Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goal of this study is to compare the transcriptome of OT-1 cells during priming (3 days after infection) and during effector phase ( 7 days after infection) in ODC-OVA mice after LCMV-OVA and Lm-OVA infection
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goal of this study is to compare the transcriptome of CNS-infiltrating OT-1 WT and Tox-deficient cells during effector phase (7 days after infection with LCMV-OVA)
Project description:Next Generation Sequencing of CNS-infiltrating Tox-sufficient and -deficient OT-1 cells in ODC-OVA mice 7 days after LCMV-OVA infection
Project description:The transcriptome of naive OT-I T cells was compared to memory CD8 T cells after 1, 2, 3, or 4 infection with ovalbumin expressing Listeria monocytogenes (LM-OVA). Naive Thy1.1 OT-I T cells were adoptively transferred into Thy1.2 naive hosts prior to infection with LM-OVA. The resulting memory CD8 T cell population was again adoptively transferred into naive hosts and the recipient mice were again infected with LM-OVA. The adoptive transfer was repeated up to four times to generate memory CD8 T cells with up to four consecutive antigen stimulations. Three individual mice were analyzed for each group. For quaternary memory CD8 T cells, spleens from two to three mice were pooled for each sample. Naive OT-I T cells served as control samples. http://dx.doi.org/10.1016/j.immuni.2010.06.014
Project description:The transcriptome of naive OT-I T cells was compared to memory CD8 T cells after 1, 2, 3, or 4 infection with ovalbumin expressing Listeria monocytogenes (LM-OVA).
Project description:Mice received DivisionRecorder+ or unmodified OT-I T cells and were challenged with LM-OVA. During memory phase we FACS sorted memory CD8+ OT-I T cells (and sub-sorted RFP+ and RFP- cells in the case of DivisionRecorder+ cells) from spleen material and performed single cell RNA sequencing.
Project description:Conditional ablation of FXR in T cells was achieved with CD4-Cre Nr1h4fl/fl mice. Twenty thousend congenically marked naive (CD44- CD62L+) WT and FXR KO T cells carrying the OT-I transgenic TCR were cotransferred into lymphoreplete hosts. Recipients were challenged with LCMV-OVA and allowed to feed Ad libitum (AL) or fasted (Fs) for 24h beginning at 6pm on D5 post-infection. Splenic CD44+ effector cells of each genotype were FACS-purified (double-sorted) at the end of the starvation period.
Project description:Purpose: ATAC-seq analysis of naive and three effector OT-I cell subsets (from a Klrg1-Cre fate reporter mouse model) isolated from the spleen of C57BL/6 mice 0 and 8 days post infection with OVA-expressing Listeria monocytogenes. The hypothesis tested in the present study was that chromatin remodeling in KLRG1+ effector CD8 T lymphocytes promotes the differentiation into KLRG1- memory CD8 T lymphocytes that provide long-lasting immunity against infectious diseases and malignancies. Methods: DNA was obtained from 50,000 FACS-purified OT-I cell subsets isolated from spleen 0 and 8 days post infection with ovalbumin-expressing Listeria monocytogenes (LM-OVA) (experiment 3). Results: Using ATAC-seq technology, we analyzed the chromatin accessibility landscape of naive and three effector OT-I cells (KLRG1+ Reporter+, KLRG1- Reporter+ (exKLRG1) and KLRG1- Reporter-). Conclusions: Our study represents the first fate mapping analysis of KLRG1+ effector OT-I cells, demonstrates that KLRG1+ effector OT-I cells differentiate into all memory T cell lineages thereby promoting protective immunity. RNA-seq identified CX3CR1 as a marker of circulating exKLRG1 early memory OT-I cells, and ATAC-seq analysis revealed that chromatin remodeling enabled exKLRG1 memory cells to exhibit both a high cytotoxic and proliferative capacity.
Project description:We report the application of TCRβ sequencing to understand T cell repertoire of matched blood and tumor samples pre and post treatment in EG7.OVA tumor bearing mice with OT-1 cell transfer. TCRβ sequencing demonstrated that OT-1 (CASSRANYEQYF) was the most abundant T-cell clone in both blood and tumors of mRBC‑OVA-4-1BBL-IL-12 treated mice. To investigate the effects of mRBC‑OVA-4-1BBL-IL-12 on immune memory, the T cell repertoire was also analyzed before and after EG7.OVA and EL4 tumor rechallenge in cured and treatment-naive mice. TCRβ sequencing on T cells in peripheral blood showed that OT-1 clones increased in frequency after EG7.OVA challenge in previously cured mice. OT-1 frequency did not increase in treatment-naïve mice after tumor challenge, indicating that the tumor alone is insufficient to drive OT-1 cell expansion. We also evaluated the frequencies of unique TCRβ sequences in T cell clones that significantly expanded post EL4 challenge (EL4 responsive TCR). Increased frequency of EL4-responsive TCRs upon each tumor challenge (EG7.OVA and EL4) was associated with complete responders (mice that rejected EL4 challenge), suggesting that T-cell-mediated protection against parental tumor antigens was generated prior to EL4 challenge. Partial responders (delayed tumor growth compared with naïve mice) had increases in EL4-responsive TCR frequencies after EL4 challenge but not during the EG7.OVA rechallenge, whereas the non-responder (tumor growth similar to naïve mice) had minimal increases in TCR frequencies upon EL4 challenge. Overall, the ability to control EL4 tumors correlated with the expansion of EL4-responsive TCR clones.
Project description:Cognate antigen signals control CD8+ T cell priming, expansion size and effector versus memory cell fates, however, it is not clear whether they can also modulate the functional features of memory CD8+ T cells. We observed that OT-I cells that were primed with weak cognate antigen signals incorporate more cytokine signals, leading to a hypothesis that CD8+ T cells that receive weak TCR signals require cytokine signals to form functional memory. Using a previously described mouse model in which IL-2 signaling via its high affinity receptor CD25 is selectively impaired, the “Il2ramut/mut” mouse, we conducted a comparative analysis of gene expression and epigenetic landscape of Il2ramut/mut and WT OT-I memory cells that were primed with strong (Lm-Ova N4) versus weak (Lm-Ova T4). RNA seq data showed that both TCR and IL-2 priming signals have minimal effect on gene expression in resting memory CD8 T cells, but they significantly modify the epigenetic landscape of the memory CD8 T cells. These findings have important contributions to the current understanding of how priming signals program memory CD8 T cells in vivo.
