgA and free secretory component were purified. Levels of the sheep and goat immunoglobulins were measured in various fluids.

Therapeutic cancer vaccines based on molecularly defined human tumor antigens.

The results of numerous phases I and II clinical trials testing the safety and immunogenicity of various cancer vaccine formulations based on cytolytic T lymphocytes (CTLs)-defined tumor antigens have been reported recently. Specific T cell responses can be detected in only a fraction of immunized patients. A smaller but significant fraction of these patients have objective tumor responses. Efficient therapeutic vaccination should aim at boosting naturally occurring anti-tumor responses and at sustaining a large contingent of tumor antigen-specific and fully functional effector T cells at tumor sites.

Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

Human Prominin-1 (CD133) Is Detected in Both Neoplastic and Non-Neoplastic Salivary Gland Diseases and Released into Saliva in a Ubiquitinated Form.

Prominin-1 (CD133) is physiologically expressed at the apical membranes of secretory (serous and mucous) and duct cells of major salivary glands. We investigated its expression in various human salivary gland lesions using two distinct anti-prominin-1 monoclonal antibodies (80B258 and AC133) applied on paraffin-embedded sections and characterized its occurrence in saliva. The 80B258 epitope was extensively expressed in adenoid cystic carcinoma, in lesser extent in acinic cell carcinoma and pleomorphic adenoma, and rarely in mucoepidermoid carcinoma. The 80B258 immunoreactivity was predominately detected at the apical membrane of tumor cells showing acinar or intercalated duct cell differentiation, which lined duct- or cyst-like structures, and in luminal secretions. It was observed on the whole cell membrane in non-luminal structures present in the vicinity of thin-walled blood vessels and hemorrhagic areas in adenoid cystic carcinoma. Of note, AC133 labeled only a subset of 80B258-positive structures. In peritumoral salivary gland tissues as well as in obstructive sialadenitis, an up-regulation of prominin-1 (both 80B258 and AC133 immunoreactivities) was observed in intercalated duct cells. In most tissues, prominin-1 was partially co-expressed with two cancer markers: carcinoembryonic antigen (CEA) and mucin-1 (MUC1). Differential centrifugation of saliva followed by immunoblotting indicated that all three markers were released in association with small membrane vesicles. Immuno-isolated prominin-1-positive vesicles contained CEA and MUC1, but also exosome-related proteins CD63, flotillin-1, flotillin-2 and the adaptor protein syntenin-1. The latter protein was shown to interact with prominin-1 as demonstrated by its co-immunoisolation. A fraction of saliva-associated prominin-1 appeared to be ubiquitinated. Collectively, our findings bring new insights into the biochemistry and trafficking of prominin-1 as well as its immunohistochemical profile in certain types of salivary gland tumors and inflammatory diseases.

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.

Expression hierarchy of T cell epitopes from melanoma differentiation antigens: unexpected high level presentation of tyrosinase-HLA-A2 Complexes revealed by peptide-specific, MHC-restricted, TCR-like antibodies.

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

Deletion of human GP1BB and SEPT5 is associated with Bernard-Soulier syndrome, platelet secretion defect, polymicrogyria, and developmental delay.

The bleeding disorder Bernard-Soulier syndrome (BSS) is caused by mutations in the genes coding for the platelet glycoprotein GPIb/IX receptor. The septin SEPT5 is important for active membrane movement such as vesicle trafficking and exocytosis in non-dividing cells (i.e. platelets, neurons). We report on a four-year-old boy with a homozygous deletion comprising not only glycoprotein Ibβ (GP1BB) but also the SEPT5 gene, located 5' to GP1BB. He presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect. The homozygous deletion of GP1BB and SEPT5, which had been identified by PCR analyses, was confirmed by Southern analyses and denaturing HPLC (DHPLC). The parents were heterozygous for this deletion. Absence of GPIbβ and SEPT5 proteins in the patient's platelets was illustrated using transmission electron microscopy. Besides decreased GPIb/IX expression, flow cytometry analyses revealed impaired platelet granule secretion. Because the bleeding disorder was extremely severe, the boy received bone marrow transplantation (BMT) from a HLA-identical unrelated donor. After successful engraftment of BMT, he had no more bleeding episodes. Interestingly, also his mental development improved strikingly after BMT. This report describes for the first time a patient with SEPT5 deficiency presenting with cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect.

IgA with "secretory piece" in bovine colostrum and saliva.

We have shown that in bovine colostrum and saliva there is a secretory IgA with a sedimentation, coefficient of 11S and a secretory piece, previously unknown and comparable with the 11S IgA described in human and rabbit external secretions. These secretory IgA contain molecules of 7S IgA with an additional protein segment called the transport or secretory piece1-5. Furthermore, the free form of the secretory piece is identified in bovine colostrum and also in mature milk which contains very little IgA.

A novel population of human melanoma-specific CD8 T cells recognizes Melan-AMART-1 immunodominant nonapeptide but not the corresponding decapeptide.

HLA-A2-restricted cytolytic T cells specific for the immunodominant human tumor Ag Melan-A(MART-1) can kill most HLA-matched melanoma cells, through recognition of two naturally occurring antigenic variants, i.e., Melan-A nonamer AAGIGILTV and decamer EAAGIGILTV peptides. Several previous studies have suggested a high degree of TCR cross-reactivity to the two peptides. In this study, we describe for the first time that some T cell clones are exclusively nonamer specific, because they are not labeled by A2/decamer-tetramers and do not recognize the decamer when presented endogenously. Functional assays with peptides gave misleading results, possibly because decamers were cleaved by exopeptidases. Interestingly, nonapeptide-specific T cell clones were rarely Valpha2.1 positive (only 1 of 19 clones), in contrast to the known strong bias for Valpha2.1-positive TCRs found in decamer-specific clones (59 of 69 clones). Molecular modeling revealed that nonapeptide-specific TCRs formed unfavorable interactions with the decapeptide, whereas decapeptide-specific TCRs productively created a hydrogen bond between CDR1alpha and glutamic acid (E) of the decapeptide. Ex vivo analysis of T cells from melanoma metastases demonstrated that both nonamer and decamer-specific T cells were enriched to substantial frequencies in vivo, and representative clones showed efficient tumor cell recognition and killing. We conclude that the two peptides should be regarded as distinct epitopes when analyzing tumor immunity and developing immunotherapy against melanoma.

Tumor-host interactions Part 1: Stromal signatures of breast and prostate cancer Part 2: GLG1 and the control of the secretory pathway in tumor cells

Tumor-host interaction is a key determinant during cancer progression, from primary tumor growth to metastatic dissemination. At each step, tumor cells have to adapt to and subvert different types of microenvironment, leading to major phenotypic and genotypic alterations that affect both tumor and surrounding stromal compartments. Understanding the molecular mechanisms that govern tumor-host interplay may be essential for better comprehension of tumorigenesis in an effort to improve current anti-cancer therapies. The present work is composed of two projects that address tumor-host interactions from two different perspectives, the first focusing on the characterization of tumor-associated stroma and the second on membrane trafficking in tumor cells. Part 1. To selectively address stromal gene expression changes during cancer progression, oligonucleotide-based Affymetrix microarray technology was used to analyze the transcriptomes of laser-microdissected stromal cells derived from invasive human breast and prostate carcinoma. Comparison showed that invasive breast and prostate cancer elicit distinct, tumor-specific stromal responses, with a limited panel of shared induced and/or repressed genes. Both breast and prostate tumor-specific deregulated stromal gene sets displayed statistically significant survival-predictive ability for their respective tumor type. By contrast, a stromal gene signature common to both tumor types did not display prognostic value, although expression of two individual genes within this common signature was found to be associated with patient survival. Part 2. GLG1 is known as an E-selectin ligand and an intracellular FGF receptor, depending on cell type and context. Immunohistochemical and immunofluorescence analyses showed that GLG1 is primarily localized in the Golgi of human tumor cells, a central location in the biosynthetic/secretory pathways. GLG1 has been shown to interact with and to recruit the ARF GEF BIGI to the Golgi membrane. Depletion of GLG1 or BIGI markedly reduced ARF3 membrane localization and activation, and altered the Golgi structure. Interestingly, these perturbations did not impair constitutive secretion in general, but rather seemed to impair secretion of a specific subset of proteins that includes MMP-9. Thus, GLG1 coordinates ARF3 activation by recruiting BIGI to the Golgi membrane, thereby affecting secretion of specific molecules. - Les interactions tumeur-hôte constituent un élément essentiel à la progression tumorale, de la croissance de la tumeur primaire à la dissémination des métastases. A chaque étape, les cellules tumorales doivent s'adapter à différents types de microenvironnement et les détourner à leur propre avantage, donnant lieu à des altérations phénotypiques et génotypiques majeures qui affectent aussi bien la tumeur elle-même que le compartiment stromal environnant. L'étude des mécanismes moléculaires qui régissent les interactions tumeur-hôte constitue une étape essentielle pour une meilleure compréhension du processus de tumorigenèse dans le but d'améliorer les thérapies anti cancer existantes. Le travail présenté ici est composé de deux projets qui abordent la problématique des interactions tumeur-hôte selon différentes perspectives, le premier se concentrant sur la caractérisation du stroma tumoral et le second sur le trafic intracellulaire des cellules tumorales. Partie 1. Pour examiner les changements d'expression des gènes dans le stroma en réponse à la progression du cancer, des puces à ADN Affymetrix ont été utilisées afin d'analyser les transcriptomes des cellules stromales issues de carcinomes invasifs du sein et de la prostate et collectées par microdissection au laser. L'analyse comparative a montré que les cancers invasifs du sein et de la prostate provoquent des réponses stromales spécifiques à chaque type de tumeur, et présentent peu de gènes induits ou réprimés de façon similaire. L'ensemble des gènes dérégulés dans le stroma associé au cancer du sein, ou à celui de la prostate, présente une valeur pronostique pour les patients atteints d'un cancer du sein, respectivement de la prostate. En revanche, la signature stromale commune aux deux types de cancer n'a aucune valeur prédictive, malgré le fait que l'expression de deux gènes présents dans cette liste soit liée à la survie des patients. Partie 2. GLG1 est connu comme un ligand des sélectines E ainsi que comme récepteur intracellulaire pour des facteurs de croissances FGFs selon le type de cellule dans lequel il est exprimé. Des analyses immunohistochimiques et d'immunofluorescence ont montré que dans les cellules tumorales, GLG1 est principalement localisé au niveau de l'appareil de Golgi, une place centrale dans la voie biosynthétique et sécrétoire. Nous avons montré que GLG1 interagit avec la protéine BIGI et participe à son recrutement à la membrane du Golgi. L'absence de GLG1 ou de BIGI réduit drastiquement le pool d'ARF3 associé aux membranes ainsi que la quantité d'ARF3 activés, et modifie la structure de l'appareil de Golgi. Il est particulièrement intéressant de constater que ces perturbations n'ont pas d'effet sur la sécrétion constitutive en général, mais semblent plutôt affecter la sécrétion spécifique d'un sous-groupe défini de protéines comprenant MMP-9. GLG1 coordonne donc l'activation de ARF3 en recrutant BIGI à la membrane du Golgi, agissant par ce moyen sur la sécrétion de molécules spécifiques.

N-Glycans on secretory component: mediators of the interaction between secretory IgA and gram-positive commensals sustaining intestinal homeostasis.

Human beings live in symbiosis with billions of microorganisms colonizing mucosal surfaces. The understanding of the mechanisms underlying this fine-tuned intestinal balance has made significant processes during the last decades. We have recently demonstrated that the interaction of SIgA with Gram-positive bacteria is essentially based on Fab-independent, glycan-mediated recognition. Results obtained using mouse hybridoma- and colostrum-derived secretory IgA (SIgA) consistently show that N-glycans present on secretory component (SC) play a crucial role in the process. Natural coating may involve specific Gram-positive cell wall components, which may explain selective recognition at the molecular level. More widely, the existence of these complexes is involved in the modulation of intestinal epithelial cell (IEC) responses in vitro and the formation of intestinal biofilms. Thus, SIgA may act as one of the pillars in homeostatic maintenance of the microbiota in the gut, adding yet another facet to its multiple roles in the mucosal environment.

The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis.

Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.

Secretory IgA, a major immunoglobulin in most bovine external secretions.

Bovine secretory IgA (SIgA), recently identified in colostrum, was shown to be homologous to human SIgA by immunologic cross-reaction. A quantitative study indicated that bovine SIgA, a minor component of colostrum, is a major immunoglobulin in most other external secretions including saliva, spermatic fluid, lacrimal, nasal and gastrointestinal secretions. SIgA was isolated from saliva. The free form of secretory component was found to be abundant in milk. A normal lactating cow produces about 1.2 g of this protein per day. Two forms of IgA were identified in serum: a normal serum IgA with no secretory antigenic determinant, and a small amount of SIgA. In vitro synthesis of SIgA by the salivary gland was studied by tissue cultures with incorporation of labeled amino acids.

Ex vivo detectable human CD8 T-cell responses to cancer-testis antigens.

Clinical trials have shown that strong tumor antigen-specific CD8 T-cell responses are difficult to induce but can be achieved for T-cells specific for melanoma differentiation antigens, upon repetitive vaccination with stable emulsions prepared with synthetic peptides and incomplete Freund's adjuvant. Here, we show in four melanoma patients that ex vivo detectable T-cells and thus strong T-cell responses can also be induced against the more universal cancer-testis antigens NY-ESO-1 and Mage-A10. Interestingly, all patients had ex vivo detectable T-cell responses against multiple antigens after serial vaccinations with three peptides emulsified in incomplete Freund's adjuvant. Antigen-specific T-cells displayed an activated phenotype and secreted IFNgamma. The robust immune responses provide a solid basis for further development of human T-cell vaccination.