Project description:Increasing evidence indicates oncogenes and tumor suppressors not only influence cell fitness but can also control the immunophenotype of cells. Here, we examined how 34 commonly mutated genes in colorectal cancer (CRC) may influence the expression of 8 key immunomodulatory proteins. To do this, we employed a functional genomics approach utilizing Pro-Code/CRISPR libraries for high-dimensional analysis. We introduced a library of 102 Pro-Code/gRNA combinations, targeting each of the 34 genes, in CT26 cells, a CRC cell model, and measured the expression of each of the immunomodulatory proteins by CyTOF mass cytometry. Notably, cells carrying a Pro-Code/CRISPR targeting the Trp53 lost expression of the immune co-stimulatory molecule CD80. Validation confirmed that Trp53 knockout resulted in the loss of CD80 and that activation of P53, through DNA damage or stabilization, resulted in CD80 upregulation. P53 ChIP-seq identified the CD80 promoter as a direct target of P53. CD80 regulation by P53 was identified in other cells, including normal epithelial cells and macrophages. Functionally, CD80 reduction caused by P53 loss led to a reduced capacity for CRC to prime antigen-specific T cells. These studies establish CD80, a canonical co-stimulatory molecule, as a direct target of the tumor suppressor and DNA damage response gene, P53.
Project description:HIV uses dendritic cells as a carrier to infect its target CD4+ T cells. At the same time, it also hampers DCs function in terms of their T cell stimulatory capacity and cytokine secretion. We have shown that HIV causes reduction in the expression of co-stimulatory molecules, CD80 and CD86 by DCs at the mRNA level irrespective of the viral subtype or the mode in DC-HIV interaction. The microarray experiments were performed to understand the changes in the transcription factor profile of HIV infected DCs which may in turn lead to reduced CD80/CD86 expression as well as the effect of HIV infection on other co-stimulatory molecules on DCs. The data suggests that TFs NFKB1, NFKB2, REL and MYB may be responsible for the observed decrease in CD80 and CD86 expression and that HIV infection also hampers the expression of other co-stimulatory molecules like CD70, CD30, 4-1BB, OX40L etc.
Project description:HIV uses dendritic cells as a carrier to infect its target CD4+ T cells. At the same time, it also hampers DCs function in terms of their T cell stimulatory capacity and cytokine secretion. We have shown that HIV causes reduction in the expression of co-stimulatory molecules, CD80 and CD86 by DCs at the mRNA level irrespective of the viral subtype or the mode in DC-HIV interaction. The microarray experiments were performed to understand the changes in the transcription factor profile of HIV infected DCs which may in turn lead to reduced CD80/CD86 expression as well as the effect of HIV infection on other co-stimulatory molecules on DCs. The data suggests that TFs NFKB1, NFKB2, REL and MYB may be responsible for the observed decrease in CD80 and CD86 expression and that HIV infection also hampers the expression of other co-stimulatory molecules like CD70, CD30, 4-1BB, OX40L etc. The total RNA was isolated from LPS matured DCs (positive control) and HIV infected DCs using Qiagen RNeasy Minikit. Agilent Quick-Amp Labelling kit (p/n 5190-0444) was used to synthesize the labeled cDNA from 3 ug of total RNA and further process the hybridized cDNA on the array. All the steps were carried out according to manufacturerM-bM-^@M-^Ys instructions (www.agilent.com/chem/dnamauals-protocol). The array slides were scanned immediately using high throughput Agilent scanner with SureScan technology. Agilent feature extraction software was used for normalization and statistical analysis,. Pathway and gene ontology analysis was done using Genespring GX v 10.0 and biointerpreter software. The experiments were performed on DCs cultured from two independent donors. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were differentially regulated genes in both the donors.
Project description:Cytotoxic T lymphocytes (CTL) and natural killer cells (NK)-mediated elimination of tumor cells is mostly dependent on Granzyme B apoptotic pathway, which is regulated by the wild type (wt) p53 protein. Because TP53 inactivating mutations, frequently found in human tumors, could interfere with Granzyme B-mediated cell death, the use of small molecules developed to reactivate wtp53 function in p53-mutated tumor cells could optimize their lysis by CTL or NK cells. Here, we analyzed the transcriptomic effect of the pharmalogical reactivation of a wt-like p53 function in p53-mutated breast cancer cells using the small molecule CP-31398.
Project description:NP-reactive murine splenic memory B cells were sorted based on the expression of the surface markers CD80 and PD-L2 AM14 tg+ X Vk8R+/- recipients of B1-8+ B cells were immunized with NP-CGG and 8 weeks later splenic EMA-, CD19+ and NP+ memory B cells were sorted based on their surface expression of CD80 and PD-L2 (DN=CD80- PDL2-, SP=CD80- PDL2+, DP=CD80+ PDL2+). 1x10E5 up to 3x10E5 cells per sample were sorted and total RNA was isolated using the RNeasy plus micro kit and cRNA was prepared using the Illumina TotalPrep RNA Amplification Kit.
Project description:Co-stimulatory molecules of the CD28 family on T lymphocytes integrate cues from innate immune system sensors, and modulate activation responses in conventional CD4+ T cells (Tconv) and their FoxP3+ regulatory counterparts (Treg). To better understand how costimulatory and co-inhibitory signals might be integrated, we profiled the changes in gene expression elicited in the hours and days after engagement of Treg and Tconv by anti-CD3 and either anti-CD28, -CTLA4, -ICOS, -PD1, -BTLA or -CD80. Total CD4+ T cells were stimulated by anti-CD3 and either anti-CD28, -CTLA4, -ICOS, -PD1, -BTLA or -CD80 antibodies for 1, 4, 20 and 48 hrs and Tconv and Treg were separated by flowcytometry. The 1 and 4 hr lysates were pooled [the 'early' samples] before RNA purification and profiling, as were the 20 and 48 hr samples [the 'late' samples] (note; for Treg cells, only the 20 hr sample was used). RNA was amplified, labeled and hybridized to Mouse Gene 1.0 ST arrays with the data generation and quality control pipeline of 19 the Immunological Genome Project (www.immgen.org), in biological triplicates (duplicates only for ICOS and CD80). Raw data were background-corrected and normalized using the RMA algorithm.