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Expression analysis of resting and stimulated naïve and MP CD8 T cells

ABSTRACT: Memory T cells respond to stimulation with more rapid expression of effector cell functions than their naïve counterparts, yet the gene expression signature underlying this enhanced recall response is not known. Therefore, we performed comprehensive, whole-genome expression profiling of murine memory CD8 T cells before and shortly after ex vivo stimulation. We compared this differential expression profile to its counterpart from stimulated naive cells. Given that memory cells arise from naive cells, the quiescent state of both cell populations prior to stimulation, and the early time point analyzed (four hours post-stimulation), it was possible that the stimulation-induced changes in gene expression were identical between the two populations. While there was a high degree of overlap, we found that the majority of up-regulated genes were more highly induced following stimulation of memory cells. This more robust increase in transcript levels was observed for a functionally diverse set of genes, including cytokines, chemokines, amino acid metabolic enzymes and transporters, transcription factors and regulators of RNA processing. We also identified the unique, stimulation-induced signatures of naive and memory CD8 T cells and found that the former was enriched for factors involved in regulating chromatin modifications. Specifically, we found that Hdac 5,7 and 8 transcript levels were rapidly down-regulated following stimulation of naive cells, which correlated with an increase in their total level of acetylated histone H3 (AcH3). This was in contrast to stimulated memory cells, which had higher levels of total AcH3 ex vivo that did not change following short-term stimulation. Furthermore, the unique stimulation-induced expression profile of memory cells was enriched for factors involved in regulating transport of molecules between the nucleus and cytoplasm, including multiple members of the nuclear pore complex. Together, these results support a model whereby the chromatin modifications that occur during the differentiation of naïve cells into memory cells are preserved in resting memory populations, facilitating their more robust re-activation of a functionally diverse set of genes that contribute to rapid recall of effector functions.

ORGANISM(S): Mus musculus

PROVIDER: GSE16145 | GEO | 2012/05/11

SECONDARY ACCESSION(S): PRJNA115049

REPOSITORIES: GEO

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Project description:Memory T cells respond to stimulation with more rapid expression of effector cell functions than their naM-CM-/ve counterparts, yet the gene expression signature underlying this enhanced recall response is not known. Therefore, we performed comprehensive, whole-genome expression profiling of murine memory CD8 T cells before and shortly after ex vivo stimulation. We compared this differential expression profile to its counterpart from stimulated naive cells. Given that memory cells arise from naive cells, the quiescent state of both cell populations prior to stimulation, and the early time point analyzed (four hours post-stimulation), it was possible that the stimulation-induced changes in gene expression were identical between the two populations. While there was a high degree of overlap, we found that the majority of up-regulated genes were more highly induced following stimulation of memory cells. This more robust increase in transcript levels was observed for a functionally diverse set of genes, including cytokines, chemokines, amino acid metabolic enzymes and transporters, transcription factors and regulators of RNA processing. We also identified the unique, stimulation-induced signatures of naive and memory CD8 T cells and found that the former was enriched for factors involved in regulating chromatin modifications. Specifically, we found that Hdac 5,7 and 8 transcript levels were rapidly down-regulated following stimulation of naive cells, which correlated with an increase in their total level of acetylated histone H3 (AcH3). This was in contrast to stimulated memory cells, which had higher levels of total AcH3 ex vivo that did not change following short-term stimulation. Furthermore, the unique stimulation-induced expression profile of memory cells was enriched for factors involved in regulating transport of molecules between the nucleus and cytoplasm, including multiple members of the nuclear pore complex. Together, these results support a model whereby the chromatin modifications that occur during the differentiation of naM-CM-/ve cells into memory cells are preserved in resting memory populations, facilitating their more robust re-activation of a functionally diverse set of genes that contribute to rapid recall of effector functions. NaM-CM-/ve (CD44lo) and memory phenotype (CD44hi) CD8 T cells from 7-wk old female B6 mice were FACS-purified and cultured in vitro for four hours in the presence or absence of PMA and ionomycin. Total RNA was purified from un-stimulated (resting) naM-CM-/ve, resting memory, stimulated naive, and stimulated memory cells and hybridized to individual single-color arrays. This purification and stimulation protocol was performed four independent times.

Project description:This lab investigates how protein-carbohydrate interactions affect cell fate in the immune system. The specific focus is on the mechanism of galectin-1 mediated death of T lymphocytes, and the role of galectin-1 in immune development, lymphocyte homeostasis, response to pathogens, autoimmunity and tumor cell evasion of the immune response. T cell memory, the ability of T cells to respond to recall antigens rapidly and effectively, is a critical component of the adaptive immune response and the essential target of vaccine development. After naive T cells are exposed to a new antigen (or pathogen), expansion of antigen-specific T cells creates an effector population; the majority of effector T cells die once the antigen or pathogen is cleared. A small subset of T cells remains after antigen is cleared, the memory T cells. These memory cells can respond more rapidly than naive cells to recall antigen, and are also capable of homeostatic self-renewal. During the development of T cell memory, there are dramatic changes in gene transcription, protein expression and post-translational modification of proteins, compared to naive and effector T cells. Our labs have described some of these changes at the phenotypic level, and have found important distinctions in the glycotypes of naive, effector and memory T cells. However, there has not been a systematic examination of differential glycosyltransferase expression among these T cell populations. Gene expression during the development of T-cell memory: After naive T cells are exposed to a new antigen (or pathogen), expansion of antigen-specific T cells creates an effector T cell population, the majority of which dies once antigen is cleared. A small subset, the memory T cells, remains after antigen is cleared. Using the LCMV system in P14 TCR transgenic mice specific for the DbGP33-41 epitope of LCMV, RNA (triplicate samples) was isolated from FACS-sorted virus-specific CD8+ T-cell populations during a time course following viral infection. The time points are day 0, day 8 (the peak of T-cell expansion following viral infection), day 15 (during the contraction or apoptotic phase), day 22 (during the transitional phase), and day 40 or later (the stable memory phase). The RNA samples were analyzed by Glyco-gene Chip analysis.

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Expression analysis of resting and stimulated naïve and MP CD8 T cells

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