Informace o publikaci
Molecular Identification of Individual T Cell Clones Specific for Graft-Versus-Leukemia Reaction
| Autoři | |
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| Rok publikování | 2008 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | Graft-verus-leukemia (GVL) effect in hematopoietic stem cell transplantation (HSCT) is usually complicated by the alloreactivity of donor T cells which leads to acute graft-versus-host (GVH) disease. GVL and GVH reactions are proved to be mediated by different T cell clones. The objective of this study was to identify and characterize T cells clones with specific antileukemia activity without mediating GVHD. We have performed primary mixed leukocyte reaction (MLR) using patient non-leukemic irradiated peripherial blood mononuclear cells (PBMC) as stimulators and donor PBMC as responders. To prepare GVL specific T cells, activated alloreactive T cells were first selectively depleted with an anti-CD25 immunotoxin. Allodepleted T cells were then stimulated in secondary MLR using irradiated leukemia cells from the same patient. Activated leukemia-reactive cells were purified by immunomagnetic selection or by FACS based on INFg or CD25 expression, respectively. Clonotypic assay was used for identification of individual leukemia-specific T cell clones. This highly sensitive assay is based on detailed analysis of T cell receptor beta VDJ unique sequence (TCRB-VDJ). mRNA was extracted from sortred activated cells and cDNA synthetized by anchored reverse transcription. Target TCRB-VDJ gene sequence was amplified by anchor PCR and used to transform bacteria. Bacterial colonies were picked for plasmid isolation and subsequent direct automated sequencing of the TCRB-VDJ sequences. We assume that the frequency of particular TCRB-VDJ sequences among bacterial clones after transformation are proportional to the frequency of those sequences in the original population of T cells activated by GVH or GVL reaction. We investigated the presence of individual antileukemic T cell clones in patients with acute myeloid leukemia (AML) and chronic lymphatic leukemia (CLL), and defined them by the TCRB-VDJ unique sequence. The sequences that occured in more than 10% bacterial colonies are likely to represent the most immunodominant clones. Populations of antileukemic T cell clones were oligoclonal, i.e. we observed limited number of individual immunodominat clones which plays important role in GVL reaction. In first CLL patient who had undergone HSCT, six antileukemic T cell clones were identified, four of them are considered to be immunodominant. In second CLL patient after HSCT, only one highly immunodominat autileukemic T cell clone was observed. This specific clone was further monitored by quantitative real-time PCR in patients peripherial blood. |
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