To identify transcription factors critically involved in the Tfh cell transcriptional network, antigen-specific Tfh and non-Tfh cells from a day 8 immune response were analyzed by RNA-seq. Overall design: Ovalbumin-specific T cells from OT-II TCR-transgenic mice were transferred into C57BL/6 recipients, which were immunized subcutaneously with nitrophenol coupled to ovalbumin. Eight days after immunization, transgenic T cells from pooled inguinal lymph nodes were sorted for a CD44hi CXCR5+ PD-1+ Tfh and CD44hi CXCR5- PD-1- non-Tfh cell phenotype for analysis by RNA-seq.
Bach2 Controls T Follicular Helper Cells by Direct Repression of Bcl-6.
Specimen part, Subject
View SamplesWe performed RNA-Seq and compared expression levels of genes of reactivated LCMV.GP66-77 specific CD4 T cells isolated from bone marrow (BM) and spleen of LCMV.GP61-80 primed C57BL/6 mice. Cells were isolated 3 days after antigenic re-challenge Overall design: C57BL/6 mice were primed at day 0 with LCMV.GP61-80-NP-MSA + poly(I:C) and immunized again at day 14 with LCMV.GP61-80 + poly(I:C). 60 days later, C57BL/6 mice were boosted with LCMV.GP61-80-NP-MSA + poly(I:C) and 3 days after the boost, LCMV specific CD4 T cells were isolated from BM and spleen
Nonfollicular reactivation of bone marrow resident memory CD4 T cells in immune clusters of the bone marrow.
Age, Specimen part, Cell line, Subject
View SamplesWe performed total RNA-Seq and compared expression levels of genes of whole blood cells isolated from patients after kidney transplantation with stable graft function, antibody mediated- and t cell mediated graft rejection. Overall design: Whole blood cells were isolated from 6 patients with stable graft function, 6 patients with histologically verified antibody mediated graft rejection episode and 4 patients with histologically verified T cell mediated graft rejection after kidney transplantation. Total RNA was extracted and cDNA libraries for total RNA sequencing were generated using “TruSeq® Stranded Total RNA Library” kit (Illumina, San Diego, CA, USA).
The regulation of interferon type I pathway-related genes RSAD2 and ETV7 specifically indicates antibody-mediated rejection after kidney transplantation.
Specimen part, Subject
View SamplesTo udnderstand the tissue-resident features of antigen-specific memory T cells of the bone marrow and spleen, we performed RNA-Seq and compared expression levels of genes of resting LCMV.GP66-77 specific CD4 T cells isolated from bone marrow (BM) and spleen of LCMV.GP61-80 primed C57BL/6 mice. Overall design: C57BL/6 mice were primed at day 0 with LCMV.GP61-80-NP-MSA + poly(I:C) and immunized again at day 14 with LCMV.GP61-80 + poly(I:C). Sixty days after the last immunization mice were sacrificed and LCMV.GP66–77-specific CD69+ and CD69- memory CD4 T cells were isolated from BM and spleen.
CD69<sup>+</sup> memory T lymphocytes of the bone marrow and spleen express the signature transcripts of tissue-resident memory T lymphocytes.
Sex, Age, Specimen part, Cell line, Subject
View SamplesWe compared gene expression profiles of Th cells, macrophages and monocytes isolated from the inflamed colon of colitis induced by the transfer of WT versus Tbx21-/- Th cells in Rag1-/- recipients.
T-bet expression by Th cells promotes type 1 inflammation but is dispensable for colitis.
Specimen part
View SamplesWe performed total RNA-Seq of murine Th1 cells which were four times reactivated in vitro in the presence of irradiated APC'srepeatedly activated in vitro. Overall design: CD4+CD62Lhi (naive) cells were isolated from C57BL/6 mice, activated with aCD3 and aCD28 an cultured under Th1 polarizing conditions in the presence of irradiated APCs. Every sixth day cells were harvested, restimulated with aCD3 and aCD28 and cultured under Th1 polarizing conditions in the presence of irradiated APCs APCs. After four rounds of restimulation, total RNA was extracted and cDNA libraries for total RNA sequencing were generated using “TruSeq® Stranded Total RNA Library” kit (Illumina, San Diego, CA, USA).
MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes.
Specimen part, Subject
View SamplesGene expression profiling on IL-10-secreting and non-secreting murine Th1 cells, stimulated in the presence or absence of the Notch ligand Delta-like 4 (Dll4), was performed to identify transcription factors co-expressed with IL-10.
Role of Blimp-1 in programing Th effector cells into IL-10 producers.
Specimen part
View SamplesThe maintenance of the TFH phenotype depends on continuous signals via ICOS. For a global assessment of differences in gene expression after interruption of the ICOS pathway a genome wide transcriptome analysis was performed. We used the OT-II adoptive transfer system to isolate antigen-specific TFH cells (day 6 after immunization) after short-term (6 hours) blockade of the ICOS pathway using a monoclonal antibody against ICOS-L.
ICOS maintains the T follicular helper cell phenotype by down-regulating Krüppel-like factor 2.
Sex, Specimen part, Treatment
View SamplesBone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1,167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non-overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identify distinct subpopulations expressing IL7, IL15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long-term maintenance of immune cells. Overall design: For single cell library preparation, ex vivo FACS sorted VCAM-1+CD45-Ter119-CD31- BM cells were applied to the 10X Genomics platform using the Single Cell 3' Reagent Kit V2 (10x Genomics) following the manufacturer's instructions. Upon adapter ligation and index PCR, the quality of the obtained cDNA library was assessed by Qubit quantification, Bioanalyzer fragment analysis (HS DNA Kit, Agilent) and KAPA library quantification qPCR (Roche). The sequencing was performed on a NextSeq500 device (Illumina) using a High Output v2 Kit (150 cycles) with the recommended sequencing conditions (read1: 26nt, read2: 98nt, index1: 8 nt, index2: n.a.).
Single-cell transcriptomes of murine bone marrow stromal cells reveal niche-associated heterogeneity.
Specimen part, Subject
View SamplesProlonged cultivation of Saccharomyces cerevisiae in aerobic, glucose-limited chemostat cultures (dilution rate, 010 h1) resulted in a progressive decrease of the residual glucose concentration (from 20 to 8 mg l1 after 200 generations). This increase in the affinity for glucose was accompanied by a fivefold decrease of fermentative capacity, and changes in cellular morphology. These phenotypic changes were retained when single-cell isolates from prolonged cultures were used to inoculate fresh chemostat cultures, indicating that genetic changes were involved. Kinetic analysis of glucose transport in an evolved strain revealed a decreased Km, while Vmax was slightly increased relative to the parental strain. Apparently, fermentative capacity in the evolved strain was not controlled by glucose uptake. Instead, enzyme assays in cell extracts of the evolved strain revealed strongly decreased capacities of enzymes in the lower part of glycolysis. This decrease was corroborated by genome-wide transcriptome analysis using DNA microarrays. In aerobic batch cultures on 20 g glucose l1, the specific growth rate of the evolved strain was lower than that of the parental strain (028 and 037 h1, respectively). Instead of the characteristic instantaneous production of ethanol that is observed when aerobic, glucose-limited cultures of wild-type S. cerevisiae are exposed to excess glucose, the evolved strain exhibited a delay of 90 min before aerobic ethanol formation set in. This study demonstrates that the effects of selection in glucose-limited chemostat cultures extend beyond glucose-transport kinetics. Although extensive physiological analysis offered insight into the underlying cellular processes, the evolutionary driving force for several of the observed changes remains to be elucidated
Prolonged selection in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae causes a partial loss of glycolytic capacity.
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