ABSTRACT: Influence of isolation procedure and storage condition on the ex vivo gene expression pattern in peripheral human effector/memory T helper lymphocytes
Project description:Gene expression profiling of cells isolated ex vivo is a unique tool to assess gene expression in vivo. Exemplified for CD4+CD45RO+ effector/memory T helper (T E/M) lymphocytes of human peripheral blood, we have analyzed different isolation procedures and storage conditions for the introduction of bias.
Project description:Previous studies revealed the abnormal lymphocytes subsets in IgA nephropathy (IgAN). Recently, emerging studies indicate that microRNA could influence the balance of T helper differentiation and function. Here we explore the underlying mechanism regarding how miRNA regulated lymphocytes subsets in IgAN, focused on T helper cell polarization.
Project description:Previous studies revealed the abnormal lymphocytes subsets in IgA nephropathy (IgAN). Recently, emerging studies indicate that microRNA could influence the balance of T helper differentiation and function. Here we explore the underlying mechanism regarding how miRNA regulated lymphocytes subsets in IgAN, focused on T helper cell polarization.
Project description:Gene expression profiling of cells isolated ex vivo is a unique tool to assess gene expression in vivo. Exemplified for CD4+CD45RO+ effector/memory T helper (T E/M) lymphocytes of human peripheral blood, we have analyzed different isolation procedures and storage conditions for the introduction of bias. Cells from clinical samples can be sorted to high purity by a flow cytometric sorter and assessed for their gene expression pattern but storage/shipping conditions and often necessary pre-enrichment due to low initial population frequencies bears the danger of introducing artificial changes. To test for changes introduced by different procedures, cells were enriched either by magnetic cell sorting (MACS) with Whole-Blood CD4 beads, density gradient centrifugation, alone or in combination with CD3 MACS, or by lysis of erythrocytes followed by MACS depletion of CD15+ cells prior to fluorescence-activated cell sorting (FACS) as CD3+CD4+CD45RO+ cells. A total of 7 different protocols were compared among each other and to T cells stimulated for 3 hours after isolation with Phorbol-12-myristate-13-acetate and Ionomycin (PMA/Iono). Preparation-induced de novo transcription was blocked in one of the protocols by addition of 2 M-BM-5g/ml Actinomycin D (ActD). To test for changes introduced by delays in sample preparation blood samples were drawn and either processed directly or kept at 4 M-BM-0C or room temperature (RT) for 2 or 6 hours before T E/M cells were isolated. Gene expression was compared among the various cell preparations and to T E/M cells isolated from 1 day old buffy coat fractions stored at RT. Sorted cells were lysed in TRIzol (Invitrogen) and frozen at -80M-BM-0C until used for RNA extraction. Total RNA was extracted using the miRNeasy kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary RNA was synthesized from 1 M-BM-5g total RNA using Message AmpII Biotin (Ambion, USA). Fifteen micrograms of fragmented cRNA of each sample were hybridized to 26 HG-U133A plus 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.4, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.
Project description:We investigated transcriptional changes in CD4CD8aa and CD4 intraepthelial lymphocytes. TCRαβ thymocytes differentiate to either CD8αβ cytotoxic T lymphocytes or CD4 T helper cells. This functional dichotomy is controlled by key transcription factors, including the T helper master regulator, ThPOK, which suppresses the cytolytic-program in MHC class II restricted CD4 thymocytes. ThPOK continues to repress CD8-lineage genes in mature CD4 T cells, even as they differentiate to T helper effector subsets. Surprisingly, we show here that the T helper-fate is not fixed and that mature antigen-stimulated CD4 T cells can switch off Thpok expression and reactivate CD8-lineage genes. This unexpected plasticity results in the post-thymic termination of the T helper program and the functional differentiation of distinct MHC class II restricted CD4 cytotoxic T lymphocytes. Intraepithelial_CD4_CD8a neg vs CD8a pos. Two sample set of CD4CD8aa and CD4 intraepthelial lymphocytes (IEL) from small intestine of RAG knockout mice ( 8 weeks after transfer of naive CD4 cells, adoptive tranfer model of colitis), were prepared via cell sorting, and RNA was prepared by TRIZol (Invitrogen, USA) . Data were analyzed in GeneSpring GX10. For microarray analysis, RNA was labeled and hybridized to GeneChip Mouse Genome 430 2.0 arrays according to the Affymetrix protocols. Data were analyzed in GeneSpring GX10.
Project description:We investigated transcriptional changes in CD4CD8aa and CD4 intraepthelial lymphocytes. TCRαβ thymocytes differentiate to either CD8αβ cytotoxic T lymphocytes or CD4 T helper cells. This functional dichotomy is controlled by key transcription factors, including the T helper master regulator, ThPOK, which suppresses the cytolytic-program in MHC class II restricted CD4 thymocytes. ThPOK continues to repress CD8-lineage genes in mature CD4 T cells, even as they differentiate to T helper effector subsets. Surprisingly, we show here that the T helper-fate is not fixed and that mature antigen-stimulated CD4 T cells can switch off Thpok expression and reactivate CD8-lineage genes. This unexpected plasticity results in the post-thymic termination of the T helper program and the functional differentiation of distinct MHC class II restricted CD4 cytotoxic T lymphocytes.
Project description:Murine lymphocytes T helper cells were obtained from spleen of C57Bl/6 mice and miR-155 deficient mice strains that have been immunised with MOG peptide 35-55 in complete Freunds adiuvant 12 days prior. Subsequently cells were stimulated for 72 hours with MOG peptide 35-55 or left unstimulated followed by T helper cell sorting using magnetic beads and RNA isolation.
Project description:Interactions between Macrophages and T-lymphocytes: Tumor
Sneaking Through Intrinsic to Helper T cell Dynamics
ROB J. DE BOER AND PAULINE HOGEWEG
Bioinformatics Group, University of Utrecht, Padualaan 8, 3584 CH Utrecht,
The Netherlands
Abstract
In a mathematical model of the cellular immune response we investigate immune
reactions to tumors that are introduced in various doses. The model represents
macrophage T-lymphocyte interactions that generate cytotoxic macrophages and
cytotoxic T-lymphocytes. In this model antigens (tumors) can induce infinitely large
T-lymphocyte effector populations because (a) effector T-lymphocytes are capable
of repeated proliferation and (b) we have omitted immunosuppression. In this
(proliferative) model small doses of weakly antigenic tumors grow infinitely large
(i.e. sneak through) eliciting an immune response of limited magnitude. Intermediate
doses of the same tumor induce larger immune responses and are hence rejected.
Large doses of the tumor break through, but their progressive growth is accompanied
by a strong immune response involving extensive lymphocyte proliferation. Similarly
a more antigenic tumor is rejected in intermediate doses and breaks through in large
doses. Initially small doses however lead to tumor dormancy. Thus although the
model is devoid of explicit regulatory mechanisms that limit the magnitude of its
response (immunosuppression is such a mechanism), the immune response to large
increasing tumors may either be (a) a stable reaction of limited magnitude (experimentally known as tolerance or unresponsiveness) or (b) a strong and ever increasing
reaction. Unresponsiveness can evolve because in this model net T-lymphocyte
proliferation requires the presence of a minimum number of helper T cells (i.e. a
proliferation threshold). Unresponsiveness is caused by depletion of helper T cell
precursors.
Project description:Murine lymphocytes T helper cells were obtained from spleen of C57Bl/6 mice and miR-155 deficient mice strains that have been immunised with MOG peptide 35-55 in complete Freunds adiuvant 12 days prior. Subsequently cells were stimulated for 72 hours with MOG peptide 35-55 or left unstimulated followed by T helper cell sorting using magnetic beads and RNA isolation. qPCR gene expression profiling. Cells from three individual mice of the same genotype were pooled and treated separately as indicated in the summary. Equal amount total RNA from each mice was pooled prior to gene expression analysis.
Project description:Using biotinylation-based ChIP-seq and microarray analysis of both NKT and PLZF-transgenic thymocytes, we identified several layers of regulation of the innate-like NKT effector program. The transcription factor PLZF is induced during the development of innate and innate-like lymphocytes to direct their acquisition of a T helper effector program, but the molecular mechanisms involved are poorly understood. Using biotinylation-based ChIP-seq and microarray analysis of both NKT and PLZF-transgenic thymocytes, we identified several layers of regulation of the innate-like NKT effector program: first, PLZF bound and regulated genes encoding cytokine receptors as well as homing and adhesion receptors; second, PLZF bound and activated T helper-specific transcription factor genes that in turn control T helper specific programs; finally, PLZF bound and suppressed the transcription of Bach2, a potent general repressor of effector differentiation in naive T cells. These findings reveal the architecture of the transcriptional program recruited by PLZF and elucidate how a single transcription factor can drive the developmental acquisition of a broad effector program.