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Title: Sensitive expression profiling of T-cell subsets reveals parallel post-thymic differentiation for CD4+ and CD8+ lineages      
keywords:
Transcriptome or Gene expression
ID:
PRJNA98961
description:
Gene expression profiling has opened new directions of investigation in scientific research, however, the need for substantial amount of biological material to perform these experiments limits significantly its application range. Here, we have developed procedures to perform microarray analysis on amplified cDNA from single cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4+ or CD8+ T-cell differentiation. Surprisingly, our data revealed that while CD4+ and CD8+ T-cells evolve differently at early stages of differentiation, they become increasingly similar as they reach late differentiation stages. This suggests that the functional heterogeneity of antigen experienced CD4+ and CD8+ T-cells is located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes. Keywords: Gene expression profiling Overall design: Experiments to test the efficacy of our method applied to low cell numbers of human lymphocytes were performed using the immortalized Jurkat T-cell and Raji (Human Burkitt's lymphoma) B-cell line. First, a set of microarrays (duplicates representing a total of 12 microarrays) was obtained with amplified material from 1, 10, 100, 1000 or 10000 Jurkat T-cells hybridized against the same numbers of Raji B-cells (all titrated by dilution) and compared with profiles obtained with non amplified material from 10^7 Jurkat cells versus Raji cells. In a second set of experiments (duplicates representing a total of 22 microarrays), the influence of the FACS sorting procedure on gene expression profiling was tested. An increasing number of Jurkat cells (1, 3, 10, 30, 100, 300, 1000) were sorted by FACS and their amplified cDNA hybridized against amplified Raji cDNA (1000 cells). Gene expression profiles from these experiments were compared with the profile from 1000 Jurkat vs. Raji cells obtained by dilution. Another set of experiments (representing a total of 20 microarrays) was performed directly using primary CD8+ T lymphocytes obtained from two donor peripheral bloods. For this purpose, naïve (CD27+, CD28+, CD45RA+ and CCR7+) as well as highly differentiated (CD27-, CD28- and CCR7-) CD8+ T-cells were sorted by flow cytometry into 10, 100, 1000, 10000 cell samples. Naïve and highly differentiated cell probes were hybridized in a competitive manner on the microarrays (in duplicate) to provide differential gene expression profiles between these two subsets. In a last set of experiments a comparative study of 10 different subpopulations, defining major steps of both CD4+ or CD8+ T-cell differentiation processes (Fig. 3a and 3b), FACS sorted from a single blood sample of 20 ml was performed. Experiments were performed on two healthy donor blood samples (a total of 20 microarrays). For both CD4+ or CD8+ lineages, the probes obtained from the different subsets of antigen experienced cells were hybridized in a competitive manner against the probes obtained from respective naïve cell pool standards (composed of three independent FACS isolated and amplified 1000 CD4+ or CD8+ cell samples).
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA98961
authentication:
none
authorization:
none
ID:
pmid:18025184
dateReleased:
06-30-2007
name:
Homo sapiens
ncbiID:
ncbitax:9606
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject