The Vacuolar Pathway of Long Peptide Cross-Presentation Can Be TAP Dependent.

The intracellular pathway of cross-presentation, which allows MHC class I-restricted presentation of peptides derived from exogenous Ags, remains poorly defined and may vary with the nature of the exogenous Ag and the type of APC. It can be cytosolic, characterized by proteasome and TAP dependency, or vacuolar, usually believed to be proteasome and TAP independent. Cross-presentation is particularly effective with long synthetic peptides, and we previously reported that the HLA-A2-restricted cross-presentation of a long peptide derived from melanoma Ag gp100 by human monocyte-derived immature dendritic cells occurred in a vacuolar pathway, making use of newly synthesized HLA-A2 molecules that follow a nonclassical secretion route. In this article, we show that the HLA-A1-restricted cross-presentation of a long peptide derived from tumor Ag MAGE-A3 by human monocyte-derived immature dendritic cells also follows a vacuolar pathway. However, as opposed to the HLA-A2-restricted peptide, cross-presentation of the HLA-A1-restricted peptide is TAP dependent. We show that this paradoxical TAP-dependency is indirect and reflects the need for TAP to load HLA-A1 molecules with peptides in the endoplasmic reticulum, to allow them to escape the endoplasmic reticulum and reach the vacuole, where peptide exchange with the cross-presented peptide likely occurs. Our results confirm and extend the involvement of the vacuolar pathway in the cross-presentation of long peptides, and indicate that TAP-dependency can no longer be used as a key criterion to distinguish the cytosolic from the vacuolar pathway of cross-presentation. They also stress the existence of an alternative secretory route for MHC class I, which will be worthy of further studies.

NNAlign: a platform to construct and evaluate artificial neural network models of receptor-ligand interactions.

Peptides are extensively used to characterize functional or (linear) structural aspects of receptor-ligand interactions in biological systems, e.g. SH2, SH3, PDZ peptide-recognition domains, the MHC membrane receptors and enzymes such as kinases and phosphatases. NNAlign is a method for the identification of such linear motifs in biological sequences. The algorithm aligns the amino acid or nucleotide sequences provided as training set, and generates a model of the sequence motif detected in the data. The webserver allows setting up cross-validation experiments to estimate the performance of the model, as well as evaluations on independent data. Many features of the training sequences can be encoded as input, and the network architecture is highly customizable. The results returned by the server include a graphical representation of the motif identified by the method, performance values and a downloadable model that can be applied to scan protein sequences for occurrence of the motif. While its performance for the characterization of peptide-MHC interactions is widely documented, we extended NNAlign to be applicable to other receptor-ligand systems as well. Version 2.0 supports alignments with insertions and deletions, encoding of receptor pseudo-sequences, and custom alphabets for the training sequences. The server is available at http://www.cbs.dtu.dk/services/NNAlign-2.0.

Gliadin-Specific CD8+ T Cell Responses Restricted by HLA Class I A*0101 and B*0801 Molecules in Celiac Disease Patients.

Initial studies associated the HLA class I A*01 and B*08 alleles with celiac disease (CD) susceptibility. Subsequent analyses showed a primary association with HLA class II alleles encoding for the HLA DQ2.5 molecule. Because of the strong linkage disequilibrium of A*01 and B*08 alleles with the DR3-DQ2.5 haplotype and a recent genome-wide association study indicating that B*08 and B*39 are predisposing genes, the etiologic role of HLA class I in CD pathogenesis needs to be addressed. We screened gliadin proteins (2α-, 2ω-, and 2γ-gliadin) using bioinformatic algorithms for the presence of peptides predicted to bind A*0101 and B*0801 molecules. The top 1% scoring 9- and 10-mer peptides (N = 97, total) were synthesized and tested in binding assays using purified A*0101 and B*0801 molecules. Twenty of ninety-seven peptides bound B*0801 and only 3 of 97 bound A*0101 with high affinity (IC50 < 500 nM). These 23 gliadin peptides were next assayed by IFN-γ ELISPOT for recognition in peripheral blood cells of CD patients and healthy controls carrying the A*0101 and/or B*0801 genes and in A*0101/B*0801- CD patients. Ten of the twenty-three peptides assayed recalled IFN-γ responses mediated by CD8+ T cells in A*0101/B*0801+ patients with CD. Two peptides were restricted by A*0101, and eight were restricted by B*0801. Of note, 50% (5/10) of CD8+ T cell epitopes mapped within the γ-gliadins. Our results highlight the value of predicted binding to HLA molecules for identifying gliadin epitopes and demonstrate that HLA class I molecules restrict the anti-gluten T cell response in CD patients.

Targeting melanoma with immunoliposomes coupled to anti-MAGE A1 TCR-like single-chain antibody.

Therapy of melanoma using T-cells with genetically introduced T-cell receptors (TCRs) directed against a tumor-selective cancer testis antigen (CTA) NY-ESO1 demonstrated clear antitumor responses in patients without side effects. Here, we exploited the concept of TCR-mediated targeting through introduction of single-chain variable fragment (scFv) antibodies that mimic TCRs in binding major histocompatibility complex-restricted CTA. We produced scFv antibodies directed against Melanoma AntiGEn A1 (MAGE A1) presented by human leukocyte antigen A1 (HLA-A1), in short M1/A1, and coupled these TCR-like antibodies to liposomes to achieve specific melanoma targeting. Two anti-M1/A1 antibodies with different ligand-binding affinities were derived from a phage-display library and reformatted into scFvs with an added cysteine at their carboxyl termini. Protein production conditions, ie, bacterial strain, temperature, time, and compartments, were optimized, and following production, scFv proteins were purified by immobilized metal ion affinity chromatography. Batches of pure scFvs were validated for specific binding to M1/A1-positive B-cells by flow cytometry. Coupling of scFvs to liposomes was conducted by employing different conditions, and an optimized procedure was achieved. In vitro experiments with immunoliposomes demonstrated binding of M1/A1-positive B-cells as well as M1/A1-positive melanoma cells and internalization by these cells using flow cytometry and confocal microscopy. Notably, the scFv with nonenhanced affinity of M1/A1, but not the one with enhanced affinity, was exclusively bound to and internalized by melanoma tumor cells expressing M1/A1. Taken together, antigen-mediated targeting of tumor cells as well as promoting internalization of nanoparticles by these tumor cells is mediated by TCR-like scFv and can contribute to melanoma-specific targeting.

Identification of cytotoxic T lymphocyte epitopes in dengue virus serotype 1.

Dengue virus (DENV) has a serious and growing impact on global health and the exact role of DENV-specific CD8(+) T-cells in DENV infection is still uncertain. In the present study, SYFPEITHI algorithm was used to screen the amino acid sequence of Dengue virus serotype 1 (DENV-1) for potential epitopes, and seven putative HLA-A*1101-restricted and five putative HLA-A*2402-restricted epitopes conserved in hundreds of DENV-1 strains were synthesized. The binding affinity of these epitope candidates to corresponding HLA molecules was evaluated using competitive peptide-binding assay. The immunogenicity and specificity of peptides were further tested in HLA-A*1101 transgenic mice, HLA-A*2402 transgenic mice and peripheral blood mononuclear cells (PBMCs) of patients infected with DENV-1. Percentage inhibition (PI) values calculated in competitive peptide-binding assay showed that six peptides (E39-47 PTLDIELLK, NS5(505-513) GVEGEGLHK, NS2b(15-23) SILLSSLLK, NS5(561-569) ALLATSIFK, NS3(99-107) AVEPGKNPK, and NS4b(159-167) VVYDAKFEK) could bind to HLA-A*1101 molecule with high affinity and five peptides (NS3472-480 QYIYMGQPL, NS4a40-48 AYRHAMEEL, NS5(880-888) DYMTSMKRF, NS3(548-556) SYKVASEGF, and NS3(22-30) IYRILQRGL) have a high affinity for HLA-A*2402 molecule. Enzyme-linked immunospot (ELISPOT) results indicated that these high-affinity peptides were recognized by splenocytes of DENV-1-infected transgenic mice and high-affinity peptide-immunized transgenic mice displayed high levels of peptide-specific IFN-γ-secreting cells. In addition, both peptide-pulsed splenocytes and DENV-1-infected splenic monocytes were efficiently killed by these peptide-specific cytotoxic T lymphocytes. Finally, except NS2b(15-23), 10 high-affinity peptides were recognized by PBMCs of patients infected with DENV-1. These identified epitopes would contribute to the understanding of the function of DENV-specific CD8(+) T-cells.

HLA class I alleles are associated with peptide-binding repertoires of different size, affinity, and immunogenicity.

Prediction of HLA binding affinity is widely used to identify candidate T cell epitopes, and an affinity of 500 nM is routinely used as a threshold for peptide selection. However, the fraction (percentage) of peptides predicted to bind with affinities of 500 nM varies by allele. For example, of a large collection of ~30,000 dengue virus-derived peptides only 0.3% were predicted to bind HLA A*0101, whereas nearly 5% were predicted for A*0201. This striking difference could not be ascribed to variation in accuracy of the algorithms used, as predicted values closely correlated with affinity measured in vitro with purified HLA molecules. These data raised the question whether different alleles would also vary in terms of epitope repertoire size, defined as the number of associated epitopes or, alternatively, whether alleles vary drastically in terms of the affinity threshold associated with immunogenicity. To address this issue, strains of HLA transgenic mice with wide (A*0201), intermediate (B*0702), or narrow (A*0101) repertoires were immunized with peptides of varying binding affinity and relative percentile ranking. The results show that absolute binding capacity is a better predictor of immunogenicity, and analysis of epitopes from the Immune Epitope Database revealed that predictive efficacy is increased using allele-specific affinity thresholds. Finally, we investigated the genetic and structural basis of the phenomenon. Although no stringent correlate was defined, on average HLA B alleles are associated with significantly narrower repertoires than are HLA A alleles.

HLA-peptide multimer selection of adenovirus-specific T cells for adoptive T-cell therapy.

Adenovirus (Ad) infection is a cause of significant morbidity and mortality in hematopoietic stem cell transplant recipients and virus-specific immunotherapy is one option for improved control. Cellular immunity is an important component in suppression of Ad replication but the frequency and population distribution of Ad-specific CD8 T cells has not been systematically investigated. This is an important question in relation to the potential use of these cells for adoptive transfer. To address this question, HLA-peptide multimers were generated for 8 HLA class I-restricted Ad epitopes, which are highly conserved across Ad species. Epitope-specific CD8 T cells from healthy donors were identified by tetramer staining and HLA class I A*01-restricted TDL peptide staining T cells were characterized in relation to frequency, phenotype, and function. The cells demonstrated a minimally differentiated central memory phenotype (CD45RA, CD45RO, CCR7, CD62L, CD27, CD28, and CD57) and were able to produce IFN-γ and proliferate extensively upon antigen stimulation in vitro. After proliferation, the phenotype switched to CD45RO, although it is interesting to note that CCR7 expression was retained. Despite their low frequency, tetramer-staining cells could be enriched with magnetic bead technology. Their characteristics should permit rapid establishment in vivo post adoptive transfer, increasing therapeutic options for patients with Ad infection. This is the first reported characterization of Ad-specific tetramer-staining T cells with a view to adoptive transfer to hematopoietic stem cell transplant patients with Ad infection. The efficacy of these cells needs to be further evaluated in the setting of a clinical trial.

Molecular epidemiology and phylodynamics of the human respiratory syncytial virus fusion protein in northern Taiwan.

BACKGROUND AND AIMS: The glycoprotein (G protein) and fusion protein (F protein) of respiratory syncytial virus (RSV) both show genetic variability, but few studies have examined the F protein gene. This study aimed to characterize the molecular epidemiology and phylodynamics of the F protein gene in clinical RSV strains isolated in northern Taiwan from 2000-2011. METHODS: RSV isolates from children presenting with acute respiratory symptoms between July 2000 and June 2011 were typed based on F protein gene sequences. Phylogeny construction and evaluation were performed using the neighbor-joining (NJ) and maximum likelihood (ML) methods. Phylodynamic patterns in RSV F protein genes were analyzed using the Bayesian Markov Chain Monte Carlo framework. Selection pressure on the F protein gene was detected using the Datamonkey website interface. RESULTS: From a total of 325 clinical RSV strains studied, phylogenetic analysis showed that 83 subgroup A strains (RSV-A) could be further divided into three clusters, whereas 58 subgroup B strains (RSV-B) had no significant clustering. Three amino acids were observed to differ between RSV-A and -B (positions 111, 113, and 114) in CTL HLA-B*57- and HLA-A*01-restricted epitopes. One positive selection site was observed in RSV-B, while none was observed in RSV-A. The evolution rate of the virus had very little change before 2000, then slowed down between 2000 and 2005, and evolved significantly faster after 2005. The dominant subtypes of RSV-A in each epidemic were replaced by different subtypes in the subsequent epidemic. CONCLUSIONS: Before 2004, RSV-A infections were involved in several small epidemics and only very limited numbers of strains evolved and re-emerged in subsequent years. After 2005, the circulating RSV-A strains were different from those of the previous years and continued evolving through 2010. Phylodynamic pattern showed the evolutionary divergence of RSV increased significantly in the recent 5 years in northern Taiwan.

The influence of HLA supertype on thymidine analogue associated with low peripheral fat in HIV.

OBJECTIVES: To examine the relationship between human leukocyte antigen (HLA) genotype and body composition changes induced by thymidine analogue nucleoside reverse transcriptase inhibitor (NtRTI) use in HIV-positive individuals. DESIGN: Data collected during the Simplification with Tenofovir-Emtricitabine (TDF-FTC) or Abacavir-Lamivudine (ABC-3TC) (STEAL) study were analysed to examine the potential association of HLA genotypes with changes in body composition in treatment-experienced HIV-positive individuals. METHODS: Demographic, HIV-related, body composition and HLA genotyping data from the STEAL study were used in this analysis. The mean percentage peripheral fat at study baseline was compared in participants with and without prior NtRTI use. Analyses were also carried out for each HLA supertype strata, for five HLA genes, within the thymidine-exposed group. These comparisons were made using Mann-Whitney rank-sum tests. RESULTS: Participants with prior NtRTI use had a significantly lower baseline mean peripheral fat percentage compared to those without NtRTI use (31.9 vs. 34.7%; P = 0.0045). However, participants carrying one or more of the three particular HLA supertype alleles, A01, B08 and DQ2, showed no significant difference in mean peripheral fat percentage at baseline by NtRTI use. Among participants with prior NtRTI exposure, there were significant differences in mean peripheral fat by HLA A01, B08 and DQ2 allele expression compared to those without expression of these alleles (A01: 34.91% vs. no A01: 30.3%; P = 0.0087; B08: 36.2% vs. no B08: 31.1%; P = 0.0317; DQ2: 35.16% vs. no DQ2: 30.06%; P = 0.0081). CONCLUSION: This analysis suggests that HIV-infected individuals carrying HLA A01, B08 or DQ2 supertype alleles may be resistant to NtRTI-induced peripheral fat loss.

Oral oocyst-induced mouse model of toxoplasmosis: effect of infection with Toxoplasma gondii strains of different genotypes, dose, and mouse strains (transgenic, out-bred, in-bred) on pathogenesis and mortality.

Humans and other hosts acquire Toxoplasma gondii infection by ingesting tissue cysts in undercooked meat, or by food or drink contaminated with oocysts. Currently, there is no vaccine to prevent clinical disease due this parasite in humans, although, various T. gondii vaccine candidates are being developed. Mice are generally used to test the protective efficacy of vaccines because they are susceptible, reagents are available to measure immune parameters in mice, and they are easily managed in the laboratory. In the present study, pathogenesis of toxoplasmosis was studied in mice of different strains, including Human Leukocyte Antigen (HLA) transgenic mice infected with different doses of T. gondii strains of different genotypes derived from several countries. Based on many experiments, the decreasing order of infectivity and pathogenicity of oocysts was: C57BL/6 background interferon gamma gene knock out (KO), HLA-A*1101, HLA-A*0201, HLA-B*0702, Swiss Webster, C57/black, and BALB/c. Mice fed as few as 1 oocyst of Type I and several atypical strains died of acute toxoplasmosis within 21 days p.i. Some Type II, and III strains were less virulent. The model developed herein should prove to be extremely useful for testing vaccines because it is possible to accurately quantitate a challenge inoculum, test the response to different strains of T. gondii using the same preparations of oocysts which are stable for up to a year, and to have highly reproducible responses to the infection.

Identification of potential human respiratory syncytial virus and metapneumovirus T cell epitopes using computational prediction and MHC binding assays.

Human respiratory syncytial virus (RSV) and human metapneumovirus (MPV) are two of the most common causes of serious viral lower respiratory tract illness in humans. CD8+ T cells have been shown to be important in animal models and human clinical studies for the clearance of viral infection, and they may contribute in part to protection against severe disease during reinfections. Precise enumeration and accurate phenotyping of RSV- or MPV-specific CD8+ T cells in humans is currently limited by the relatively small number of T cell epitopes that have been mapped with accompanying identification of MHC restriction patterns. We sought to expand the number of potential RSV and MPV epitopes for use in clinical and translational studies by identifying an expanded set of MHC-binding peptides based on RSV and MPV wild-type virus strain protein sequences. We interrogated the full protein sequences of all 9 or 11 proteins of MPV or RSV respectively using four established epitope prediction algorithms for human HLA A*0101, A*0201, or B*0702 binding and attempted to synthesize the top-scoring 150-152 peptides for each of the two viruses. Synthesis resulted in 442 synthesized and soluble peptides of the 452 predicted epitopes for MPV or RSV. We then determined the binding of the synthetic peptides to recombinant human HLA A*0101, A*0201 or B*0702 molecules with the predicted restriction using a commercially available plate-based assay, iTopia. A total of 230 of the 442 peptides tested exhibited binding to the appropriate MHC molecule. The binding results suggested that existing algorithms for prediction of MHC A*0201 binding are particularly robust. The binding results also provided a large benchmarking data collection for comparison of new prediction algorithms.

A "silent" nucleotide substitution in exon 4 is responsible for the "alternative expression" of HLA-A*01:01:38L through aberrant splicing.

A Welsh Bone Marrow Donor Registry donor was serologically typed, using both alloantisera and monoclonal antibodies, as human leukocyte antigen (HLA)-A2, A-, but typed by polymerase chain reaction sequence-specific priming as HLA-A*01, A*02. Full gene sequencing of the A*01 separated allele indicated an apparently normal A*01:01:01:01 apart from a silent change at nucleotide 705 in exon 4, codon 211 (alanine: normally GCG but GCA in this donor). Sequence analysis of the amplified A*01 allele in cDNA synthesized from RNA indicated that exons 1, 2, 3, and 5 had typical A*01:01 sequences. However, exon 4 was truncated in this allele (87 nucleotides shorter), beginning just after the single nucleotide polymorphism (SNP) identified in genomic DNA sequencing. The nucleotide sequence up to, and 1 nucleotide after, the SNP is homologous with the 3' end of human leukocyte antigen (HLA)-A intron 3 and thus resembles a splice site. However, a small amount of "normal" HLA-A1 was detected on the surface of cells from an Epstein-Barr virus transformed B-cell line (BCL), but not on peripheral blood mononuclear cells, by flow cytometry. Additionally, a trace amount of "normal sized" A*01 was amplified from cDNA. We suggest that in this A*01 variant allele (A*01:01:38L) intron 3 is largely spliced out with a part of exon 4; exon 4 is still in-frame but the protein is smaller than the wild type. This is likely to affect folding and assembly of the "wild type" mature protein on the cell surface, thus explaining the apparent null phenotype when assayed by conventional serology. However, a small amount of A1 protein is made from correctly spliced A*01 mRNA and is detectable on BCLs using flow cytometry.

Modeling major histocompatibility complex binding by nonparametric averaging of multiple predictors and sequence encodings.

There has been considerable interest in statistical approaches that leverage the large volumes of experimental data to predict the binding of Major Histocompatibility Complex class I (MHC-I) molecules to peptides. Here we present our method for averaging together multiple predictors for MHC-peptide binding, where given a particular MHC molecule, a set of predictors and a set of training peptides, our method will average multiple simple predictors for MHC binding to produce a final prediction of the binding affinity between a given MHC molecule and a test peptide. The averaging of predictors is done using a nonparametric method, whereby for any test peptide, we identify similar peptides in the training set and average the predictions on the training set, weighted by each predictor's average accuracy for similar peptides in the training set. We show that our method significantly improves on individual predictors based on held-out data and also produces a predictor whose accuracy is competitive with state-of-the-art techniques based on the results from the Machine Learning in Immunology competition in which 21 submitted techniques were assessed on their accuracy in predicting the binding of HLA-A*0101, HLA-A*0201 and HLA-B*0702 molecules to 9-mer and 10-mer peptides.

Ensemble approaches for improving HLA class I-peptide binding prediction.

Accurately predicting peptides binding to major histocompatibility complex (MHC) I molecules is of great importance to immunologists for elucidating the underlying mechanism of immune recognition and facilitating the design of peptide-based vaccine. Various computational methods have been developed for MHC I-peptide binding prediction, and several of them are reported to achieve high accuracy in recent evaluation on benchmark datasets. For attending the machine learning in immunology competition (MLIC) in prediction of human leukocyte antigen (HLA)-binding peptides, we (FudanCS) have made use of ensemble approaches to further improve the prediction performance by integrating the outputs of several leading predictors. Two ensemble approaches, PM and AvgTanh, have been implemented for attending MLIC. AvgTanh and PM achieved the fourth and the seventh out of all 20 submissions in MLIC in terms of the average AUC. In addition, AvgTanh was awarded the winner in the category of HLA-A*0101 of 9-mer. Overall, the competition results validate the effectiveness of ensemble approaches.

Comparison of membrane fraction proteomic profiles of normal and cancerous human colorectal tissues with gel-assisted digestion and iTRAQ labeling mass spectrometry.

The aim of this study was to uncover the membrane protein profile differences between colorectal carcinoma and neighboring normal mucosa from colorectal cancer patients. Information from cellular membrane proteomes can be used not only to study the roles of membrane proteins in fundamental biological processes, but also to discover novel targets for improving the management of colorectal cancer patients. We used solvent extraction and a gel-assisted digestion method, together with isobaric tags with related and absolute quantitation (iTRAQ) reagents to label tumoral and adjacent normal tissues in a pairwise manner (n = 8). For high-throughput quantification, these digested labeled peptides were combined and simultaneously analyzed using LC-MS/MS. Using the shotgun approach, we identified a total of 438 distinct proteins from membrane fractions of all eight patients. After comparing protein expression between cancerous and corresponding normal tissue, we identified 34 upregulated and eight downregulated proteins with expression changes greater than twofold (Student's t-test, P < 0.05). Among these, the overexpression of well-established biomarkers such as carcinoembryonic antigens (CEACAM5, CEACAM6), as well as claudin-3, HLA class I histocompatibility antigen A-1, tapasin and mitochondrial solute carrier family 25A4 were confirmed by western blotting. We conclude that gel-assisted digestion and iTRAQ labeling MS is a potential approach for uncovering and comparing membrane protein profiles of tissue samples that has the potential to identify novel biomarkers.

Production of an antigenic peptide by insulin-degrading enzyme.

Most antigenic peptides presented by major histocompatibility complex (MHC) class I molecules are produced by the proteasome. Here we show that a proteasome-independent peptide derived from the human tumor protein MAGE-A3 is produced directly by insulin-degrading enzyme (IDE), a cytosolic metallopeptidase. Cytotoxic T lymphocyte recognition of tumor cells was reduced after metallopeptidase inhibition or IDE silencing. Separate inhibition of the metallopeptidase and the proteasome impaired degradation of MAGE-A3 proteins, and simultaneous inhibition of both further stabilized MAGE-A3 proteins. These results suggest that MAGE-A3 proteins are degraded along two parallel pathways that involve either the proteasome or IDE and produce different sets of antigenic peptides presented by MHC class I molecules.

Conformational changes within the HLA-A1:MAGE-A1 complex induced by binding of a recombinant antibody fragment with TCR-like specificity.

Although there is X-ray crystallographic evidence that the interaction between major histocompatibility complex (MHC, in humans HLA) class I molecules and T cell receptors (TCR) or killer cell Ig-like receptors (KIR) may be accompanied by considerable changes in the conformation of selected residues or even entire loops within TCR or KIR, conformational changes between receptor-bound and -unbound MHC class I molecules of comparable magnitude have not been observed so far. We have previously determined the structure of the MHC class I molecule HLA-A1 bound to a melanoma antigen-encoding gene (MAGE)-A1-derived peptide in complex with a recombinant antibody fragment with TCR-like specificity, Fab-Hyb3. Here, we compare the X-ray structure of HLA-A1:MAGE-A1 with that complexed with Fab-Hyb3 to gain insight into structural changes of the MHC molecule that might be induced by the interaction with the antibody fragment. Apart from the expulsion of several water molecules from the interface, Fab-Hyb3 binding results in major rearrangements (up to 5.5 A) of heavy chain residues Arg65, Gln72, Arg145, and Lys146. Residue 65 is frequently and residues 72 and 146 are occasionally involved in TCR binding-induced conformational changes, as revealed by a comparison with MHC class I structures in TCR-liganded and -unliganded forms. On the other hand, residue 145 is subject to a reorientation following engagement of HLA-Cw4 and KIR2DL1. Therefore, conformational changes within the HLA-A1:MAGE-A1:Fab-Hyb3 complex include MHC residues that are also involved in reorientations in complexes with natural ligands, pointing to their central importance for the peptide-dependent recognition of MHC molecules.

An oncolytic adenovirus redirected with a tumor-specific T-cell receptor.

To improve safety and specificity of oncolytic adenoviruses, we introduced T-cell receptors (TCR) specific for a unique class of truly tumor-specific antigens into the adenoviral fiber protein. The adenoviral fiber knob responsible for attachment to the coxsackie-adenoviral receptor (CAR) on target cells was replaced by a single-chain TCR (scTCR) molecule with specificity for the melanoma-associated cancer-testis antigen MAGE-A1, presented by HLA-A1, and an extrinsic trimerization motif in a replicating Ad5 vector (Ad5.R1-scTCR). The production of the recombinant virus was initiated in a novel producer cell line that expressed an antibody-based hexon-specific receptor (293T-AdR) in the cell membrane. This new production system allowed CAR-independent and target antigen-independent propagation of Ad5.R1-scTCR. Infection with adenovirus bearing the scTCR-based fiber resulted in an efficient killing of target tumor cells. The infection was cell type specific because only HLA-A1(+)/MAGE-A1(+) melanoma cells were killed, and thus, this retargeting strategy provides a versatile tool for future clinical application.

Membrane-bound CD95 ligand expressed on human antigen-presenting cells prevents alloantigen-specific T cell response without impairment of viral and third-party T cell immunity.

Genetically modified antigen-presenting cells (APC) represent an attractive strategy for in vitro immunomodulation. In the human system, APC expressing HLA-A1 and a membrane-bound form of CD95L (m-CD95L) were used for selective depletion of HLA-A1-specific T cells. In short-term assays, m-CD95L-expressing APC-induced apoptosis in activated T cells and the constitutive presence of m-CD95L and HLA-A1 expressing APC in long-term T cell cultures prevented the expansion of CD4(+) and CD8(+) HLA-A1-specific T cells and the development of HLA-A1-specific cytotoxicity. However, immunity towards third party, viral and bacterial antigens was maintained and T cells spared from depletion could be induced to develop cytotoxicity towards unrelated antigens. Interestingly, inhibition of HLA-A1-specific T cell response absolutely requires the coexpression of m-CD95L and HLA-A1 antigen on the same APC. Thus, m-CD95L expressing APC might be used in clinical settings to obtain tolerance induction in allogeneic transplantation systems or autoimmune diseases.

Phenotypic and functional changes of human melanoma xenografts induced by DNA hypomethylation: immunotherapeutic implications.

Emerging in vitro evidence points to an immunomodulatory activity of DNA hypomethylating drugs in human malignancies. We investigated the potential of 5-aza-2'-deoxycytidine (5-AZA-CdR) to modulate the expression of cancer testis antigens (CTA) and of HLA class I antigens by melanoma xenografts, and the resulting modifications in immunogenicity of neoplastic cells. Three primary cultures of melanoma cells, selected for immune phenotype and growth rate, were grafted into BALB/c nu/nu mice that were injected intraperitoneally with different dose- and time-schedules of 5-AZA-CdR. Molecular analyses demonstrated a de novo long-lasting expression of the CTA MAGE-1, -2, -3, -4, -10, GAGE 1-6, NY-ESO-1, and the upregulation of MAGE-1, MAGE-3, and NY-ESO-1 levels in melanoma xenografts from 5-AZA-CdR-treated mice. Serological and biochemical analyses identified a de novo expression of NY-ESO-1 protein and a concomitant and persistent upregulation of HLA class I antigens and of HLA-A1 and -A2 alleles. Immunization of BALB/c mice with 5-AZA-CdR-treated melanoma cells generated high titer circulating anti-NY-ESO-1 antibodies. Altogether, the data obtained identify an immunomodulatory activity of 5-AZA-CdR in vivo and strongly suggest for its clinical use to design novel strategies of CTA-based chemo-immunotherapy for melanoma patients.