Molecular insights for optimizing T cell receptor specificity against cancer.

Cytotoxic CD8 T cells mediate immunity to pathogens and they are able to eliminate malignant cells. Immunity to viruses and bacteria primarily involves CD8 T cells bearing high affinity T cell receptors (TCRs), which are specific to pathogen-derived (non-self) antigens. Given the thorough elimination of high affinity self/tumor-antigen reactive T cells by central and peripheral tolerance mechanisms, anti-cancer immunity mostly depends on TCRs with intermediate-to-low affinity for self-antigens. Because of this, a promising novel therapeutic approach to increase the efficacy of tumor-reactive T cells is to engineer their TCRs, with the aim to enhance their binding kinetics to pMHC complexes, or to directly manipulate the TCR-signaling cascades. Such manipulations require a detailed knowledge on how pMHC-TCR and co-receptors binding kinetics impact the T cell response. In this review, we present the current knowledge in this field. We discuss future challenges in identifying and targeting the molecular mechanisms to enhance the function of natural or TCR-affinity optimized T cells, and we provide perspectives for the development of protective anti-tumor T cell responses.

HTPSELEX--a database of high-throughput SELEX libraries for transcription factor binding sites.

HTPSELEX is a public database providing access to primary and derived data from high-throughput SELEX experiments aimed at characterizing the binding specificity of transcription factors. The resource is primarily intended to serve computational biologists interested in building models of transcription factor binding sites from large sets of binding sequences. The guiding principle is to make available all information that is relevant for this purpose. For each experiment, we try to provide accurate information about the protein material used, details of the wet lab protocol, an archive of sequencing trace files, assembled clone sequences (concatemers) and complete sets of in vitro selected protein-binding tags. In addition, we offer in-house derived binding sites models. HTPSELEX also offers reasonably large SELEX libraries obtained with conventional low-throughput protocols. The FTP site contains the trace archives and database flatfiles. The web server offers user-friendly interfaces for viewing individual entries and quality-controlled download of SELEX sequence libraries according to a user-defined sequencing quality threshold. HTPSELEX is available from ftp://ftp.isrec.isb-sib.ch/pub/databases/htpselex/ and http://www.isrec.isb-sib.ch/htpselex.

The yield of high-density electric source imaging in focal epilepsy

Background: EEG is the cornerstone of epilepsy diagnostics and mandatory to determine the underlying epilepsy syndrome (e.g. focal vs idiopathic generalized). However, its potential as imaging tool is still underrecognized. In the present study, we aim to determine the prerequisites of maximal benefit of electric source imaging (ESI) to localize the irritative zone in patients with focal epilepsy. Methods: 150 patients suffering from focal epilepsy and with minimum 1 year post-operative follow-up were studied prospectively by reviewers blinded to the underlying diagnosis and outcome. We evaluated the influence of two important factors on sensitivity and specificity of ESI: the number of electrodes (low resolution, LR-ESI: \30 vs. high resolution, HR-ESI: 128-256 electrodes), and the use of individual MRI (i-MRI) vs. template MRI (t-MRI) as head model.Results: ESI had a sensitivity of 85% and a specificity of 87% when HR-ESI with i-MRI was used. Using LR-ESI, sensitivity decreased to 68%, or even 57% when only t-MRI was available. The sensitivity of HR-ESI/i-MRI compared favorably with those of MRI (76%), PET (69%) and ictal/interictal SPECT (64%).Interpretation: This study on a large patient group shows excellent sensitivity and specificity of ESI if 128 EEG channels or more are used for ESI and if the results are co-registered to the patient's individual MRI. Localization precision is as high as or even higher than established brain imaging techniques, providing excellent costeffectiveness in epilepsy evaluation. HR-ESI appears to be a valuable additional imaging tool, given that larger electrode arrays are easily and rapidly applied with modern EEG equipment and that structural MRI is nearly always available for these patients.

Reliable tidal volume estimates at the airway opening with an infant monitor during high-frequency oscillatory ventilation.

OBJECTIVE: To assess the suitability of a hot-wire anemometer infant monitoring system (Florian, Acutronic Medical Systems AG, Hirzel, Switzerland) for measuring flow and tidal volume (Vt) proximal to the endotracheal tube during high-frequency oscillatory ventilation. DESIGN: In vitro model study. SETTING: Respiratory research laboratory. SUBJECT: In vitro lung model simulating moderate to severe respiratory distress. INTERVENTION: The lung model was ventilated with a SensorMedics 3100A ventilator. Vt was recorded from the monitor display (Vt-disp) and compared with the gold standard (Vt-adiab), which was calculated using the adiabatic gas equation from pressure changes inside the model. MEASUREMENTS AND MAIN RESULTS: A range of Vt (1-10 mL), frequencies (5-15 Hz), pressure amplitudes (10-90 cm H2O), inspiratory times (30% to 50%), and Fio2 (0.21-1.0) was used. Accuracy was determined by using modified Bland-Altman plots (95% limits of agreement). An exponential decrease in Vt was observed with increasing oscillatory frequency. Mean DeltaVt-disp was 0.6 mL (limits of agreement, -1.0 to 2.1) with a linear frequency dependence. Mean DeltaVt-disp was -0.2 mL (limits of agreement, -0.5 to 0.1) with increasing pressure amplitude and -0.2 mL (limits of agreement, -0.3 to -0.1) with increasing inspiratory time. Humidity and heating did not affect error, whereas increasing Fio2 from 0.21 to 1.0 increased mean error by 6.3% (+/-2.5%). CONCLUSIONS: The Florian infant hot-wire flowmeter and monitoring system provides reliable measurements of Vt at the airway opening during high-frequency oscillatory ventilation when employed at frequencies of 8-13 Hz. The bedside application could improve monitoring of patients receiving high-frequency oscillatory ventilation, favor a better understanding of the physiologic consequences of different high-frequency oscillatory ventilation strategies, and therefore optimize treatment.

Role of high normal gamma-glutamyltransferase level in identifying heavy alcohol use in young men.

The objective of the current study was to determine the predictive value of high normal gamma-glutamyltransferase (GGT) level as an indication of heavy drinking in young men. In a sample of 577 men attending a one-day army recruitment process mandatory for all Swiss men at age 19 years, GGT level was evaluated as the dependent variable for each of eight dichotomous classifications of individuals on the basis of meeting cut-off criteria for five indexes of alcohol use, two indexes of alcohol-related problems, and one index of body mass. The sensitivity, specificity, and predictive values of GGT level in identifying subjects as either heavy drinkers or being overweight were determined. Compared with findings for their counterparts, GGT level was higher in subjects reporting consumption of more than 14 drinks per week (20.5 +/- 7.81 vs. 18.9 +/- 7.60, P <.05), in those reporting being drunk at least once during the past 30 days (20.3 +/- 7.80 vs. 18.3 +/- 7.43, P <.001), and in individuals with body mass indexes >or=25 kg/m(2) (25.8 +/- 10.84 vs. 18.3 +/- 6.59, P <.001). At a GGT level cut-off of 20 U/l, the sensitivity, specificity, and positive and negative predictive values of either being a heavy drinker or overweight were 48.2%, 70.2%, 67.7%, and 51.2%, respectively. Exclusion of subjects with body mass indexes of >or=25 kg/m(2) revealed similar results. High normal GGT level in young men is indicative of heavy alcohol use or being overweight; when present, subjects should be screened further for potential concomitant drinking problems.

Combinations of prognostic tools for identification of high-risk normotensive patients with acute symptomatic pulmonary embolism.

Background: In haemodynamically stable patients with acute symptomatic pulmonary embolism (PE), studies have not evaluated the usefulness of combining the measurement of cardiac troponin, transthoracic echocardiogram (TTE), and lower extremity complete compression ultrasound (CCUS) testing for predicting the risk of PE-related death. Methods: The study assessed the ability of three diagnostic tests (cardiac troponin I (cTnI), echocardiogram, and CCUS) to prognosticate the primary outcome of PE-related mortality during 30 days of follow-up after a diagnosis of PE by objective testing. Results: Of 591 normotensive patients diagnosed with PE, the primary outcome occurred in 37 patients (6.3%; 95% CI 4.3% to 8.2%). Patients with right ventricular dysfunction (RVD) by TTE and concomitant deep vein thrombosis (DVT) by CCUS had a PE-related mortality of 19.6%, compared with 17.1% of patients with elevated cTnI and concomitant DVT and 15.2% of patients with elevated cTnI and RVD. The use of any two-test strategy had a higher specificity and positive predictive value compared with the use of any test by itself. A combined three-test strategy did not further improve prognostication. For a subgroup analysis of high-risk patients, according to the pulmonary embolism severity index (classes IV and V), positive predictive values of the two-test strategies for PE-related mortality were 25.0%, 24.4% and 20.7%, respectively. Conclusions: In haemodynamically stable patients with acute symptomatic PE, a combination of echocardiography (or troponin testing) and CCUS improved prognostication compared with the use of any test by itself for the identification of those at high risk of PE-related death.

A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

New infectious mammary tumor virus superantigen with V beta-specificity identical to staphylococcal enterotoxin B (SEB).

Only few infectious mouse mammary tumor viruses (MMTV) have been characterized which induce a potent superantigen response in vivo. Here we describe the characterization of an MMTV which was isolated from milk of the highly mammary tumor-prone SHN mouse strain. Exposure of newborn mice to milk-borne MMTV (SHN) results in a very slow deletion of V beta 7, 8.1, 8.2 and 8.3 expressing peripheral T cells. Subcutaneous injection of adult mice with this virus induces a rapid and strong stimulation of all four affected V beta-subsets in vivo. Besides the strong T cell effect we observed an early proliferation and activation of the local B cell pool leading to the initial secretion of IgM followed by preferential secretion of IgG2a by day 6. Sequence comparison of the polymorphic C terminus with known open reading frames revealed high homology to the endogenous provirus Mtv-RCS. This is the first report of a virus having a complete overlap in V beta-specificity with a bacterial superantigen stimulating as many as 35% of the whole CD4+ T cell repertoire including V beta 8.2.

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

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

High-throughput SELEX SAGE method for quantitative modeling of transcription-factor binding sites.

The ability to determine the location and relative strength of all transcription-factor binding sites in a genome is important both for a comprehensive understanding of gene regulation and for effective promoter engineering in biotechnological applications. Here we present a bioinformatically driven experimental method to accurately define the DNA-binding sequence specificity of transcription factors. A generalized profile was used as a predictive quantitative model for binding sites, and its parameters were estimated from in vitro-selected ligands using standard hidden Markov model training algorithms. Computer simulations showed that several thousand low- to medium-affinity sequences are required to generate a profile of desired accuracy. To produce data on this scale, we applied high-throughput genomics methods to the biochemical problem addressed here. A method combining systematic evolution of ligands by exponential enrichment (SELEX) and serial analysis of gene expression (SAGE) protocols was coupled to an automated quality-controlled sequence extraction procedure based on Phred quality scores. This allowed the sequencing of a database of more than 10,000 potential DNA ligands for the CTF/NFI transcription factor. The resulting binding-site model defines the sequence specificity of this protein with a high degree of accuracy not achieved earlier and thereby makes it possible to identify previously unknown regulatory sequences in genomic DNA. A covariance analysis of the selected sites revealed non-independent base preferences at different nucleotide positions, providing insight into the binding mechanism.

Toward high-resolution myocardial tagging.

Magnetic resonance imaging with preceding tissue tagging is a robust method for assessing cardiac motion of the entire heartbeat cycle with a high degree of accuracy. One limitation of this technique, however, is the low resolution of the obtained displacement map of the labeled points within the myocardium. By a new tagging technique, which is based on the combination of two or more measurements of the same slice but with different grid positions, a highly improved resolution of cardiac motion data can be achieved. In combination with a multi-heart-phase echo-planar imaging sequence, such images with doubled grid frequency can be acquired in two short breath-hold periods.

Nonspecific PCR amplification by high-fidelity polymerases: implications for next-generation sequencing of AFLP markers.

High-fidelity 'proofreading' polymerases are often used in library construction for next-generation sequencing projects, in an effort to minimize errors in the resulting sequence data. The increased template fidelity of these polymerases can come at the cost of reduced template specificity, and library preparation methods based on the AFLP technique may be particularly susceptible. Here, we compare AFLP profiles generated with standard Taq and two versions of a high-fidelity polymerase. We find that Taq produces fewer and brighter peaks than high-fidelity polymerase, suggesting that Taq performs better at selectively amplifying templates that exactly match the primer sequences. Because the higher accuracy of proofreading polymerases remains important for sequencing applications, we suggest that it may be more effective to use alternative library preparation methods.

High-resolution ultrasonography of subretinal structure and assessment of retina degeneration in rat.

The purpose of this work was to evaluate the ability of 80 MHz ultrasonography to differentiate intra-retinal layers and quantitatively assess photoreceptor dystrophy in small animal models. Four groups of 10 RCS rats each (five dystrophic and five controls) were explored at 25, 35, 45 and 55 days post-natal (PN). A series of retina cross-sections were obtained ex vivo from outside intact eyes using an 80 MHz three-dimensional ultrasound backscatter microscope (20-microm-axial resolution). Ultrasound features of normal retina were correlated to those of corresponding histology and thickness measurements of photoreceptor segment and nuclear layers were performed on all groups. To show the ability of 80 MHz ultrasonography to distinguish the retinal degeneration in vivo, one RCS rat was explored at 25 and 55 days post-natal. Ultrasound image of normal retina displayed four distinct layers marked by reflections at neurites/nuclei interfaces and permitted to differentiate the photoreceptor segment and nuclear layers. The backscatter level from the retina was shown to be related to the size, density and organization of the intra-layer structure. Ultrasound thickness measurements highly correlated with histologic measurements. A thinning (p<0.05) of outer nuclear layer (ONL) was detected over time for controls and was thought to be assigned to retina maturation. Retinal degeneration started at PN35 and resulted in a more pronounced ONL thinning (p<0.05) over time. ONL degeneration was accompanied by segment layer thickening (p<0.05) at PN35 and thinning thereafter. These changes may indicate accumulation of outer segment debris at PN35 then progressive destruction. In vivo images of rat intra-retinal structure showed the ability of the method to distinguish the photoreceptor layer changes. Our results indicate that 80 MHz ultrasonography reveals intra-retinal layers and is sensitive to age and degenerative changes of photoreceptors. This technique has great potential to follow-up retinal dystrophy and therapeutic effects in vivo.

Combinatorial peptide library-based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity.

A novel approach for the identification of tumor antigen-derived sequences recognized by CD8(+) cytolytic T lymphocytes (CTL) consists in using synthetic combinatorial peptide libraries. Here we have screened a library composed of 3.1 x 10(11) nonapeptides arranged in a positional scanning format, in a cytotoxicity assay, to search the antigen recognized by melanoma-reactive CTL of unknown specificity. The results of this analysis enabled the identification of several optimal peptide ligands, as most of the individual nonapeptides deduced from the primary screening were efficiently recognized by the CTL. The results of the library screening were also analyzed with a mathematical approach based on a model of independent and additive contribution of individual amino acids to antigen recognition. This biometrical data analysis enabled the retrieval, in public databases, of the native antigenic peptide SSX-2(41-49), whose sequence is highly homologous to the ones deduced from the library screening, among the ones with the highest stimulatory score. These results underline the high predictive value of positional scanning synthetic combinatorial peptide library analysis and encourage its use for the identification of CTL ligands.

Specificity and affinity of monoclonal antibodies against carcinoembryonic antigen.

The binding specificities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen (CEA) from 12 different research groups were studied by immunohistochemistry and immuno flow cytometry. In addition, the binding constant for the interaction between Mab and CEA was determined by a solution-phase assay. Cryostat sections of colon carcinoma and normal colon, stomach, liver, pancreas, and spleen were studied by immunohistochemistry. Peripheral blood granulocytes, monocytes, and lymphocytes were assayed by immuno flow cytometry. The Mabs used here have previously been classified into five essentially nonoverlapping epitope groups (GOLD 1-5) (Cancer Res., 49: 4852-4858, 1989). Most Mabs cross-reacted with different normal tissues, ranging from highly cross-reactive Mabs (positive reaction with 8 of 9 discriminating tissues) to relatively specific Mabs (positive reaction with 1 of 9 discriminating tissues). Five Mabs (10%) were specific, reacting only with colon carcinoma, normal colon mucosa, and normal gastric foveola. There was a correlation between epitope group and binding specificity. Mabs with a high degree of CEA specificity almost exclusively belonged to epitope groups 1, 2, and 3, while highly cross-reactive Mabs belonged to epitope groups 4 and 5. There was no correlation between antibody specificity and affinity for CEA. Specific Mabs with high as well as low affinity were found.