22 results on '"Laske K"'
Search Results
2. Case report: defibrotide combined with N-acetylcysteine for the treatment of a patient with hepatic veno-occlusive disease after myelablative therapy with busulfan and melphalane and autologous stem cell transplantation
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Gerecke, C., Hildebrandt, M., Laske, K., Dörffel, W., Ludwig, W.-D., and Bargou, R.
- Published
- 2005
3. Effects of Categorization Training in Patients With TBI During Postacute Rehabilitation
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Constantinidou, Fofi, Thomas, Robin D., Scharp, V. L., Laske, K. M., Hammerly, M. D., Guitonde, S., and Constantinidou, Fofi [0000-0002-7928-8363]
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Adult ,Male ,medicine.medical_specialty ,Multivariate analysis ,Traumatic brain injury ,medicine.medical_treatment ,Physical Therapy, Sports Therapy and Rehabilitation ,Community integration ,Thinking ,Cognition ,Physical medicine and rehabilitation ,Intervention (counseling) ,Task Performance and Analysis ,medicine ,Humans ,Cognitive rehabilitation therapy ,Rehabilitation ,business.industry ,Recovery of Function ,Middle Aged ,medicine.disease ,Categorization ,Brain Injuries ,Multivariate Analysis ,Physical therapy ,Female ,Neurology (clinical) ,Cognition Disorders ,business - Abstract
Background: Previous research suggests that traumatic brain injury (TBI) interferes with the ability to extract and use attributes to describe objects. This study explored the effects of a systematic Categorization Program (CP) in participants with TBI and noninjured controls. Participants: Ten persons with moderate to severe TBI who received comprehensive postacute rehabilitation services and 13 matched noninjured controls participated in the study. Intervention: All participants received CP training for 3 to 5 hours per week for 10 to 12 weeks that consisted of 8 levels and targeted concept formation, object categorization, and decision-making abilities. Main outcome measures: The Mayo-Portland Adaptability Inventory-3 (MPAI-3) and the Community Integration Questionnaire (CIQ). Two Categorization Tests (administered pretraining and posttraining) and 3 Probe Tasks (administered at specified intervals during training) assessed skills relating to categorization. Results: Both groups showed significant improvement in categorization performance after the CP training on the 2 Categorization Tests related to the CP They also were able to generalize and apply categorization and sorting skills in new situations (as measured by the Probe Tasks). Participants with TBI had improved functional outcome performance measured by the MPAI-3 and the CIQ. Conclusions: The systematic and hierarchical structure of the CP is beneficial to participants with TBI during postacute rehabilitation. This study contributes to the growing body of evidence supporting cognitive rehabilitation after moderate to severe TBI. © 2005 Lippincott Williams & Wilkins, Inc. 20 2 143 157 Cited By :22
- Published
- 2005
4. 762 Results of a phase I/II study in metastatic renal cell carcinoma patients treated with an adjuvant HLA personalized peptide vaccine after resection of metastases and comparison to a contemporary cohort of patients with mRCC
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Bedke, J., primary, Rausch, S., additional, Gouttefangeas, C., additional, Kruck, S., additional, Walter, K., additional, Feyerabend, S., additional, Hennenlotter, J., additional, Laske, K., additional, Stevanovic, S., additional, Rammensee, H-G., additional, and Stenzl, A., additional
- Published
- 2016
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5. 549 Results of a prospective phase I/II randomized trial of peptide-specific vaccination in HLA-A*0201 positive prostate carcinoma patients with biochemical recurrence after radical prostatectomy
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Bedke, J., primary, Gouttefangeas, C., additional, Feyerabend, S., additional, Hennenlotter, J., additional, Avilés Escobar, C.M., additional, Laske, K., additional, Widenmeyer, M., additional, Griesemann, H., additional, Stevanovic, S., additional, Rammensee, H-G., additional, and Stenzl, A., additional
- Published
- 2016
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6. 76 Clinical and immunological monitoring of a multi-peptide vaccination trial in patients with stage III renal cell carcinoma
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Bedke, J., primary, Laske, K., additional, Feyerabend, S., additional, Gouttefangeas, C., additional, Dröge, A., additional, Hennelotter, J., additional, Hrstić, P., additional, Stevanović, S., additional, Rammensee, H.G., additional, and Stenzl, A., additional
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- 2012
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7. Adjuvant multipeptide vaccination in high-risk renal cell carcinoma.
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Feyerabend, S., primary, Gouttefangeas, C., additional, Stevanovic, S., additional, Wernet, D., additional, Hennenlotter, J., additional, Bedke, J., additional, Droege, A., additional, Laske, K., additional, Rammensee, H., additional, and Stenzl, A., additional
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- 2010
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8. Personalized peptide-vaccination for pediatric acute lymphoblastic leukemia patients based on patient-individual tumor-specific variants (iVacALL)
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Lang, P., Rabsteyn, A., Kyzirakos, C., Schroeder, C., Sturm, M., Mohr, C., Walzer, M., Pflueckhahn, U., Walter, M., Feldhahn, M., Laske, K., Bonin, M., Martin Ebinger, Stevanovic, S., Bauer, P., Kohlbacher, O., Gouttefangeas, C., Handgretinger, R., and Rammensee, H. -G
9. Impact of thrombus surface on first pass reperfusion in contact aspiration and stent retriever thrombectomy.
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Kaiser D, Laske K, Winzer R, Hädrich K, Wahl H, Krukowski P, Daubner D, Pallesen LP, Linn J, Puetz V, and Gerber JC
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- Aged, Aged, 80 and over, Angiography, Digital Subtraction methods, Brain Ischemia diagnostic imaging, Female, Humans, Male, Middle Aged, Prospective Studies, Retrospective Studies, Stroke diagnostic imaging, Thrombosis diagnostic imaging, Thrombosis surgery, Treatment Outcome, Brain Ischemia surgery, Paracentesis methods, Reperfusion methods, Stents adverse effects, Stroke surgery, Thrombectomy methods
- Abstract
Background: To assess whether thrombus surface morphology has an impact on first pass reperfusion in contact aspiration (CA) and stent retriever (SR) thrombectomy., Methods: From January 2016 to December 2018, consecutive stroke patients with an occlusion of the middle cerebral artery and thrombectomy (CA or SR) were examined in this retrospective study. We assessed patients' characteristics, procedural data and clinical outcome. Thrombus surface on pretreatment digital subtraction angiography (DSA) was categorized into regular versus irregular phenotype by blinded three-reader-consensus. Primary outcome was successful reperfusion (modified treatment in cerebral ischemia (mTICI) 2b-3) after first pass. Data analysis was stratified according to thrombectomy technique and thrombus phenotype., Results: Among 203 patients (76 years (IQR 65.5-81.9), 47.3% male, National Institutes of Health Stroke Scale Score 16 (IQR 12-20)), 155 patients were treated primarily with CA and 48 with SR. 40% (n=62/155) CA and 41.7% (n=20/48) SR-treated patients had a regular thrombus phenotype. In the CA group, successful reperfusion after first pass was more frequently obtained in patients with regular compared with irregular phenotype (69.4% (n=43/62) vs 34.4% (n=32/93); P<0.0001). In contrast, in the SR group, reperfusion after first pass was achieved in 35% (n=7/20; P=0.01) of patients with regular phenotypes. In the CA group, median number of passes (1 (1-2) vs 2 (1-4); P<0.00001) and time from reaching the thrombus to reperfusion (19±27 vs 38±36 min; P=0.0001) were lower among patients with a regular phenotype., Conclusion: Direct CA is associated with higher rates of successful first pass reperfusion in patients with a regular thrombus phenotype in pretreatment DSA., Competing Interests: Competing interests: JCG received speaking fees from Penumbra Inc., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)
- Published
- 2021
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10. Optimization in Detection of Antigen-Specific T Cells Through Differentially Labeled MHC Multimers.
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Pedersen NW, Laske K, Maurer D, Welters M, Walter S, Gouttefangeas C, and Hadrup SR
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- Antigens, CD8-Positive T-Lymphocytes, Flow Cytometry, Humans, Staining and Labeling, Major Histocompatibility Complex, T-Lymphocytes
- Abstract
A large variety of fluorescent molecules are used on a regular basis to tag major histocompatibility complex (MHC) multimers for detection of antigen-specific T cells. We have evaluated the way in which the choice of fluorescent label can impact the detection of MHC multimer binding T cells in an exploratory proficiency panel where detection of MHC multimer binding T cells was assessed across 16 different laboratories. We found that the staining index (SI) of the multimer reagent provided the best direct correlation with the value of a given fluorochrome for T cell detection studies. The SI is dependent on flow cytometer settings and chosen antibody panel; hence, the optimal fluorochrome selection may differ from lab to lab. Consequently, we describe a strategy to evaluate performance of the detection channels and optimize the SI for selected fluorescent molecules. This approach can easily be used to test and optimize fluorescence detection in relation to MHC multimer staining and in general, for antibody-based identification of rare cell populations. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry., (© 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.)
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- 2020
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11. A new synthetic toll-like receptor 1/2 ligand is an efficient adjuvant for peptide vaccination in a human volunteer.
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Rammensee HG, Wiesmüller KH, Chandran PA, Zelba H, Rusch E, Gouttefangeas C, Kowalewski DJ, Di Marco M, Haen SP, Walz JS, Gloria YC, Bödder J, Schertel JM, Tunger A, Müller L, Kießler M, Wehner R, Schmitz M, Jakobi M, Schneiderhan-Marra N, Klein R, Laske K, Artzner K, Backert L, Schuster H, Schwenck J, Weber ANR, Pichler BJ, Kneilling M, la Fougère C, Forchhammer S, Metzler G, Bauer J, Weide B, Schippert W, Stevanović S, and Löffler MW
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- B-Lymphocytes immunology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Dendritic Cells immunology, Granuloma immunology, HEK293 Cells, Healthy Volunteers, Humans, Killer Cells, Natural immunology, Ligands, Male, Middle Aged, Vaccination, Adjuvants, Immunologic therapeutic use, Peptides therapeutic use, Toll-Like Receptor 1 immunology, Toll-Like Receptor 2 immunology
- Abstract
Background: We previously showed that the bacterial lipopeptide Pam
3 Cys-Ser-Ser, meanwhile established as a toll-like receptor (TLR) 1/2 ligand, acts as a strong adjuvant for the induction of virus specific CD8+ T cells in mice, when covalently coupled to a synthetic peptide., Case Presentation: We now designed a new water-soluble synthetic Pam3 Cys-derivative, named XS15 and characterized it in vitro by a TLR2 NF-κB luciferase reporter assay. Further, the capacity of XS15 to activate immune cells and stimulate peptide-specific CD8+ T and NK cells by 6-sulfo LacNAc+ monocytes was assessed by flow cytometry as well as cytokine induction using immunoassays. The induction of a functional immune response after vaccination of a volunteer with viral peptides was assessed by ELISpot assay and flow cytometry in peripheral blood cells and infiltrating cells at the vaccination site, as well as by immunohistochemistry and imaging. XS15 induced strong ex vivo CD8+ and TH 1 CD4+ responses in a human volunteer upon a single injection of XS15 mixed to uncoupled peptides in a water-in-oil emulsion (Montanide™ ISA51 VG). A granuloma formed locally at the injection site containing highly activated functional CD4+ and CD8+ effector memory T cells. The total number of vaccine peptide-specific functional T cells was experimentally assessed and estimated to be 3.0 × 105 in the granuloma and 20.5 × 106 in peripheral blood., Conclusion: Thus, in one volunteer we show a granuloma forming by peptides combined with an efficient adjuvant in a water-in-oil-emulsion, inducing antigen specific T cells detectable in circulation and at the vaccination site, after one single vaccination only. The ex vivo T cell responses in peripheral blood were detectable for more than one year and could be strongly boosted by a second vaccination. Hence, XS15 is a promising adjuvant candidate for peptide vaccination, in particular for tumor peptide vaccines in a personalized setting.- Published
- 2019
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12. Results of a Phase 1/2 Study in Metastatic Renal Cell Carcinoma Patients Treated with a Patient-specific Adjuvant Multi-peptide Vaccine after Resection of Metastases.
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Rausch S, Gouttefangeas C, Hennenlotter J, Laske K, Walter K, Feyerabend S, Chandran PA, Kruck S, Singh-Jasuja H, Frick A, Kröger N, Stevanović S, Stenzl A, Rammensee HG, and Bedke J
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- Carcinoma, Renal Cell surgery, Cohort Studies, Humans, Metastasectomy, Vaccines, Subunit, Adjuvants, Immunologic therapeutic use, Cancer Vaccines therapeutic use, Carcinoma, Renal Cell drug therapy, Carcinoma, Renal Cell secondary, Kidney Neoplasms drug therapy, Kidney Neoplasms pathology
- Abstract
Treatment of metastatic renal cell carcinoma comprises metastasectomy±systemic medical treatment. Specific immunotherapy after metastasectomy could be a complementary option. In this phase 1/2 study, safety and tolerability of an adjuvant multi-peptide vaccine (UroRCC) after metastasectomy was evaluated together with immune response and efficacy, compared with a contemporary cohort of patients (n=44) treated with metastasectomy only. Nineteen metastatic renal cell carcinoma patients received UroRCC via intradermal or subcutaneous application randomized to immunoadjuvants (granulocyte-macrophage colony-stimulating factor or Montanide). Adverse events of UroRCC were mainly grade I and II; frequency of immune response was higher for major histocompatibility complex class II peptides (17/19, 89.5%) than for major histocompatibility complex class I peptides (8/19, 42.1%). Median overall survival was not reached in the UroRCC group (mean: 112.6 mo, 95% confidence interval [CI]: 92.1-133.1) and 58.0 mo (95% CI: 32.7-83.2) in the control cohort (p=0.015). UroRCC was an independent prognosticator of overall survival (hazard ratio=0.19, 95% CI: 0.05-0.69, p=0.012). Adjuvant UroRCC multi-peptide vaccine after metastasectomy was well tolerated, immunogenic, and indicates potential clinical benefit when compared with a contemporary control cohort (NCT02429440). PATIENT SUMMARY: The application of a patient-specific peptide vaccine after complete resection of metastases in metastatic renal cell carcinoma patients resulted in favorable tolerability and outcome., (Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.)
- Published
- 2019
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13. Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.
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Bidmon N, Kind S, Welters MJP, Joseph-Pietras D, Laske K, Maurer D, Hadrup SR, Schreibelt G, Rae R, Sahin U, Gouttefangeas C, Britten CM, and van der Burg SH
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- Biological Assay methods, Blood Buffy Coat cytology, Cell Culture Techniques methods, Cell Engineering instrumentation, Electroporation instrumentation, Electroporation methods, Feasibility Studies, HLA-A2 Antigen immunology, Humans, Immunomagnetic Separation instrumentation, Immunomagnetic Separation methods, RNA Stability, Receptors, Chimeric Antigen immunology, Receptors, Chimeric Antigen metabolism, Reference Standards, T-Lymphocytes metabolism, Biological Assay standards, Cell Engineering methods, RNA, Messenger metabolism, Receptors, Chimeric Antigen genetics, T-Lymphocytes immunology
- Abstract
Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2018
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14. Validation of Immunomonitoring Methods for Application in Clinical Studies: The HLA-Peptide Multimer Staining Assay.
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Chandran PA, Laske K, Cazaly A, Rusch E, Schmid-Horch B, Rammensee HG, Ottensmeier CH, and Gouttefangeas C
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- CD8-Positive T-Lymphocytes immunology, Humans, Immunoassay methods, Immunotherapy methods, Reproducibility of Results, Sensitivity and Specificity, Staining and Labeling methods, HLA Antigens immunology, Peptides immunology
- Abstract
Background: Validated assays are essential to generate data with defined specificity, consistency, and reliability. Although the process of validation is required for applying immunoassays in the context of clinical studies, reports on systematic validation of in vitro T cell assays are scarce so far. We recently validated our HLA-peptide multimer staining assay in a systematic manner so as to qualify the method for monitoring antigen-specific T cell responses after immunotherapy., Methods: Parameters of the assay, specificity, precision, linearity, sensitivity, and robustness were assessed systematically. Experiments were designed to address specifically each parameter and are detailed., Results: Nonspecific multimer staining was below the acceptance limit of 0.02% multimer
(+) CD8(+) cells. The assay showed acceptable precision in all dimensions it was repeated (CV < 10%) and also demonstrated a linear detection (R2 > 0.99) of antigen specific cells., Conclusions: We succeeded in validating the HLA-multimer staining assay in a systematic manner. Additionally, we propose a technical framework and recommendations that can be applied for validating other T cell assessment methods. © 2016 International Clinical Cytometry Society., (© 2016 International Clinical Cytometry Society.)- Published
- 2018
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15. Erratum to "Personalized peptide vaccine-induced immune response associated with long-term survival of a metastatic cholangiocarcinoma patient".
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Löffler MW, Chandran PA, Laske K, Schroeder C, Bonzheim I, Walzer M, Hilke FJ, Trautwein N, Kowalewski DJ, Schuster H, Günder M, Carcamo Yañez VA, Mohr C, Sturm M, Nguyen HP, Riess O, Bauer P, Nahnsen S, Nadalin S, Zieker D, Glatzle J, Thiel K, Schneiderhan-Marra N, Clasen S, Bösmüller H, Fend F, Kohlbacher O, Gouttefangeas C, Stevanović S, Königsrainer A, and Rammensee HG
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- 2017
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16. Personalized peptide vaccine-induced immune response associated with long-term survival of a metastatic cholangiocarcinoma patient.
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Löffler MW, Chandran PA, Laske K, Schroeder C, Bonzheim I, Walzer M, Hilke FJ, Trautwein N, Kowalewski DJ, Schuster H, Günder M, Carcamo Yañez VA, Mohr C, Sturm M, Nguyen HP, Riess O, Bauer P, Nahnsen S, Nadalin S, Zieker D, Glatzle J, Thiel K, Schneiderhan-Marra N, Clasen S, Bösmüller H, Fend F, Kohlbacher O, Gouttefangeas C, Stevanović S, Königsrainer A, and Rammensee HG
- Subjects
- Bile Duct Neoplasms, Cancer Vaccines, Humans, Neoplasm Recurrence, Local, Vaccines, Subunit, Cholangiocarcinoma
- Abstract
Background & Aims: We report a novel experimental immunotherapeutic approach in a patient with metastatic intrahepatic cholangiocarcinoma. In the 5year course of the disease, the initial tumor mass, two local recurrences and a lung metastasis were surgically removed. Lacking alternative treatment options, aiming at the induction of anti-tumor T cells responses, we initiated a personalized multi-peptide vaccination, based on in-depth analysis of tumor antigens (immunopeptidome) and sequencing., Methods: Tumors were characterized by immunohistochemistry, next-generation sequencing and mass spectrometry of HLA ligands., Results: Although several tumor-specific neo-epitopes were predicted in silico, none could be validated by mass spectrometry. Instead, a personalized multi-peptide vaccine containing non-mutated tumor-associated epitopes was designed and applied. Immunomonitoring showed vaccine-induced T cell responses to three out of seven peptides administered. The pulmonary metastasis resected after start of vaccination showed strong immune cell infiltration and perforin positivity, in contrast to the previous lesions. The patient remains clinically healthy, without any radiologically detectable tumors since March 2013 and the vaccination is continued., Conclusions: This remarkable clinical course encourages formal clinical studies on adjuvant personalized peptide vaccination in cholangiocarcinoma., Lay Summary: Metastatic cholangiocarcinomas, cancers that originate from the liver bile ducts, have very limited treatment options and a fatal prognosis. We describe a novel therapeutic approach in such a patient using a personalized multi-peptide vaccine. This vaccine, developed based on the characterization of the patient's tumor, evoked detectable anti-tumor immune responses, associating with long-term tumor-free survival., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)
- Published
- 2016
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17. Managing Multi-center Flow Cytometry Data for Immune Monitoring.
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White S, Laske K, Welters MJ, Bidmon N, van der Burg SH, Britten CM, Enzor J, Staats J, Weinhold KJ, Gouttefangeas C, and Chan C
- Abstract
With the recent results of promising cancer vaccines and immunotherapy1-5, immune monitoring has become increasingly relevant for measuring treatment-induced effects on T cells, and an essential tool for shedding light on the mechanisms responsible for a successful treatment. Flow cytometry is the canonical multi-parameter assay for the fine characterization of single cells in solution, and is ubiquitously used in pre-clinical tumor immunology and in cancer immunotherapy trials. Current state-of-the-art polychromatic flow cytometry involves multi-step, multi-reagent assays followed by sample acquisition on sophisticated instruments capable of capturing up to 20 parameters per cell at a rate of tens of thousands of cells per second. Given the complexity of flow cytometry assays, reproducibility is a major concern, especially for multi-center studies. A promising approach for improving reproducibility is the use of automated analysis borrowing from statistics, machine learning and information visualization21-23, as these methods directly address the subjectivity, operator-dependence, labor-intensive and low fidelity of manual analysis. However, it is quite time-consuming to investigate and test new automated analysis techniques on large data sets without some centralized information management system. For large-scale automated analysis to be practical, the presence of consistent and high-quality data linked to the raw FCS files is indispensable. In particular, the use of machine-readable standard vocabularies to characterize channel metadata is essential when constructing analytic pipelines to avoid errors in processing, analysis and interpretation of results. For automation, this high-quality metadata needs to be programmatically accessible, implying the need for a consistent Application Programming Interface (API). In this manuscript, we propose that upfront time spent normalizing flow cytometry data to conform to carefully designed data models enables automated analysis, potentially saving time in the long run. The ReFlow informatics framework was developed to address these data management challenges.
- Published
- 2015
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18. Cryopreservation of MHC multimers: Recommendations for quality assurance in detection of antigen specific T cells.
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Hadrup SR, Maurer D, Laske K, Frøsig TM, Andersen SR, Britten CM, van der Burg SH, Walter S, and Gouttefangeas C
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- Cryoprotective Agents chemistry, Fluorescent Dyes chemistry, Humans, Protein Multimerization, Quality Control, Quantum Dots chemistry, Reproducibility of Results, T-Lymphocytes immunology, T-Lymphocytes pathology, Cryopreservation, Flow Cytometry standards, Histocompatibility Antigens Class I chemistry, Indicators and Reagents standards, Peptides chemistry, Staining and Labeling standards
- Abstract
Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5-16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability., (© 2014 International Society for Advancement of Cytometry.)
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- 2015
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19. Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.
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Chudley L, McCann KJ, Coleman A, Cazaly AM, Bidmon N, Britten CM, van der Burg SH, Gouttefangeas C, Jandus C, Laske K, Maurer D, Romero P, Schröder H, Stynenbosch LF, Walter S, Welters MJ, and Ottensmeier CH
- Subjects
- Clinical Laboratory Techniques, Germany, Humans, Leukocytes, Mononuclear immunology, Netherlands, Reproducibility of Results, Staining and Labeling, Switzerland, United Kingdom, CD8-Positive T-Lymphocytes cytology, Enzyme-Linked Immunospot Assay methods, HLA Antigens chemistry
- Abstract
Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.
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- 2014
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20. Alternative variants of human HYDIN are novel cancer-associated antigens recognized by adaptive immunity.
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Laske K, Shebzukhov YV, Grosse-Hovest L, Kuprash DV, Khlgatian SV, Koroleva EP, Sazykin AY, Penkov DN, Belousov PV, Stevanovic S, Vass V, Walter S, Eisel D, Schmid-Horch BD, Nedospasov SA, Rammensee HG, and Gouttefangeas C
- Subjects
- Alternative Splicing, Animals, Antigens, Neoplasm genetics, Antigens, Neoplasm metabolism, Chromosomes, Human, Pair 1, Chromosomes, Human, Pair 16, HLA-A2 Antigen metabolism, Humans, Mice, Microfilament Proteins genetics, Microfilament Proteins metabolism, Mutation, Neoplasms immunology, RNA, Messenger immunology, RNA, Messenger metabolism, Adaptive Immunity genetics, Antigens, Neoplasm immunology, CD8-Positive T-Lymphocytes immunology, Microfilament Proteins immunology
- Abstract
A mutation in the hydin gene has been recently described as one possible mechanism leading to lethal congenital hydrocephalus in mice, and a similar defect is proposed to be involved in an autosomal recessive form of hydrocephalus in human. Here, we report for the first time on the cancer association and immunogenicity of two HYDIN variants in humans. One is a previously described sequence derived from the chromosome 1 gene copy, that is, KIAA1864. The second is encoded by a novel alternative transcript originating from the chromosome 16, which we identified by immunoscreening of a testis-derived cDNA expression library with sera of patients with colorectal cancer, and called MO-TES391. Both variants are targeted by immunoglobulin G antibodies in a significant subset of cancer patients but only rarely in healthy donors. Moreover, we identify HLA-A*0201-restricted sequences derived from MO-TES391 and KIAA1864, which are specifically recognized by human cytotoxic CD8(+) T cells. Taken together, these results suggest frequent and coordinated adaptive immune responses against HYDIN variants in patients with cancer and propose HYDIN as a novel cancer-associated antigen., (©2013 AACR.)
- Published
- 2013
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21. The development of standard samples with a defined number of antigen-specific T cells to harmonize T cell assays: a proof-of-principle study.
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Singh SK, Tummers B, Schumacher TN, Gomez R, Franken KL, Verdegaal EM, Laske K, Gouttefangeas C, Ottensmeier C, Welters MJ, Britten CM, and van der Burg SH
- Subjects
- Biological Assay methods, HLA Antigens immunology, HLA-A2 Antigen immunology, Humans, Observer Variation, Receptors, Antigen, T-Cell immunology, Staining and Labeling, Transduction, Genetic, Transgenes, Antigens, Neoplasm immunology, Biological Assay standards, CD8-Positive T-Lymphocytes immunology, Leukocytes, Mononuclear immunology, Monitoring, Immunologic standards, Reference Values
- Abstract
The validation of assays that quantify antigen-specific T cell responses is critically dependent on cell samples that contain clearly defined measurable numbers of antigen-specific T cells. An important requirement is that such cell samples are handled and analyzed in a comparable fashion to peripheral blood mononuclear cells (PBMC). We performed a proof-of-principle study to show that retrovirally TCR-transduced T cells spiked at defined numbers in autologous PBMC can be used as standard samples for HLA/peptide multimer staining. NY-ESO-1157-165-specific, TCR-transduced CD8+ T cell batches were successfully generated from PBMC of several HLA-A*0201 healthy donors, purified by magnetic cell sorting on the basis of HLA tetramer (TM) staining and expanded with specific antigen in vitro. When subsequently spiked into autologous PBMC, the detection of these CD3+CD8+TM+ T cells was highly accurate with a mean accuracy of 91.6 %. The standard cells can be preserved for a substantial period of time in liquid nitrogen. Furthermore, TM staining of fresh and cryopreserved standard samples diluted at decreasing concentrations into autologous cryopreserved unspiked PBMC revealed that the spiked CD3+CD8+TM+ T cells could be accurately detected at all dilutions in a linear fashion with a goodness-of-fit of over 0.99 at a frequency of at least 0.02 % among the CD3+CD8+ T cell population. Notably, the CD3+CD8+TM+ cells of the standard samples were located exactly within the gates used to analyze patient samples and displayed a similar scatter pattern. The performance of the cryopreserved standard samples in the hands of 5 external investigators was good with an inter-laboratory variation of 32.9 % and the doubtless identification of one outlier.
- Published
- 2013
- Full Text
- View/download PDF
22. CIMT 2010: report on the eighth annual meeting of the Association for Cancer Immunotherapy, May 26-28, 2010, Mainz, Germany.
- Author
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Singh SK, Laske K, Gouttefangeas C, Britten CM, and Welters MJ
- Subjects
- Germany, Humans, Cancer Vaccines therapeutic use, Immunotherapy, Neoplasms therapy
- Published
- 2011
- Full Text
- View/download PDF
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