Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen

Human cancer vaccines are often prepared with altered "analog" or "heteroclitic" antigens that have been optimized for HLA class I binding, resulting in enhanced immunogenicity. Here, we take advantage of CpG oligodeoxynucleotides as powerful vaccine adjuvants and demonstrate the induction of high T cell frequencies in melanoma patients, despite the use of natural (unmodified) tumor antigenic peptide. Compared with vaccination with analog peptide, natural peptide induced T cell frequencies that were approximately twofold lower. However, T cells showed superior tumor reactivity because of (i) increased functional avidity for natural antigen and (ii) enhancement of T cell activation and effector function. Thus, novel vaccine formulations comprising potent immune stimulators may allow to circumvent the need for modified antigens and can induce highly functional T cells with precise antigen specificity

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.

Glucose and leptin induce apoptosis in human beta-cells and impair glucose-stimulated insulin secretion through activation of c-Jun N-terminal kinases.

c-Jun N-terminal kinases (SAPK/JNKs) are activated by inflammatory cytokines, and JNK signaling is involved in insulin resistance and beta-cell secretory function and survival. Chronic high glucose concentrations and leptin induce interleukin-1beta (IL-1beta) secretion from pancreatic islets, an event that is possibly causal in promoting beta-cell dysfunction and death. The present study provides evidence that chronically elevated concentrations of leptin and glucose induce beta-cell apoptosis through activation of the JNK pathway in human islets and in insulinoma (INS 832/13) cells. JNK inhibition by the dominant inhibitor JNK-binding domain of IB1/JIP-1 (JNKi) reduced JNK activity and apoptosis induced by leptin and glucose. Exposure of human islets to leptin and high glucose concentrations leads to a decrease of glucose-induced insulin secretion, which was partly restored by JNKi. We detected an interplay between the JNK cascade and the caspase 1/IL-1beta-converting enzyme in human islets. The caspase 1 gene, which contains a potential activating protein-1 binding site, was up-regulated in pancreatic sections and in isolated islets from type 2 diabetic patients. Similarly, cultured human islets exposed to high glucose- and leptin-induced caspase 1 and JNK inhibition prevented this up-regulation. Therefore, JNK inhibition may protect beta-cells from the deleterious effects of high glucose and leptin in diabetes.

Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance.

Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.

Human thymus exports naive CD8 T cells that can home to nonlymphoid tissues.

Functionally naive CD8 T cells in peripheral blood from adult humans can be fully described by their CD45RA(bright)CCR7(+)CD62L(+) cell surface phenotype. Cord blood lymphocytes, from healthy newborns, are homogenously functionally naive. Accordingly, the majority of cord blood CD8 T cells express the same pattern of cell surface molecules. Unexpectedly, however, a significant fraction of cord blood CD8 T cells express neither CCR7 nor CD62L. Yet these cells remain functionally naive as they contain high levels of TCR excision circles, have long telomeres, display highly polyclonal TCRs, and do not exhibit immediate effector functions. In addition, these CD8 T cells already represent a significant fraction of the mature naive CD8 single-positive thymocyte repertoire and may selectively express the cutaneous lymphocyte Ag. We suggest that CD8 single-positive thymocytes comprise two pools of naive precursors that exhibit distinct homing properties. Once seeded in the periphery, naive CCR7(+)CD62L(+) CD8 T cells patrol secondary lymphoid organs, whereas naive CCR7(-)CD62L(-) CD8 T cells selectively migrate to peripheral tissues such as skin.

A phenotypical study of vascular smooth muscle cells in human arterial and venous stenosis

Abstract Introduction The primary function of the contractile vascular smooth muscle cells (cVSMCs) is the regulation of the vascular contractility which means the adaptation of the vascular tonus in response to the modulation of the blood pressure and blood flow. The cVSMCs are essentially quiescent, and therefore their synthesis rate is very limited. They are characterized by the expression of contractile proteins specific to the muscular tissue including myosin, h-­‐caldesmon and <-­‐smooth muscle actin (〈-­‐SMA). These contractile cells are strongly represented in the media layer of the arterial wall and, in a smaller proportion, of the vein wall. Their typical stretched-­‐out morphology allows recognizing them by a histological analysis. They do not produce any extracellular matrix (ECM), and do not migrate through the different layers of the vessel wall, and are not directly involved in the development of intimal hyperplasia (IH). Neointimal formation occurs after endothelial disruption leading to complex molecular and biological mechanisms. The de-­‐differentiation of cVSMCs into synthetic VSMCs (sVSMCs) is mentioned as a key element. These non mature cells are able to proliferate and produce ECM. The characterization of the vascular smooth muscle cells (VSMCs) from healthy and stenosed vascular tissues will contribue to the understanding of the different biological processes leading to IH and will be useful for the development of new therapies to interfere with the cVSMCs growth and migration. The aim of our research was to quantify the proportion of cVSMCs and sVSMCs into the healthy and pathologic human blood vessel wall and to characterize their phenotype. Methods We selected 23 specimens of arterial and venous segments from 18 patients. All these specimens were stored in the biobank from the thoracic and vascular surgery departement. 4 groups were designed (group 1 :arteries without lesions (n=3) ;group 2 : veins without lesions (n=1); group 3: arteries with stenosis (n=9); group 4: veins with stenosis (n=10)). Histology: 5µm-­‐sections were made from each sample embedded in paraffin wax and further stained with hematoxylin & eosin (HE), Van Gieson's stain (VGEL) and Masson's Trichrome (TMB). Pathologic tissues were defined using the label that was given to the macroscopic samples by the surgeon and also, based on the histological analysis with HE and VGEL evaluating the presence of a thickened intima. The same was done to the control samples evaluating the absence of thickening. Immunohistochemistry : The primary antibodies were used :〈-­‐SMA, vimentin, h-­‐ caldesmon, calponin, smooth muscle-myosin heavy chain (SM-­‐MHC), tropomyosin-­‐4, retinol binding protein-­‐1 (RBP-­‐1), nonmuscle-­‐myosin heavy chain-­‐B (NM-­‐MHC-­‐B), Von Willebrand factor (VWF). A semi-­‐quantitative assessment of the intensity of each sample stained was performed. Western Blot : Segments of arteries and veins were analyzed using the following primary antibodies :〈-­‐SMA, Calponin, SM-­‐MHC, NM-­‐MHC-­‐B. The given results were then normalized with tubulin. Results Our data showed that, when using immunohistochemistry analysis we found that〈-­‐SMA was mostly expressed in control arteries, whereas NM-­‐MHC-­‐B in the pathologic ones. Using SM-­‐MHC, calponin, vimentin and caldesmon we found no significative differences in the expression of these proteins in the control and in the pathologic samples. Western Blot analysis showed an inverse correlation between healthy and pathological samples as <-­‐ SMA was more expressed in the pathological samples, while NM-­‐MHC-­‐B in the control group; SM-­‐MHC and calponin were mostly expressed in the pathologic samples. Conclusion Our study showed no clear differences between stenotic and control arterial and venous segments using semi-­‐quantitative assessement by immunohistochemistry. Western Blot showed a significant increased expression of 〈-­‐SMA, calponin and SM-­‐MHC in the arteries with stenosis, while NM-­‐MHC-­‐B was mostly expressed in the arteries without lesions. Further studies are needed to track the lineage of VSMCs to understand the mechanisms leading toIH.

Clonotype selection and composition of human CD8 T cells specific for persistent herpes viruses varies with differentiation but is stable over time.

Protection from reactivation of persistent herpes virus infection is mediated by Ag-specific CD8 T cell responses, which are highly regulated by still poorly understood mechanisms. In this study, we analyzed differentiation and clonotypic dynamics of EBV- and CMV-specific T cells from healthy adults. Although these T lymphocytes included all subsets, from early-differentiated (EM/CD28(pos)) to late-differentiated (EMRA/CD28(neg)) stages, they varied in the sizes/proportions of these subsets. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28(neg) subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, because some clonotypes were selected with differentiation while others were not. Late-differentiated CMV-specific clonotypes were mostly characterized by TCR with lower dependency on CD8 coreceptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of 4 years. Remarkably, clonotypic distribution was stable not only in late-differentiated but also in less-differentiated T cell subsets. Thus, T cell clonotypes segregate with differentiation, but the clonal composition once established is kept constant for at least several years. These findings reveal novel features of the highly sophisticated control of steady state protective T cell activity in healthy adults.

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.

Functionalization of microstructured open-porous bioceramic scaffolds with human fetal bone cells.

Bone substitute materials allowing trans-scaffold migration and in-scaffold survival of human bone-derived cells are mandatory for development of cell-engineered permanent implants to repair bone defects. In this study, we evaluated the influence on human bone-derived cells of the material composition and microstructure of foam scaffolds of calcium aluminate. The scaffolds were prepared using a direct foaming method allowing wide-range tailoring of the microstructure for pore size and pore openings. Human fetal osteoblasts (osteo-progenitors) attached to the scaffolds, migrated across the entire bioceramic depending on the scaffold pore size, colonized, and survived in the porous material for at least 6 weeks. The long-term biocompatibility of the scaffold material for human bone-derived cells was evidenced by in-scaffold determination of cell metabolic activity using a modified MTT assay, a repeated WST-1 assay, and scanning electron microscopy. Finally, we demonstrated that the osteo-progenitors can be covalently bound to the scaffolds using biocompatible click chemistry, thus enhancing the rapid adhesion of the cells to the scaffolds. Therefore, the different microstructures of the foams influenced the migratory potential of the cells, but not cell viability. Scaffolds allow covalent biocompatible chemical binding of the cells to the materials, either localized or widespread integration of the scaffolds for cell-engineered implants.

Targeted expression of human CD1d in transgenic mice reveals independent roles for thymocytes and thymic APCs in positive and negative selection of Valpha14i NKT cells.

CD1d-dependent invariant Valpha14 (Valpha14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Valpha14-Jalpha18 TCR alpha-chain paired mostly with Vbeta8.2 and Vbeta7. The cellular requirements for thymic positive and negative selection of Valpha14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Valpha14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Valpha14i NKT cells that proved functional when activated ex vivo with the Ag alpha-galactosyl ceramide. Valpha14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vbeta8.2. The low number of Vbeta8.2+ Valpha14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vbeta7+ Valpha14i NKT cells. Vbeta8.2+, but not Vbeta7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Valpha14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.

Epigenetic features of human mesenchymal stem cells determine their permissiveness for induction of relevant transcriptional changes by SYT-SSX1.

BACKGROUND: A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism. METHODOLOGY/PRINCIPAL FINDINGS: We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression. CONCLUSIONS/SIGNIFICANCE: Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.

A soluble hexameric form of CD40 ligand activates human dendritic cells and augments memory T cell response.

A strategy to improve the immunogenicity of candidate vaccines is to trigger the innate immune system. Triggering of CD40 at the surface of dendritic cells (DC) is essential in the induction of an efficient immune response. Although CD40 agonist antibodies have been shown to be potent inducers of immune responses in experimental models, serious safety concerns have been raised for their use in humans. In addition, the production of soluble functional CD40 ligand has been challenging and the soluble form existing so far is not developed anymore. Here, we have evaluated the potency of a new soluble form of hexameric CD40 ligand (sCD40L) to serve as an adjuvant for anti-viral T cell responses. sCD40L was able to activate human DC and to enhance virus-specific memory T cell responses. These results demonstrate that this soluble form of CD40 ligand may serve as an adjuvant for T cell response and thus provide the rationale for its potential use in T cell based vaccine strategies.

Reconstituted human polyclonal plasma-derived secretory-like IgM and IgA maintain the barrier function of epithelial cells infected with an enteropathogen.

Intravenous administration of polyclonal and monoclonal antibodies has proven to be a clinically valid approach in the treatment, or at least relief, of many acute and chronic pathologies, such as infection, immunodeficiency, and a broad range of autoimmune conditions. Plasma-derived IgG or recombinant IgG are most frequently used for intravenous or subcutaneous administration, whereas a few IgM-based products are available as well. We have established recently that secretory-like IgA and IgM can be produced upon association of plasma-derived polymeric IgA and IgM with a recombinant secretory component. As a next step toward potential future mucosal administration, we sought to unravel the mechanisms by which these secretory Igs protect epithelial cells located at the interface between the environment and the inside of the body. By using polarized epithelial Caco-2 cell monolayers and Shigella flexneri as a model enteropathogen, we found that polyspecific plasma-derived SIgA and SIgM fulfill many protective functions, including dose-dependent recognition of the antigen via formation of aggregated immune complexes, reduction of bacterial infectivity, maintenance of epithelial cell integrity, and inhibition of proinflammatory cytokine/chemokine production by epithelial cells. In this in vitro model devoid of other cellular or molecular interfering partners, IgM and secretory IgM showed stronger bacterial neutralization than secretory IgA. Together, these data suggest that mucosally delivered antibody preparations may be most effective when combining both secretory-like IgA and IgM, which, together, play a crucial role in preserving several levels of epithelial cell integrity.

Molecular design of the Calphabeta interface favors specific pairing of introduced TCRalphabeta in human T cells.

A promising approach to adoptive transfer therapy of tumors is to reprogram autologous T lymphocytes by TCR gene transfer of defined Ag specificity. An obstacle, however, is the undesired pairing of introduced TCRalpha- and TCRbeta-chains with the endogenous TCR chains. These events vary depending on the individual endogenous TCR and they not only may reduce the levels of cell surface-introduced TCR but also may generate hybrid TCR with unknown Ag specificities. We show that such hybrid heterodimers can be generated even by the pairing of human and mouse TCRalpha- and TCRbeta-chains. To overcome this hurdle, we have identified a pair of amino acid residues in the crystal structure of a TCR that lie at the interface of associated TCR Calpha and Cbeta domains and are related to each other by both a complementary steric interaction analogous to a "knob-into-hole" configuration and the electrostatic environment. We mutated the two residues so as to invert the sense of this interaction analogous to a charged "hole-into-knob" configuration. We show that this inversion in the CalphaCbeta interface promotes selective assembly of the introduced TCR while preserving its specificity and avidity for Ag ligand. Noteworthily, this TCR modification was equally efficient on both a Mu and a Hu TCR. Our data suggest that this approach is generally applicable to TCR independently of their Ag specificity and affinity, subset distribution, and species of origin. Thus, this strategy may optimize TCR gene transfer to efficiently and safely reprogram random T cells into tumor-reactive T cells.