High-throughput sequencing analysis of the T-cell receptor repertoire in chronic lymphocytic leukemia
Greece (HelMSIC) - Aristotle University of Thessaloniki, Thessaloniki
Institute of Applied Biosciences, Center for Research and Technology Hellas, Thessaloniki
Kostas Stamatopoulos
Anna Vardi, Evangelia Stalika
4 weeks
Cities/Months Jan Feb Mar Apr May Jun Jul Augt Sep Oct Nov Dec
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Type of Research Project
- Basic science
What is the background of the project?
Chronic lymphocytic leukemia (CLL), the most common neoplasm of the elderly in the West, stands at a crossroad between cancer and autoimmunity, as antigen(s) have been found to play an important role in the selection and expansion of the malignant B cell clone. T cells of CLL patients appear to interact with the malignant B cells to provide survival and proliferation signals, while being incapable of mounting effective anti-tumor responses due to exhaustion, however immunogenetic data are scarce. Our previous studies of T cell receptor beta chain gene repertoire (TCRβ) based on Sanger sequencing and classic subcloning identified shared T cell clonotypes among different CLL patients, alluding to selection by common antigenic elements.

We employ high-throughput, next-generation sequencing (NGS) technologies, combined with bioinformatics data management, evaluation and correction pipeline, in order to perform a detailed and reliable analysis of TCRβ gene repertoire in CLL. This immunogenetic approach will provide information that may prove particularly relevant in the context of T cell manipulation for therapeutic interventions.

Techniques that will be used include:
1. T helper cell (CD4+) and cytotoxic T cell (CD8+) isolation from the peripheral blood of CLL patients
2. RNA isolation and cDNA synthesis
3. RT-PCR of TRBV-TRBD-TRBJ gene rearrangements according to the BIOMED-2 collaborative effort, separation of PCR products on a 3% low melting agarose gel, excision and gel purification
4. Library preparation for the Illumina NGS platform
5. Sequence data analysis using the International imMunoGenetics information system ( and more particularly IMGT/HighV-QUEST tool.
What is the aim of the project?
What techniques and methods are used?
What is the role of the student?
What are the tasks expected to be accomplished by the student?
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student(s) may be involved in any of the aforementioned experimental steps that is more relevant to their interests.
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Biology, genetics and hematology knowledge, if medical student (graduate level). Bioinformatics, if students in biomedical fields.
Are there any legal limitations in the student’s involvement

Type of students accepted
This project accepts:
- Medical students
- Stamatopoulos K et al. Over 20% of patients with CLL carry stereotyped receptors: Pathogenetic implications and clinical correlations. Blood. 2007;109:259-270.
- Darzentas N et al. A different ontogenesis for CLL cases carrying stereotyped antigen receptors: molecular and computational evidence. Leukemia. 2010;24:125-132.
- Agathangelidis A et al. Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies. Blood. 2012;119:4467-4475.
- Ramsay AG. Clear AJ. Fatah R. Gribben JG. Multiple inhibitory ligands induce impaired T cell immunological function in CLL that can be blocked with lenalidomide. Blood. 2012;120:1412-1421
- Bagnara D et al. A novel adoptive transfer model of CLL suggests a key role for T lymphocytes in the disease. Blood. 2011;117:5463-5472