Follicular helper T cell signature in Type 1 Diabetes
ABSTRACT: This study set out to examine CD4 T cell differentiation in a mouse model of diabetes based on transgenic expression of ovalbumin under the control of the rat insulin promoter and co-expression of the DO11.10 transgene (DO11 x rip-mOVA mice). The transcriptome of T cells isolated from the pancreatic lymph nodes (lymph nodes draining the site of self antigen expression) was compared with that of T cells isolated from inguinal lymph nodes (non-draining lymph nodes). T cells were sorted based on expression of CD4, DO11.10 TCR (KJ-126), CD25 and CD69. Primary cells from 6 week old DO11 x rip-mOVA mice were isolated ex-vivo from the pancreatic lymph nodes or inguinal lymph nodes. Cells were sorted by flow cytometry using antibodies to CD4, DO11.10 TCR (KJ-126+), CD25 and CD69. 3-6 replicates were collected per experimental group with each replicate deriving from 14 mice. RNA was isolated using the RNeasy micro kit (Qiagen).
Project description:The aim of the dataset was to study on a genome-wide level the impact of Lat deficiency on gene expression in resting and activated CD4+ T cells Lat+ and Lat− CD4+ T cells were isolated from lymph nodes and spleen of Latfl-dtr Tmat-Cre mice using a Dynabeads untouched mouse CD4 cells kit (Life Technology) and further purified by cell sorting. Lat+ CD4+ T cells were defined as: CD5+, hDTR+, CD8−, TCRγδ−, CD25−, CD69−, CD62L+, lineage (CD11c, CD11b, CD19, CD45R, CD161)− and Lat− CD4+ Tcells were defined as: CD5+, hDTR−, CD8−, TCRγδ−, CD25−, CD69−, CD62L+, lineage (CD11c, CD11b, CD19, CD45R, CD161)−. Sorted Lat+ or Lat− CD4+ T cells were then kept in vitro for 4 hours without stimulation or activated for 4 hours using anti-CD3 antibody and anti-CD28 antibody. Cell samples corresponding to three biological replicates were analyzed and gene expression profiles were obtained from total RNA.
Project description:Expression data from mouse tumor-specific CD4+ T cells The central role of tumor-specific Th1 cells in fighting cancer is becoming increasingly appreciated. However, little is known about how these cells are generated in vivo. Here, we used flow cytometry and gene expression microarrays to characterize the primary activation and Th1 differentiation of naïve tumor-specific CD4+ T cells in a mouse model for cancer immunosurveillance. We took advantage of T cell receptor-transgenic mice where CD4+ T cells recognize a tumor-specific antigen secreted by the major histocompatibility complex (MHC) class II-negative MOPC315 myeloma. Cancer cells were injected subcutaneously and T cell activation was analyzed in draining lymph nodes and at the incipient tumor site at day +8. After activation and migration to the incipient tumor site, tumor-specific CD4+ T cells had 29 up-regulated molecules (CD2, CD5, CD11a, CD18, CD25, CD28, CD44, CD45, CD49d, CD51, CD54, CD69, CD71, CD83, CD86, CD90, CD95, CD102, CD122, CD153, CD166, CD200, CD249, CD254, CD274, CD279, Ly6C, MHC class I, and CCR7) and 5 down-regulated molecules (CD27, CD31, CD45RB, CD62L, and CD126) on the surface. The activated CD4+ T cells produced interferon , a cytokine consistent with Th1 polarization, and also interleukin 2 (IL-2), IL-3, IL-10, and tumor necrosis factor . Activation of naïve tumor-specific CD4+ T cells in draining lymph node resulted in the up-regulation of 609 genes and down-regulation of 284 genes. Bioinformatics analysis of genes differentially expressed identified functional pathways related to tumor-specific Th1 cell activation. This study may represent a useful resource to guide the development of Th1-based immunotherapy for cancer. TCR-transgenic SCID mice (n = 12-15) were injected s.c. on the flank with 10(5) MOPC315 cells suspended in 250 µl ice-cold growth factor-reduced Matrigel. At day +8, draining axillary LN were dissected out and pooled, single-cell suspensions were made and staining with mAb was carried out. Activated tumor-specific CD4+ GB113+ T cells were sorted by FACSAria (≥ 95% pure) in three independent experiments. Naïve tumor-specific CD4+ control T cells were obtained from pooled LN from naive TCR-transgenic SCID mice and treated identically in two independent experiments.
Project description:Experimental autoimmune encephalomyelitis (EAE)-susceptible DA and EAE-resistant PVG rats were immunized with myelin oligodendrocyte glycoprotein (MOG) to induce an autoimmune response.<br>Seven days later draining inguinal lymph nodes were removed. 2 conditions were examined: 'ex vivo' and 'MOG restimulated', which involved 24hrs of incubation with an encephalogenic MOG 91-108 peptide.
Project description:Experimental autoimmune encephalomyelitis (EAE)-susceptible DA and EAE-resistant congenic R23 rats were immunized with myelin oligodendrocyte glycoprotein (MOG) to induce an autoimmune response.<br><br>Seven days later draining inguinal lymph nodes were removed. 2 conditions were examined: 'ex vivo' and 'MOG restimulated', which involved 24hrs of incubation with an encephalogenic MOG 91-108 peptide.<br><br>
Project description:To investigate the influence of CNS3, a cis-regulatory element in the Foxp3 locus, on the selection of T cell antigen receptor (TCR) repertoire of regulator CD4+ T cells (Treg), we crossed Foxp3ΔCNS3-gfp or control Foxp3gfp mice to DO11.10 TCRβ transgene and Tcra-/+ background. We isolated Treg and conventional CD4+T cells from thymus, spleen and lymph nodes of Foxp3ΔCNS3-gfp DO11.10 TCRβ Tcra-/+ or Foxp3gfp DO11.10 TCRβ Tcra-/+ male littermates, and sequenced the TCRα chains. Analysis of the diversity of Complementary Determining Region 3 (CDR3) of TCRα showed a distinct clustering of CNS3-deficient Treg cells from the CNS3-sufficient ones. Overall design: DO11.10 TCRβ transgene inhibits the recombination of endogenous Tcrb loci thus restricting TCR repertoire to TCRα chains expressed by T cells. Further limitation of the TCR repertoire was achieved by the presence of one functional Tcra gene. With restricted TCR repertoire, mRNA of TCRα was extracted from Treg and conventional CD4+ T cells for library preparation and high throughput sequencing.
Project description:T cells are the main responding arm of the immune system to allografts. One mechanism that may be encouraging tolerance to allografts is T regulatory cells, a CD4+ T cell phenotype that displays antigen-directed immune suppression. T regulatory cells are reduced after allografting in the graft draining lymph node compared to the syngeneic graft draining lymph node, and miRNAs may be responsible for this decrease in suppressive cells. We used microarrays to detail what miRNAs are dysregulated after allografting in purified CD4+ T cells to identify what miRNAs are hindering the expansion of pro-tolerogenic T regulatory cells. Overall design: Female C57BL/6 mice were given either tail-skin syngeneic grafts (C57BL/6 --> C57BL/6) or allogenic grafts (C3H --> C57BL/6) on the dorsal lateral surface of the recipient mouse. Allograft directed inflammation or syngeneic graft acceptance proceeded for 10 days. Mice were sacrificed and their draining lymph nodes (axillary and brachial) were harvested. CD4+ T cells were purified from single cell suspensions of this population via magnetic bead isolation. Purity was verified to be > 90% CD4+ by flow cytometry before the purified cells were subjected to total RNA extraction before hybridization on Affymetrix arrays. Naive CD4+ T cells from axillary and brachial lymph nodes were extracted and purified as well for a non-surgery control. Thus, samples were from three groups: Naive CD4+ T cells (N4), allograft-responding CD4+ cells (Al4) or syngeneic graft-responding CD4+ cells (Au4).
Project description:We grouped Foxp3+ cells from Foxp3 Timer reporter mice into Blue1, Blue2, Red1, and Red2, and sorted these cells according to their Timer fluorescence expression, and analysed the transcriptional profiles of those cells, comparing them with those of effector and naive T cells using RNA-seq. Foxp3 Timer reporter mice were sensitised by a hapten, and 5 days later, the draining LNs of the skin were analysed. Overall design: Foxp3Timer mice were sensitised with application of 3% Oxazolone to abdominal skin. 5 days later inguinal and axillary Lymph nodes were harvested and pooled within the 3 biological replicates. The following populations of T-cells were sorted from each mouse: 1. CD44loFoxp3Timer- (Naïve T-cells) 2. CD44hiFoxp3Timer- (Activated effector T-cells) 3. CD4+ Pers1 (BL1, persistent Foxp3 transcription) 4. CD4+ Pers2 (BL2, persistent Foxp3 transcription) 5. CD4+ PAt (RD1, quiescent Treg) 6. CD4+ Arrested (RD2, arrested Foxp3 transcription) RNA was extracted and RNA-seq data obtained, generating a triplicate dataset for the 6 T-cell populations.
Project description:To determine the influence of primary tumors on pre-metastatic lymph nodes, we have employed whole genome microarray expression profiling as a discovery platform to identify gene signatures of stromal cells from tumor-draining lymph nodes, compared with normal lymph nodes. We subcutaneously inoculated C57BL/6 mice with the 4T1 mammary carcinoma. Two weeks later, tumor-draining lymph nodes were dissociated and stromal cells (CD45-) were sorted. Lymph nodes stromal cells from normal mice without tumor bearing were set as controls. Overall design: Primary tumor induced gene expression in stromal cells from tumor-draining lymph nodes was measured at 2 weeks after tumor inoculation subcutaneously. Lymph nodes stromal cells from normal mice without tumor bearing were set as controls.
Project description:To determine the influence of primary tumors on pre-metastatic lymph nodes, we have employed whole genome microarray expression profiling as a discovery platform to identify gene signatures of B cells from tumor-draining lymph nodes, compared with normal lymph nodes. We subcutaneously inoculated C57BL/6 mice with the 4T1 mammary carcinoma. Two weeks later, tumor-draining lymph nodes were dissociated and B cells (CD19+) were sorted. Lymph nodes B cells from normal mice without tumor bearing were set as controls. Overall design: Primary tumor induced gene expression in B cells from tumor-draining lymph nodes was measured at 2 weeks after tumor inoculation subcutaneously. Lymph node B cells from normal mice without tumor bearing were set as controls.
Project description:Investigating the gene expression profile changes between 4T1LN and 4T1PT. Overall design: Balb/c mice were injected with 4T1 cells at both site of 4th mammary fat pad. Three weeks later, mice were sacrificed and primary tumors and inguinal tumor-draining lymph nodes were harvested. Primary tumors from both 4th fat pads were excised, minced with scissors, and then digested in collagenase type I (150 U⁄ ml) plus hyaluronidase (50 U/ml) for 16 hr at humidified 37 °C incubator supplemented with 5% CO2. Inguinal tumor-draining LNs were excised and dissociated by mechanical disruption. Tissues were dissociated by 100 μm cell strainers from BD Biosciences (San Jose, CA, USA) and maintained in complete RPMI 1640 medium containing 60 μM 6-thioguanine (Sigma-Aldrich, St. Louis, MO, USA) and seeded into 10 cm dishes. After 10-14 days, cells were harvested and stained with APC-conjugated rat anti-mouse CD24 (#101814, Biolegend, San Diego, CA, USA) and PE-conjugated hamster anti-mouse CD29 antibodies (#102208, Biolegend, San Diego, CA, USA) for 30 mins at 4℃. The CD24+CD29+ 4T1 cell population was sorted on a FACS Aria II cell sorter. CD24+CD29+ 4T1 cell sorted from Inguinal tumor-draining LNs called 4T1LN cells, CD24+CD29+ 4T1 cell sorted from primary tumors called 4T1PT cells.