Project description:Next Generation Sequencing of Immunized Mouse Splenocytes to Develop an anti-TIM3 Chimeric Antigen Receptor for Acute Myeloid Leukemia
Project description:Spike-specific T and B cells from splenocytes of immunized mice were profiled with 10x sequencing for gene expression (Chromium Next Automated GEM 5’ v2 kit), cell surface marker (Chromium Automated 5’ Feature Barcode kit) and immune receptor (Chromium Automated Mouse BCR/TCR Amplification and Library Construction kit)
Project description:mouse 4T1 breast cancer stem cell spheres were co-culutred with in vivo tumor antigen primed splenocytes, with in vivo tumor antigen primed splenocytes plus ex vivo reinforced activation via anti-CD3/CD28 beads or without co-culturing with splenocytes. Stem cell spheres were then collected and sunjected for gene expression analyses using RNA sequencing.
Project description:To explore TNF-related genes in GPI-induced arthritis, we performed GeneChip analysis using arthritic splenocytes and control-immunized splenocytes. Among the arrayed TNFalpha-related genes, TIARP mRNA was highly expressed in arthritic splenocytes, with levels exceeding more than 20-times the control splenocytes
Project description:The aim of the study was to compare and contrast cytokine production by CD4+ chimeric antigen receptor + T-cells and putative myeloid derived suppressor cell populations (CD11b+Gr-1 hi and lo) in the spleens of Balb/c mice which had received a transfer of CD19 specific second-generation CAR T-cells 56 days previously following cyclophosphamide pre-conditioning. Splenocytes from four individual mice were pooled, incubated with antibodies to CD4, CD34 (CAR), CD11b, Gr-1 and sorted using flow cytometric cell sorting for CD4+CD34+, Gr-1 and the negative cell fraction. Total RNA was isolated and samples loaded in duplicate in the array. Data was normalized to global expression levels.
Project description:Chimeric antigen receptor (CAR) therapy targeting CD19 yielded remarkable outcomes in patients with acute lymphoblastic leukemia. To identify potential CAR targets in acute myeloid leukemia (AML), we probed the AML surfaceome for over-expressed molecules with potentially tolerable systemic expression. We integrated large transcriptomics and proteomics data sets from malignant and normal tissues, and developed an algorithm to identify potential targets expressed in leukemia stem cells, but not in normal CD34+CD38– hematopoietic cells, T cells or vital tissues. As these investigations did not uncover candidate targets with a profile as favorable as CD19, we developed a generalizable combinatorial targeting strategy fulfilling stringent efficacy and safety criteria. Our findings indicate that several target pairings hold great promise for CAR therapy of AML.
Project description:The major goal of this experiment was to identify the overall changes in gene expression of human CD4+CD28 null T-cells that develop after repetitive [xeno] antigen stimulation, in comparison to normal CD4+CD28+ T-cells. These cells were derived by adoptive transfer of human CD4 T-cells from a normal donor (all CD28+) into immunodeficient mice. Chimeric human CD4+CD28+ were isolated from 4 mice, and chimeric human CD4+CD28 null cells from two of these animals after expansion and T-cell differentiation in vivo. Animals were euthanized, and single cell suspensions of splenocytes derived. CD4 T-cell subpopulations were isolated by fluoresence activated cell sorting, RNA was extrated, labeled and hybridized to whole human gene expression arrays. Overall changes in the gene expression were identified using GEDI (gene expression dynamic inspector). 603 genes were found to be statistically significant (P<0.05) between the CD4+CD28+ and CD4+CD28null cells. Candidate genes were validated using qRT-PCR
Project description:Several next-generation cancer immunotherapies are designed to target LAG3 and TIM3 checkpoints, though the molecular and cellular mechanisms by which both receptors operate to mediate their anti-tumour effects are still poorly understood. Here, we examined the phenotypical and transcriptional consequences of treatment with bispecific antibodies (bsAbs), currently being tested in clinical trials, which co-target PD1 and either TIM3 or LAG3, respectively, using scRNAseq.
Project description:To explore TNF-related genes in GPI-induced arthritis, we performed GeneChip analysis using arthritic splenocytes and control-immunized splenocytes. Among the arrayed TNFalpha-related genes, TIARP mRNA was highly expressed in arthritic splenocytes, with levels exceeding more than 20-times the control splenocytes The spleens of three GPI-GST (MW=89 kD) (300 µg) -immunized DBA/1 mice were harvested on day 10. As a control, the spleens of three GST (MW=26 kd) (100 µg) -immunized DBA/1 mice were used. Total RNA was extracted from the splenocytes using Isogen (Nippon gene), then 15 ug of RNA was utilized for cDNA synthesis by reverse transcription followed by synthesis of biotinylated cRNA through in vitro transcription. After cRNA fragmentation, hybridization with mouse 430A2.0 GeneChip (Affymetrix, Santa Clara, CA) with probes for 43,000 mouse genes ESTs was performed according to the protocol provided by the manufacturer. Analysis was performed by gene expression software
Project description:Although the landscape for treating acute myeloid leukemia (AML) patients has changed substantially in recent years, the majority of patients will eventually relapse and succumb to their disease. Allogeneic stem cell transplantation provides the best anti-leukemia treatment strategy, but is only suitable in a minority of patients. In contrast to B-cell neoplasias, chimeric antigen receptor (CAR) T-cell therapy in AML has encountered challenges in target antigen heterogeneity, safety, and T-cell dysfunction. We developed a Fab-based adapter CAR (AdCAR) T-cell platform with flexibility of targeting and control of AdCAR T-cell activation. Utilizing AML cell lines and a long-term culture assay for primary AML cells, we were able to demonstrate AML-specific cytotoxicity using anti-CD33, anti-CD123, and anti-CLL1 adapter molecules in vitro and in vivo. Notably, we show for the first time the feasibility of sequential application of adapter molecules of different specificity in primary AML co-cultures. Importantly, utilizing the AML platform, we were able to demonstrate that continuous adapter molecule exposure led to AdCAR T-cell exhaustion through chronic AdCAR stimulation, which was counteracted through treatment-free intervals. As CAR T-cell exhaustion is a well-known cause of resistance, the AdCAR platform might ameliorate CAR T-cell dysfunction.