Peptide-antibody conjugates for tumour therapy: a MHC-class-II-restricted tetanus toxin peptide coupled to an anti-Ig light chain antibody can induce cytotoxic lysis of a human B-cell lymphoma by specific CD4 T cells.

Anti-idiotype antibody therapy of B-cell lymphomas, despite numerous promising experimental and clinical studies, has so far met with limited success. Tailor-made monoclonal anti-idiotype antibodies have been injected into a large series of lymphoma patients, with a few impressive complete tumour remissions but a large majority of negative responses. The results presented here suggest that, by coupling to antilymphoma idiotype antibodies a few molecules of the tetanus toxin universal epitope peptide P2 (830-843), one could markedly increase the efficiency of this therapy. We show that after 2-hr incubation with conjugates consisting of the tetanus toxin peptide P2 coupled by an S-S bridge to monoclonal antibodies directed to the lambda light chain of human immunoglobulin, human B-lymphoma cells can be specifically lysed by a CD4 T-lymphocyte clone specific for the P2 peptide. Antibody without peptide did not induce B-cell killing by the CD4 T-lymphocyte clone. The free cysteine-peptide was also able to induce lysis of the B-lymphoma target by the T-lymphocyte clone, but at a molar concentration 500 to 1000 times higher than that of the coupled peptide. Proliferation assays confirmed that the antibody-peptide conjugate was antigenically active at a much lower concentration than the free peptide. They also showed that antibody-peptide conjugates required an intact processing function of the B cell for peptide presentation, which could be selectively inhibited by leupeptin and chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)

HLA class I - associated immunodominance affects CTL responsiveness to an ESO recombinant protein tumor antigen vaccine.

PURPOSE: Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region (ESO 81-110). The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders. EXPERIMENTAL DESIGN: We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL. RESULTS: CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them. CONCLUSIONS: Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness.

Clinical effectiveness of direct class II restorations: a meta-analysis.

Purpose: More than five hundred million direct dental restorations are placed each year worldwide. In about 55% of the cases, resin composites or compomers are used, and in 45% amalgam. The longevity of posterior resin restorations is well documented. However, data on resin composites that are placed without enamel/dentin conditioning and resin composites placed with self-etching adhesive systems are missing. Material and Methods: The database SCOPUS was searched for clinical trials on posterior resin composites without restricting the search to the year of publication. The inclusion criteria were: (1) prospective clinical trial with at least 2 years of observation; (2) minimum number of restorations at last recall = 20; (3) report on dropout rate; (4) report of operative technique and materials used; (5) utilization of Ryge or modified Ryge evaluation criteria. For amalgam, only those studies were included that directly compared composite resin restorations with amalgam. For the statistical analysis, a linear mixed model was used with random effects to account for the heterogeneity between the studies. P-values under 0.05 were considered significant. Results: Of the 373 clinical trials, 59 studies met the inclusion criteria. In 70% of the studies, Class II and Class I restorations had been placed. The overall success rate of composite resin restorations was about 90% after 10 years, which was not different from that of amalgam. Restorations with compomers had a significantly lower longevity. The main reason for replacement were bulk fractures and caries adjacent to restorations. Both of these incidents were infrequent in most studies and accounted only for about 6% of all replaced restorations after 10 years. Restorations with macrofilled composites and compomer suffered significantly more loss of anatomical form than restorations with other types of material. Restorations that were placed without enamel acid etching and a dentin bonding agent showed significantly more marginal staining and detectable margins compared to those restorations placed using the enamel-etch or etch-and-rinse technique; restorations with self-etching systems were between the other groups. Restorations with compomer suffered significantly more chippings (repairable fracture) than restorations with other materials, which did not statistically differ among each other. Restorations that were placed with a rubber-dam showed significantly fewer material fractures that needed replacement, and this also had a significant effect on the overall longevity. Conclusion: Restorations with hybrid and microfilled composites that were placed with the enamel-etching technique and rubber-dam showed the best overall performance; the longevity of these restorations was similar to amalgam restorations. Compomer restorations, restorations placed with macrofilled composites, and resin restorations with no-etching or self-etching adhesives demonstrated significant shortcomings and shorter longevity.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.

MHC Class II Transactivator Is an In Vivo Regulator of Osteoclast Differentiation and Bone Homeostasis Co-opted From Adaptive Immunity.

The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co-activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c-fms and receptor activator of NF-κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation, plays a novel, unexpected function in skeletal homeostasis, independent of MHC Class II expression and T cells, by exerting a selective and intrinsic control of osteoclast differentiation and bone resorption in vivo. © 2014 American Society for Bone and Mineral Research.

Short-term HIIT and Fatmax training increase aerobic and metabolic fitness in men with class II and III obesity.

OBJECTIVE: To compare the effects of two different 2-week-long training modalities [continuous at the intensity eliciting the maximal fat oxidation (Fatmax ) versus high-intensity interval training (HIIT)] in men with class II and III obesity. METHODS: Nineteen men with obesity (BMI ≥ 35 kg(.) m(-2) ) were assigned to Fatmax group (GFatmax ) or to HIIT group (GHIIT ). Both groups performed eight cycling sessions matched for mechanical work. Aerobic fitness and fat oxidation rates (FORs) during exercise were assessed prior and following the training. Blood samples were drawn to determine hormones and plasma metabolites levels. Insulin resistance was assessed by the homeostasis model assessment of insulin resistance (HOMA2-IR). RESULTS: Aerobic fitness and FORs during exercise were significantly increased in both groups after training (P ≤ 0.001). HOMA2-IR was significantly reduced only for GFatmax (P ≤ 0.001). Resting non-esterified fatty acids (NEFA) and insulin decreased significantly only in GFatmax (P ≤ 0.002). CONCLUSIONS: Two weeks of HIIT and Fatmax training are effective for the improvement of aerobic fitness and FORs during exercise in these classes of obesity. The decreased levels of resting NEFA only in GFatmax may be involved in the decreased insulin resistance only in this group.

Absence of nonhematopoietic MHC class II expression protects mice from experimental autoimmune myocarditis.

Experimental autoimmune myocarditis (EAM) is a CD4(+) T-cell-mediated model of human inflammatory dilated cardiomyopathies. Heart-specific CD4(+) T-cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation-induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV-/- K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α-myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV-/- K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4(+) T cells and in expression of IFN-γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN-γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN-γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis.

Distribution of HLA-DRB1 alleles in a mixed population with insulin-dependent diabetes mellitus from the Southeast of Brazil

HLA class II genes are strongly associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM). The present study reports the HLA-DRB1 genotyping of 41 IDDM patients and 99 healthy subjects from the Southeast of Brazil (Campinas region). Both groups consisted of an ethnic mixture of Caucasian, African Negro and Amerindian origin. HLA-DRB1*03 and *04 alleles were found at significantly higher frequencies among IDDM patients compared to the controls (DRB1*03: 48.8% vs 18.2%, P<0.005, RR = 4.27; DRB1*04: 43.9% vs 15.1%, P<0.008, RR = 4.37) and were associated with a susceptibility to the disease. DRB1*03/*04 heterozygosity conferred a strong IDDM risk (RR = 5.44). In contrast, the HLA-DRB1*11 allele frequency was lower among IDDM patients (7.3% vs 26.3% in controls), but the difference was not significant. These data agree with those described for other populations and allow genetic characterization of IDDM in Brazil

Idiopathic focal segmental glomerulosclerosis and HLA antigens

The objective of the present study was to investigate a possible association between HLA class II antigens and idiopathic focal segmental glomerulosclerosis (FSGS). HLA-A, -B, -DR and -DQ antigens were determined in 19 Brazilian patients (16 white subjects and three subjects of Japanese origin) with biopsy-proven FSGS. Comparison of the HLA antigen frequencies between white patients and white local controls showed a significant increase in HLA-DR4 frequency among FSGS patients (37.7 vs 17.2%, P<0.05). In addition, the three patients of Japanese extraction, not included in the statistical analysis, also presented HLA-DR4. In conclusion, our data confirm the association of FSGS with HLA-DR4 previously reported by others, thus providing further evidence for a role of genes of the HLA complex in the susceptibility to this disease

HLA-DQA1 allele typing by nonisotopic PCR-LIS-SSCP

In the present study we used a simple and reliable method for HLA-DQA1 allele typing based on the single-stranded conformation polymorphism (SSCP) properties of DNA molecules obtained by PCR. The technique consists of PCR amplification of a DNA fragment comprising the second exon of the HLA-DQA1 gene, amplicon denaturation using a low ionic strength solution (LIS), and electrophoresis on a small native polyacrylamide gel, followed by a rapid silver staining procedure. In order to validate the technique and to obtain the allele patterns for the DQA1 gene, 50 cervical samples were typed using this methodology and the commercial Amplitype® HLA DQA1 Amplification and Typing kit. All the alleles detected with the kit were characterized by the LIS-SSCP approach. This procedure proved to be useful for population screening and typing of the DQA1 gene as well as for detecting new alleles or mutations in the donor-recipient molecular matching of HLA class II genes.

Melanosomal targeting sequences from gp100 are essential for MHC class II-restricted endogenous epitope presentation and mobilization to endosomal compartments

CD4+ T lymphocytes play an important role in CD8+ T cell-mediated responses against tumors. Considering that about 20% of melanomas express major histocompatibility complex (MHC) class II, it is plausible that concomitant antigenic presentation by MHC class I and class II complexes shapes positive (helper T cells) or negative (regulatory T cells) anti-tumor responses. Interestingly, gp100, a melanoma antigen, can be presented by both MHC class I and class II when expressed endogenously, suggesting that it can reach endosomal/MHC class II compartments (MIIC). Here, we demonstrated that the gp100 putative amino-terminal signal sequence and the last 70 residues in carboxy-terminus, are essential for MIIC localization and MHC class II presentation. Confocal microscopy analyses confirmed that gp100 was localized in LAMP-1+ endosomal/MIIC. Gp100-targeting sequences were characterized by deleting different sections in the carboxy-terminus (residues 590 to 661). Transfection in 293T cells, expressing MHC class I and class II molecules, revealed that specific deletions in carboxy-terminus resulted in decreased MHC class II presentation, without effects on MHC class I presentation, suggesting a role in MIIC trafficking for these deleted sections. Then, we used these gp100-targeting sequences to mobilize the green fluorescent protein (GFP) to endosomal compartments, and to allow MHC class II and class I presentation of minimal endogenous epitopes. Thus, we concluded that these specific sequences are MIIC targeting motifs. Consequently, these sequences could be included in expression cassettes for endogenously expressed tumor or viral antigens to promote MHC class II and class I presentation and optimize in vivo T cell responses, or as an in vitro tool for characterization of new MHC class II epitopes.

Asociación del HLA Clase II con la Hepatitis Autoinmune en Latinoamérica: Meta-análisis

Introducción: La Hepatitis Autoinmune (AIH) es una enfermedad hepática crónica en la cual se han asociado diferentes alelos de susceptibilidad del antígeno leucocitario humano (HLA) de acuerdo a grupos étnicos, geográficos afectados, así como edad de presentación pronóstico y perfil serológico. El objetivo de este trabajo es identificar alelos del HLA de Clase II que contribuyen a la susceptibilidad de la HAI en pacientes latinoamericanos. Metodología: El presente desarrolló una revisión sistemática de la literatura seguida por un meta-análisis de 694 casos y 1769 controles de estudios del tipo casos y controles que brindaron información suficiente para calcular el odds ratio y el intervalo de confianza del 95% desarrollados en América Latina Resultados: El grupo serológico DQ2 fue encontrado como factor de riesgo, mientras los grupos DR5 y DQ3 fueron encontrados como factores protectores en esta población. En el nivel alélico, el DQB1*02, DQB1*0603, DRB1*0405, y DRB1*1301, se encontraron como factores de riesgo, mientras que los alelos DRB1*1302 y DQB1*0301 fueron factores protectores. Las similitudes físico químicas de los aminoácidos críticos que codifican los péptidos de la fosa de anclaje en los bolsillos P1, P4, y P6 de estas moléculas del HLA, permiten dilucidar su influencia en el desarrollo de la enfermedad. Conclusión: El presente estudio fortalece el conocimiento del componente del HLA en el desarrollo de la HAI en Latinoamérica y su relación con otras poblaciones a nivel mundial.

Are class II phosphoinositide 3-kinases potential targets for anticancer therapies?

Of the three classes of true phosphoinositide (PI) 3-kinases, the class II subdivision, which consists of three isoforms, PI3K-C2alpha, PI3K-C2beta and PI3K-C2gamma, is the least well understood. There are a number of reasons for this. This class of PI 3-kinase was identified exclusively by PCR and homology cloning approaches and not on the basis of cellular function. Like class I PI 3-kinases, class II PI 3-kinases are activated by diverse receptor types. To complicate the elucidation of class II PI 3-kinase function further, their in vitro substrate specificity is intermediate between the receptor activated class I PI 3-kinases and the housekeeping class III PI 3-kinase. The class II PI 3-kinases are inhibited by the two commonly used PI 3-kinase family selective inhibitors, wortmannin and LY294002, and there are no widely available, specific inhibitors for the individual classes or isoforms. Here the current state of understanding of class II PI 3-kinase function is reviewed, followed by an appraisal as to whether there is enough evidence to suggest that pharmaceutical companies, who are currently targeting the class I PI 3-kinases in an attempt to generate anticancer agents, should also consider targeting the class II PI 3-kinases.

Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration, and survival. Disregulation of PI3K-activated pathways is implicated in different diseases including cancer and diabetes. Among the three classes of PI3Ks, class I is the best characterized, whereas class II has received increasing attention only recently and the precise role of these isoforms is unclear. Similarly, the role of phosphatidylinositol-3-phosphate (PtdIns-3-P) as an intracellular second messenger is only just beginning to be appreciated. Here, we show that lysophosphatidic acid (LPA) stimulates the production of PtdIns-3-P through activation of a class II PI3K (PI3K-C2β). Both PtdIns-3-P and PI3K-C2β are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2β as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2β- PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.