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Background
Germinal center-derived B cell lymphomas are tumors of the lymphoid tissues representing one of the most heterogeneous malignancies. Here we characterize the variety of transcriptomic phenotypes of this disease based on 873 biopsy specimens collected in the German Cancer Aid MMML (Molecular Mechanisms in Malignant Lymphoma) consortium. They include diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt’s lymphoma, mixed FL/DLBCL lymphomas, primary mediastinal large B cell lymphoma, multiple myeloma, IRF4-rearranged large cell lymphoma, MYC-negative Burkitt-like lymphoma with chr. 11q aberration and mantle cell lymphoma.
Methods
We apply self-organizing map (SOM) machine learning to microarray-derived expression data to generate a holistic view on the transcriptome landscape of lymphomas, to describe the multidimensional nature of gene regulation and to pursue a modular view on co-expression. Expression data were complemented by pathological, genetic and clinical characteristics.
Results
We present a transcriptome map of B cell lymphomas that allows visual comparison between the SOM portraits of different lymphoma strata and individual cases. It decomposes into one dozen modules of co-expressed genes related to different functional categories, to genetic defects and to the pathogenesis of lymphomas. On a molecular level, this disease rather forms a continuum of expression states than clearly separated phenotypes. We introduced the concept of combinatorial pattern types (PATs) that stratifies the lymphomas into nine PAT groups and, on a coarser level, into five prominent cancer hallmark types with proliferation, inflammation and stroma signatures. Inflammation signatures in combination with healthy B cell and tonsil characteristics associate with better overall survival rates, while proliferation in combination with inflammation and plasma cell characteristics worsens it. A phenotypic similarity tree is presented that reveals possible progression paths along the transcriptional dimensions. Our analysis provided a novel look on the transition range between FL and DLBCL, on DLBCL with poor prognosis showing expression patterns resembling that of Burkitt’s lymphoma and particularly on ‘double-hit’ MYC and BCL2 transformed lymphomas.
Conclusions
The transcriptome map provides a tool that aggregates, refines and visualizes the data collected in the MMML study and interprets them in the light of previous knowledge to provide orientation and support in current and future studies on lymphomas and on other cancer entities.
Cytotoxic T lymphocytes are effector CD8\(^{+}\) T cells that eradicate infected and malignant cells. Here we show that the transcription factor NFATc1 controls the cytotoxicity of mouse cytotoxic T lymphocytes. Activation of Nfatc1\(^{-/-}\) cytotoxic T lymphocytes showed a defective cytoskeleton organization and recruitment of cytosolic organelles to immunological synapses. These cells have reduced cytotoxicity against tumor cells, and mice with NFATc1-deficient T cells are defective in controlling Listeria infection. Transcriptome analysis shows diminished RNA levels of numerous genes in Nfatc1\(^{-/-}\) CD8\(^{+}\) T cells, including Tbx21, Gzmb and genes encoding cytokines and chemokines, and genes controlling glycolysis. Nfatc1\(^{-/-}\), but not Nfatc2\(^{-/-}\) CD8\(^{+}\) T cells have an impaired metabolic switch to glycolysis, which can be restored by IL-2. Genome-wide ChIP-seq shows that NFATc1 binds many genes that control cytotoxic T lymphocyte activity. Together these data indicate that NFATc1 is an important regulator of cytotoxic T lymphocyte effector functions.
Background
Measles virus (MV) causes T cell suppression by interference with phosphatidylinositol-3-kinase (PI3K) activation. We previously found that this interference affected the activity of splice regulatory proteins and a T cell inhibitory protein isoform was produced from an alternatively spliced pre-mRNA.
Hypothesis
Differentially regulated and alternatively splice variant transcripts accumulating in response to PI3K abrogation in T cells potentially encode proteins involved in T cell silencing.
Methods
To test this hypothesis at the cellular level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) upon T cell suppression.
Results
Applying our algorithm to the data, 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulation, and were found to be enriched in different functional groups. AS genes targeted extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, while RG genes were mainly enriched in cytokine-receptor interaction and Jak-STAT. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry.
Conclusions
PI3K abrogation interferes with key T cell activation processes through both differential expression and alternative splicing, which together actively contribute to T cell suppression.
In this thesis we have investigated the effect of NFAT (Nuclear Factor of Activated T Cell) transcription factors on the expression of Rag-(Recombination Activating Genes) genes in murine thymus. The protein products of Rag genes, RAG1 and RAG2, are critical for the recombination and generation of the TCR (T Cell Receptor) repertoire during thymocyte development, and their expression can be suppressed by the activity of NFAT factors. In thymus, the expression of Rag1 and Rag2 genes is induced at the double-negative (DN, CD4-8-) 3 stage, down-regulated at the DN4 stage, re-induced at the double-positive (DP, CD4+8+) stage, and suppressed again at the single-positive (SP, CD4+8- or CD4-8+) stage. Although it is known that TCR signaling suppresses the expression of Rag1 and Rag2 at the SP stage, the signals that mediate the Rag gene down-reulation remain elusive. Here we report that both the calcineurin-NFAT-signaling and MAPKinase signaling pathways, which are activated by TCR signaling during positive selection, mediate the Rag gene down-regulation in DP thymocytes. The calcineurin-NFAT pathway suppresses both the Rag1 and the Rag2 gene expression. This pathway has a stronger suppressive effect on the Rag1 than the Rag2 gene. A synergistic activity between the two NFAT factors NFATc2 and NFATc3 is essential for calcineurin-NFAT signaling to efficiently suppress the Rag gene expression in DP thymocytes. It is likely that the calcineurin-NFAT signaling down-regulates Rag gene expression by suppressing both the Rag anti-silencer element (ASE) activity and the Rag promoter activity. Similarly, MEK-ERK signaling of MAPK signaling pathway mediates the Rag gene suppression in DP thymocytes although the mechanism through which MEK-ERK mediates the Rag gene down-regulation has to be elucidated. In DN thymocytes, it appears that neither the calcineurin-NFAT signaling nor MAPK signaling is involved in the Rag gene down-regulation. However, a role for these two signaling pathways in the Rag gene up-regulation in DN thymocytes is not excluded. In DN thymocytes, pre-TCR signaling stimulates the expression both Nfatc1 and Nfatc2 genes but has no effect on Nfatc3 gene expression. In DN thymocytes, pre-TCR signaling activates Nfatc1α expression but not Nfatc1ß expression, i.e. the two promoters controling Nfatc1 gene xpression are differently controled by pre-TCR signals. Nfatc1α gene expression in DN thymocytes is mainly regulated by the MAPK signaling pathway because activation of Nfatc1α is mediated by MEK-ERK signaling but opposed by JNK signaling. Calcineuirn-NFAT and p38 signaling pathways are not involved in Nfatc1α promoter regulation in DN thymocytes. In DP thymocytes, TCR signaling up-regulates Nfatc1 and Nfatc2 expression but down-regulates Nfatc3 expression. In DP thymocytes, TCR signaling activates Nfatc1α expression. The activation of Nfatc1α in DP thymocytes is mediated by NFATc1, but not or to a less degree by NFATc2 and NFATc3. MEK-ERK, JNK, and p38 signaling pathways are involved in Nfatc1α gene activation in DP thymocytes, probably by activating NFAT trans-activation activity. All these findings illustrate that in thymocytes the expression of NFAT transcription factors – which are essential for thymic development - is controled at multiple levels.