A global transcriptional view of apoptosis in human T-cell activation.

BACKGROUND: T-cell activation is an essential step of immune response. The process of proper T-cell activation is strictly monitored and regulated by apoptosis signaling. Yet, regulation of apoptosis, an integral and crucial facet during the process of T-cell activation, is not well understood. METHODS: In this study, a Gene-Ontology driven global gene expression analysis coupled with protein abundance and activity assays identified genes and pathways associated with regulation of apoptosis in primary human CD3+ T cells and separately CD4+ and CD8+ T cells. RESULTS: We identified significantly regulated apoptotic genes in several protein families, such as BCL2 proteins, CASPASE proteins, and TNF receptors, and detailed their transcriptional kinetics during the T-cell activation process. Transcriptional patterns of a few select genes (BCL2A1, BBC3 and CASP3) were validated at the protein level. Many of these apoptotic genes are involved in NF-kappaB signaling pathway, including TNFRSF10A, TNFRSF10B, TRAF4, TRAF1, TRAF3, and TRAF6. Upregulation of NF-kappaB and IkappaB family genes (REL, RELA, and RELB, NFKBIA, NFKBIE and NFKB1) at 48 to 96 hours, supported by the increase of phosphorylated RELA (p65), suggests that the involvement of the NF-kappaB complex in the process of T-cell proliferation is not only regulated at the protein level but also at the transcriptional level. Examination of genes involved in MAP kinase signalling pathway, important in apoptosis, suggests an induction of p38 and ERK1 cascades in T-cell proliferation (at 48 to 96 hours), which was explored using phosphorylation assays for p38 (MAPK14) and ERK1 (MAPK3). An immediate and short-lived increase of AP-1 activity measured by DNA-binding activity suggests a rapid and transient activation of p38 and/or JNK cascades upon T-cell activation. CONCLUSION: This comparative genome-scale, transcriptional analysis of T-cell activation in the CD4+ and CD8+ subsets and the mixed CD3+ population identified many apoptosis genes not previously identified in the context of T-cell activation. Furthermore, it provided a comprehensive temporal analysis of the transcriptional program of apoptosis associated with T-cell activation.

Comparative analysis of transcriptional profiling of CD3+, CD4+ and CD8+ T cells identifies novel immune response players in T-cell activation.

BACKGROUND: T-cell activation is an essential step of the immune response and relies on the tightly controlled orchestration of hundreds of genes/proteins, yet the cellular and molecular events underlying this complex process are not fully understood, especially at the genome-scale. Significantly, a comparative genome-scale transcriptional analysis of two T-cell subsets (CD4+ and CD8+) against each other and against the naturally mixed population (CD3+ cells) remains unexplored. RESULTS: Comparison of the microarray-based gene expression patterns between CD3+ T cells, and the CD4+ and CD8+ subsets revealed largely conserved, but not identical, transcriptional patterns. We employed a Gene-Ontology-driven transcriptional analysis coupled with protein abundance assays in order to identify novel T-cell activation genes and cell-type-specific genes associated with the immune response. We identified potential genes involved in the communication between the two subsets (including IL23A, NR4A2, CD83, PSMB2, -8, MIF, IFI16, TNFAIP1, POU2AF1, and OTUB1) and would-be effector-function-specific genes (XCL2, SLAMF7, TNFSF4, -5, -9, CSF3, CD48 and CD244). Chemokines induced during T-cell activation, but not previously identified in T cells, include CCL20, CXCL9, -10, -11 (in all three populations), and XCL2 (preferentially in CD8+ T cells). Increased expression of other unexpected cytokines (GPI, OSM and MIF) suggests their involvement in T-cell activation with their functions yet to be examined. Differential expression of many receptors, not previously reported in the context of T-cell activation, including CCR5, CCR7, IL1R2, IL1RAP, IL6R, TNFRSF25 and TNFRSF1A, suggests their role in this immune process. Several receptors involved in TCR activation (CD3D, CD3G, TRAT1, ITGAL, ITGB1, ITGB2, CD8A and B (CD8+ T-cell specific) along with LCK, ZAP70 and TYROBP were synchronously downregulated. Members of cell-surface receptors (HLA-Ds and KLRs), none previously identified in the context of T-cell activation, were also downregulated. CONCLUSION: This comparative genome-scale, transcriptional analysis of T-cell activation in the CD4+ and CD8+ subsets and the mixed CD3+ populations made possible the identification of many immune-response genes not previously identified in the context of T-cell activation. Significantly, it made possible to identify the temporal patterns of many previously known T-cell activation genes, and also identify genes implicated in effector functions of and communication between CD4+ and CD8+ T cells.

Molecular insights into the pleiotropic effects of plasma on ex vivo-expanded T cells using DNA-microarray analysis.

OBJECTIVE: Immunotherapy with ex vivo-expanded T cells depends on a large supply of biologically active cells. Understanding the effects of culture parameters is essential for improving cell expansion and efficacy. We used DNA-microarray and flow-cytometric analysis coupled with functional assays to investigate mechanistic aspects of plasma supplementation in ex vivo T-cell expansion. METHODS: The effect of plasma supplementation on 18 primary T-cell cultures over a 15-day expansion was investigated. Transcriptional analysis of 5 samples was done with time points every 2 to 3 days throughout the 15-day expansion. Quantitative RT-PCR analysis was used to confirm selected microarray data. The expression of granzyme A and vimentin were analyzed using intracellular flow cytometry. T-cell functionality was assessed using a mixed leukocyte reaction (MLR). RESULTS: We show that the increased expansion of plasma-supplemented cultures of primary human T cells is mostly due to increased cell survival. T cells from plasma-supplemented cultures show higher expression of immunoglobulin genes, integrins, and genes of cytotoxic granules, suggesting a possible enhanced immune function. This was confirmed using a mixed leukocyte reaction and intracellular granzyme-A measurements. A distinct gene expression pattern was correlated to viability differences between plasma-supplemented and serum-free cultures. Ontological analysis of genes in this pattern suggests that the decreased viability of serum-free cultures correlates with higher expression of actin-cytoskeleton and lipid-metabolism genes. Vimentin was found to be expressed higher in serum-free cultures. CONCLUSIONS: These results indicate that the observed decreased cytotoxicity of T cells cultured in serum-free media may be due to increased oxidative stress and cytoskeleton degradation.

Molecular understanding of oxygen-tension and patient-variability effects on ex vivo expanded T cells.

Immunotherapy with ex vivo cultured T cells depends on a large supply of biologically active cells. Understanding the effects of culture parameters is essential for improving the proliferation and efficacy of the expanded cells. Low oxygen tension (5% pO(2)) was previously reported to improve T-cell expansion and alter cellular phenotypic characteristics compared to T cells cultured at 20% pO(2). Here we report the use of DNA-array based transcriptional analysis coupled with protein-level analysis to provide molecular insights into pO(2) and patient-variability effects on expanded primary human T cells. Analysis of seven blood samples showed that reduced pO(2) results in higher expression of genes important in lymphocyte biology, immune function, and cell-cycle progression. 20% pO(2) resulted in higher expression of genes involved in stress response, cell death, and cellular repair. Expression of granzyme A (gzmA) was found to be significantly regulated by oxygen tension with cells at 5% pO(2) having greater gzmA expression than at 20% pO(2). Protein-level analysis of gzmA was consistent with transcriptional analysis. Granzyme K (gzmK) was coexpressed with gzmA, whereas Granzyme B (gzmB) expression was found to precede the expression of both gzmA and gzmK in 15-day cultures. Temporal gene expression patterns for seven blood samples demonstrate that most genes are expressed by all patient samples in similar temporal patterns. However, several patient-specific gene clusters were identified, and one cluster was found to correlate well with cell proliferation and may potentially be used to predict patient-specific T-cell expansion.

Liver-specific physiology of immortal, functionally differentiated hepatocytes and of deficient hepatocyte-like variants.

Five different immortalized transgenic hepatocyte cell lines derived from mice were investigated with respect to their potential to maintain the physiological properties of primary hepatocytes using chemically defined medium. This research completes a previous study by Klocke and coworkers in 2002, using gene expression analysis of the same cell lines by the respective physiological analysis for investigating the hepatocyte-like function. Three transgenic cell lines harboring a fusion gene derivative (construct 202) consisting of the complete SV40 early region, including the coding sequences for the transforming large and small tumor antigens, placed under the control of the murine metallothioneine 1-promotor/enhancer element, showed a hepatocyte-like function and physiology. They grew as a monolayer with a polygonal cell shape, consumed lactate, and secreted albumin at a cell-specific rate of 1.5 pg/h, which is in the range of primary hepatocytes. In addition, the potential of detoxifying ammonium could be maintained. Ammonium was metabolized and urea was produced and released into the medium. A complete urea cycle could be determined. A cell line established from neonatal transgenic mice and expressing a secretory variant of the human epidermal growth factor (IgEGF) under the control of the albumin promoter was characterized by an incomplete urea cycle. Another cell line isolated from the liver of homozygote neonatal p53-knockout mice showed no hepatocyte-specific functions but only properties of continuous cell lines. Specific nucleoside triphosphate (NTP) and uridine (U) ratios were used to characterize the differentiation status of the particular cell lines. A low NTP-U value was found for the three cell lines containing construct 202, which was identical to that observed for primary hepatocytes. In contrast, the cell line harvested from the liver of homozygote neonatal p53-knockout mice presented a NTP-U ratio characteristic for continuous cell lines. This study demonstrates that the four transgenic and the p53-knockout hepatocyte-derived cell lines can be used as models for investigating the conservation of tissue-specific functions in immortalized cells.