Monitoring of vaccine-specific gamma interferon induction in genital mucosa of mice by real-time reverse transcription-PCR.

Monitoring of T-cell responses in genital mucosa has remained a major challenge because of the absence of lymphoid aggregates and the low abundance of T cells. Here we have adapted to genital tissue a sensitive real-time reverse transcription-PCR (TaqMan) method to measure induction of gamma interferon (IFN-gamma) mRNA transcription after 3 h of antigen-specific activation of CD8 T cells. For this purpose, we vaccinated C57BL/6 mice subcutaneously with human papillomavirus type 16 L1 virus-like particles and monitored the induction of CD8 T cells specific to the L1(165-173) H-2D(b)-restricted epitope. Comparison of the responses induced in peripheral blood mononuclear cells and lymph nodes (LN) by L1-specific IFN-gamma enzyme-linked immunospot assay and TaqMan determination of the relative increase in L1-specific IFN-gamma mRNA induction normalized to the content of CD8b mRNA showed a significant correlation, despite the difference in the readouts. Most of the cervicovaginal tissues could be analyzed by the TaqMan method if normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA was used and a significant L1-specific IFN-gamma induction was found in one-third of the immunized mice. This local response did not correlate with the immune responses measured in the periphery, with the exception of the sacral LN, an LN draining the genital mucosa, where a significant correlation was found. Our data show that the TaqMan method is sensitive enough to detect antigen-specific CD8 T-cell responses in the genital mucosa of individual mice, and this may contribute to elaborate effective vaccines against genital pathogens.

A novel derivative of the chelon desferrioxamine for site-specific conjugation to antibodies.

We describe the preparation of the modified chelator aminooxyacetyl-ferrioxamine, and the replacement of its iron atom by 67Ga at high specific activity. The aminooxy function of this compound was allowed to react with the aldehyde groups generated by the periodate oxidation of the oligosaccharide of a mouse IgG1 monoclonal antibody (MAb) directed against carcino-embryonic antigen (CEA). The use of the aminooxy group allowed a stable bond to be formed between the chelon and the antibody with no need for reduction. Iron was removed from the ferrioxamine moiety and replaced by 67Ga either before or after conjugation of the chelon to the antibody. In either case the labelled antibody was injected into nude mice bearing a human colon carcinoma having the appropriate antigenicity. Unoxidized antibody, labelled with 125I by conventional methods, was co-injected as an internal control. Additional control experiments were carried out with a non-immune IgG using the same 67Ga-labelled modified chelon as above. The in vivo distribution of the modified antibodies was evaluated at various times between 24 and 96 hr after injection. The methods used were gamma-camera imaging and, more quantitatively, gamma-counting of the various organs after dissection. Interestingly, with the metal-chelon-labelled antibody, the intensity and specificity of tumor labelling was comparable and in some cases superior to the results obtained with radio-iodinated antibody. In particular, there was almost no increase in liver and spleen uptake of radioactive metal relative to radio-iodine, contrary to what has been observed with most antibodies labelled with 111In after conjugation with DTPA.

Lassa virus entry in antigen presenting cells and evaluation of a novel nanoparticle vaccine platform against arena viruses

Arenaviruses are a large and diverse family of viruses that merit significant attention as causative agents of severe hemorrhagic fevers in humans. Lassa virus (LASV) in Africa and the South American hemorrhagic fever viruses Junin (JUNV), Machupo (MACV), and Guanarito (GTOV) have emerged as important human pathogens and represent serious public health problems in their respective endemic areas. A hallmark of fatal arenaviruses hemorrhagic fevers is a marked immunosuppression of the infected patients. Antigen presenting cells (APCs) such as macrophages and in particular dendritic cells (DCs) are early and preferred targets of arenaviruses infection. Instead of being recognized and presented as foreign antigens by DCs, arenaviruses subvert the normal mechanisms of pathogen recognition, invade DCs and establish a productive infection. Viral replication perturbs the DCs' ability to present antigens and to activate T and B cells, contributing to the marked virus-induced immunosuppression observed in fatal disease. Considering their crucial role in the development of an anti-viral immune response, the mechanisms by which arenaviruses, and in particular LASV, invade DCs are of particular interest. The C-type lectin DC-specific Intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) was recently identified as a potential entry receptor for LASV. The first project of my thesis focused therefore on the investigation of the role of DC-SIGN in LASV entry into primary human DCs. My data revealed that DC-SIGN serves as an attachment factor for LASV on human DCs and can facilitate capture of free virus and subsequent cell entry. However, in contrast to other emerging viruses, of the phlebovirus family, I found that DC-SIGN does likely not function as an authentic entry receptor for LASV. Moreover, I was able to show that LASV enters DCs via an unusually slow pathway that depends on actin, but is independent of clathrin and dynamin. Considering the lack of effective treatments and the limited public health infrastructure in endemic regions, the development of protective vaccines against arenaviruses is an urgent need. To address this issue, the second project of my thesis aimed at the development of a novel recombinant arenavirus vaccine based on a nanoparticle (NPs) platform and its evaluation in a small animal model. During the first phase of the project I designed, produced, and characterized suitable vaccine antigens. In the second phase of the project, I generated antigen-conjugated NPs, developed vaccine formulations, and tested the NPs for their ability to elicit anti-viral T cell responses as well as anti-viral antibodies. I demonstrated that the NPs platform is able to activate both cellular and humoral branches of the adaptive anti-viral immunity, providing proof-of-principle. In sum, my first project will allow, in a long term perspective, a better understanding of the viral pathogenesis and contribute to the development of novel antiviral strategies. The second project will expectidly offer a new treatment option against arenaviruses.

Proliferation capacity and cytotoxic activity are mediated by functionally and phenotypically distinct virus-specific CD8 T cells defined by interleukin-7R{alpha} (CD127) and perforin expression.

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127(+) perforin(-)/CD127(-) perforin(+), and CD127(-)/perforin(-) CD8 T cells, respectively. CD127(-)/perforin(-) and CD127(-)/perforin(+) cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin(+)/IL-2(-) or perforin(-)/IL-2(+) cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127(+)/IL-2-secreting) and cytotoxic (perforin(+)) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.

Ex vivo detectable activation of Melan-A-specific T cells correlating with inflammatory skin reactions in melanoma patients vaccinated with peptides in IFA.

The purpose of this study was to test melanoma vaccines consisting of peptides and immunological adjuvants for optimal immunogenicity and to evaluate laboratory immune monitoring for in vivo relevance. Forty-nine HLA-A2 positive patients with Melan-A positive melanoma were repeatedly vaccinated with Melan-A peptide, with or without immune adjuvant AS02B (QS21 and MPL) or IFA. Peptide-specific CD8 T cells in PBLs were analyzed ex vivo using fluorescent HLA-A2/Melan-A multimers and IFN-gamma ELISPOT assays. The vaccines were well tolerated. In vivo expansion of Melan-A-specific CD8 T cells was observed in 13 patients (1/12 after vaccination with peptide in AS02B and 12/17 after vaccination with peptide in IFA). The T cells produced IFN-gamma and downregulated CD45RA and CD28. T-cell responses correlated with inflammatory skin reactions at vaccine injection sites (P < 0.001) and with DTH reaction to Melan-A peptide (P < 0.01). Twenty-six of 32 evaluable patients showed progressive disease, whereas 4 patients had stable disease. The two patients with the strongest Melan-A-specific T-cell responses experienced regression of metastases in skin, lymph nodes, and lung. We conclude that repeated vaccination with Melan-A peptide in IFA frequently leads to sustained responses of specific CD8 T cells that are detectable ex vivo and correlate with inflammatory skin reactions.

Deciphering the unusual HLA-A2/Melan-A/MART-1-specific TCR repertoire in humans.

The Melan-A/MART-1(26-35) antigenic peptide is one of the best studied human tumor-associated antigens. It is expressed in healthy melanocytes and malignant melanoma and is recognized by CD8(+) T cells in the context of the MHC class I molecule HLA-A*0201. While an unusually large repertoire of CD8(+) T cells specific for this antigen has been documented, the reasons for its generation have remained elusive. In this issue of the European Journal of Immunology, Pinto et al. [Eur. J. Immunol. 2014. 44: 2811-2821] uncover one important mechanism by comparing the thymic expression of the Melan-A gene to that in the melanocyte lineage. This study shows that medullary thymic epithelial cells (mTECs) dominantly express a truncated Melan-A transcript, the product of misinitiation of transcription. Consequently, the protein product in mTECs lacks the immunodominant epitope spanning residues 26-35, thus precluding central tolerance to this antigen. In contrast, melanocytes and melanoma tumor cells express almost exclusively the full-length Melan-A transcript, thus providing the target antigen for efficient recognition by HLA-A2-restricted CD8(+) T cells. The frequency of these alternative gene transcription modes may be more common than previously appreciated and may represent an important factor modulating the efficiency of central tolerance induction in the thymus.

Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T-cell activation in vivo.

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c(high) DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44(+) CD62(-)) CD4(+) and CD8(+) T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC(+) DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0.95% versus 0.47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.

Distinct profiles of cytotoxic granules in memory CD8 T cells correlate with function, differentiation stage, and antigen exposure.

Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules. Degranulation activity and cytotoxic granules (perforin plus granzymes) generally define CD8 T cells with cytotoxic function. In this study, we have investigated the expression of granzyme K (GrmK) in comparison to that of GrmA, GrmB, and perforin. The expression of the cytotoxic granules was assessed in virus-specific CD8 T cells specific to influenza virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), or human immunodeficiency virus type 1 (HIV-1). We observed a dichotomy between GrmK and perforin expression in virus-specific CD8 T cells. The profile in influenza virus-specific CD8 T cells was perforin(-) GrmB(-) GrmA(+/-) GrmK(+); in CMV-specific cells, it was perforin(+) GrmB(+) GrmA(+) GrmK(-/+); and in EBV- and HIV-1-specific cells, it was perforin(-/+) GrmB(+) GrmA(+) GrmK(+). On the basis of the delineation of memory and effector CD8 T cells with CD45RA and CD127, the GrmK(+) profile was associated with early-stage memory CD8 T-cell differentiation, the perforin(+) GrmB(+) GrmA(+) profile with advanced-stage differentiation, and the GrmB(+) GrmA(+) Grmk(+) profile with intermediate-stage differentiation. Furthermore, perforin and GrmB but not GrmA and GrmK correlated with cytotoxic activity. Finally, changes in antigen exposure in vitro and in vivo during primary HIV-1 infection and vaccination modulated cytotoxic granule profiles. These results advance our understanding of the relationship between distinct profiles of cytotoxic granules in memory CD8 T cells and function, differentiation stage, and antigen exposure.

Conditioned polarized Caco-2 cell monolayers allow to discriminate for the ability of gut-derived microorganisms to modulate permeability and antigen-induced basophil degranulation.

BACKGROUND: Food allergy is a common allergic disorder--especially in early childhood. The avoidance of the allergenic food is the only available method to prevent further reactions in sensitized patients. A better understanding of the immunologic mechanisms involved in this reaction would help to develop therapeutic approaches applicable to the prevention of food allergy. OBJECTIVE: To establish a multi-cell in vitro model of sensitized intestinal epithelium that mimics the intestinal epithelial barrier to study the capacity of probiotic microorganisms to modulate permeability, translocation and immunoreactivity of ovalbumin (OVA) used as a model antigen. METHODS: Polarized Caco-2 cell monolayers were conditioned by basolateral basophils and used to examine apical to basolateral transport of OVA by ELISA. Activation of basophils with translocated OVA was measured by beta-hexosaminidase release assay. This experimental setting was used to assess how microorganisms added apically affected these parameters. Basolateral secretion of cytokine/chemokines by polarized Caco-2 cell monolayers was analysed by ELISA. RESULTS: Basophils loaded with OVA-specific IgE responded to OVA in a dose-dependent manner. OVA transported across polarized Caco-2 cell monolayers was found to trigger basolateral basophil activation. Microorganisms including lactobacilli and Escherichia coli increased transepithelial electrical resistance while promoting OVA passage capable to trigger basophil activation. Non-inflammatory levels of IL-8 and thymic stromal lymphopoietin were produced basolaterally by Caco-2 cells exposed to microorganisms. CONCLUSION: The complex model designed in here is adequate to learn about the consequence of the interaction between microorganisms and epithelial cells vis-a-vis the barrier function and antigen translocation, two parameters essential to mucosal homeostasis. It can further serve as a direct tool to search for microorganisms with anti-allergic and anti-inflammatory properties.

High frequencies of functionally impaired cytokeratin 18-specific CD8+ T cells in healthy HLA-A2+ donors.

Combining cell surface phenotyping with functional analysis, human CD8+ T cells have been divided into several subsets which are being studied extensively in diverse physiological situations, such as viral infection, cancer and ageing. In particular, so-called terminally differentiated effector cells possess a CD45RA+ CCR7- CD27- CD28- phenotype, contain perforin and, in different models, have been shown to exert direct ex vivo killing and to release interleukins upon both antigen-nonspecific and -specific stimulation. Using HLA class I multimers, we have identified a high frequency of peripheral CD8+ T cells that recognize a peptide derived from the self protein cytokeratin 18 presented by the HLA-A*0201 molecule. These cells can be detected in approximately 15% of the HLA-A2-positive healthy donors tested. A detailed analysis revealed that they must have divided extensively in vivo, have an effector cell phenotype and express various natural killer cell-associated receptors. Interestingly, however, they remained unresponsive to antigen-specific stimulation in vitro in terms of cytotoxicity and cytokine secretion. Thus, cytokeratin 18-specific cells constitute a frequently encountered, new CD8+ T lymphocyte subpopulation without classical effector status and with so far unknown function.

High antigen concentration inhibits T cell proliferation but not interleukin 2 production: examination of limiting dilution microcultures and T cell clones.

The effect of high antigen dose on the activation of cytochrome c peptide-primed lymph node cells was determined in several strains of mice by a limiting dilution analysis. It was found that proliferation of cytochrome c peptide-specific T cells was completely inhibited at high antigen concentration in C57BL/6 but only partially in DBA mice and had no effect in SJL mice. Clones derived from DBA mice showed a differential capacity to be inhibited by high antigen dose. On the other hand, interleukin 2 production by these clones was not impaired regardless of the antigen concentrations used.

Carcinoembryonic antigen (CEA): demonstration of a partial identity between CEA and a normal glycoprotein.

The carcinoembryonic antigen of the human digestive tract (CEA), described by Gold as a glycoprotein specific for digestive carcinomatous and foetal tissues, was found to have common antigenic determinants with a glycoprotein of smaller size extracted from normal adult tissues. This observation suggests that only a part of the CEA molecule carries the onco-foetal specificity. It also has practical implications regarding the radioimmunoassay for CEA used for the diagnosis of certain carcinomas.

Identification of proteoglycans as the APRIL-specific binding partners.

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

Leishmania major-specific B cells are necessary for Th2 cell development and susceptibility to L. major LV39 in BALB/c mice.

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (microMT). Whereas BALB/c microMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c microMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c microMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c microMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.