Biomedical subjects

Lydia Tesfa

Publications and source records attributed to Lydia Tesfa.

4 recordsLinked to original sources

PubMed2005

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy.

Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4(+) and CD8(+) T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-gamma-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4(+) and CD8(+) lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.

Cell Proliferation↗

PubMed2004

Confirmation of Mycobacterium tuberculosis infection by flow cytometry after ex vivo incubation of peripheral blood T cells with an ESAT-6-derived peptide pool.

BACKGROUND: The presence of a T-cell response to the early secretory antigenic target-6 (ESAT-6) indicates previous infection with or exposure to Mycobacterium tuberculosis. Measuring this response is useful for identifying individuals infected with M. tuberculosis. It was also reported that the frequencies of ESAT-6-specific T cells correlate with disease state. Established procedures measure secreted T-cell cytokines following whole blood stimulation with recombinant ESAT-6 protein or use Elispot as a read-out. METHODS: A single ESAT-6- spanning pool of overlapping peptides (15 amino acids length with 11 overlaps) was used for overnight stimulation of peripheral blood mononuclear cells (PBMCs) from 15 patients infected with Mycobacterium tuberculosis and 11 healthy controls. T-cell responses were rated positive if interferon-gamma (IFN-gamma)-producing T cells were identified above background level, using 4-color cytokine flow cytometry. RESULTS: Thirteen of 15 (87%) patients, but none of the healthy controls, had a positive CD4 T-cell response to the ESAT-6 spanning peptide pool. The frequencies of IFN-gamma-producing cells varied between 1 and 167 per 10,000 CD4 T cells. The test performed as well as the tests described in the literature. CONCLUSIONS: Cytokine flow cytometry following PBMC stimulation with an ESAT-6 spanning peptide pool is a useful laboratory test for ESAT-6-specific T cells combining precise counting and multi-parameter phenotyping.

Adult↗

PubMed2003

Comparison of proliferation and rapid cytokine induction assays for flow cytometric T-cell epitope mapping.

BACKGROUND: T-cell epitope mapping by flow cytometry based on rapid ex vivo peptide-specific cytokine induction in T cells is very efficient and time saving compared with traditional assays. We investigated whether the same epitopes could be identified by proliferation studies. METHODS: An assay based on rapid interferon-gamma induction in T cells (6 h of ex vivo stimulation) was run in parallel with a proliferation assay based on the incremental loss of carboxy-fluorescein diacetate succinimidyl ester staining in proliferating cells. The proliferation assay was chosen because it can be evaluated by high-resolution modern multiparameter flow cytometry. In both cases, T cells were stimulated with the same cytomegalovirus-derived peptides. The peptides identified by the rapid induction of interferon-gamma were compared with those inducing T-cell proliferation. RESULTS: Most epitopes were identified by proliferation and rapid cytokine induction methods; however, each method also identified epitopes that the other one did not. In general, rapid cytokine induction was associated with considerably less background noise, making epitope identification easier, and, owing to the short stimulation time necessary, several identification steps could be carried out on material stored in the incubator. CONCLUSIONS: Even though most epitopes were identified by both approaches, the rapid cytokine induction method had major logistic advantages. However, it may be best to use both assays, particularly in situations in which the identification of epitopes may depend on prior clonal T-cell expansion.

Adult↗

PubMed2003

Mapping T cell epitopes by flow cytometry.

Epitope mapping by flow cytometry is a very modern approach that not only identifies T-cell epitopes but simultaneously allows for detailed analysis of the responding T-cell subsets including lineage, activation marker expression, and other markers of interest. The most frequently used approach is based on the identification of intracellular cytokines in secretion-inhibited activated T cells following stimulation with peptides or peptide pools. A more recently developed assay analyzes T-cell proliferation by measuring the decrease in carboxyfluorescein diacetate succinimidyl ester staining in proliferated cells. This article includes information on peptide configuration, a section on the design and efficient application of peptide pools, and working laboratory protocols for both assays.

Epitope Mapping↗