Anticancer immunity 2013


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Anticancer immunity 2013

  1. 1. Anti-Cancer Immunity Jesse Liang, Ph.D. 1 Sample & Assay Technologies
  2. 2. Cancer from an Immune Perspective Cancers are not infectious agents and therefore lack the molecular signatures that normally enable the immune system to recognize and react. This appears to be the root of the problem. This is not to say that tumors are invisible to the immune system. Tumors are frequently heavily infiltrated by macrophages and neutrophils. But such cells are often actively recruited by the tumor and can promote tumor proliferation, angiogenesis, and progression through the production of inflammatory mediators and reactive oxygen or nitrogen species that provoke DNA damage and thus additional mutations. -2- Sample & Assay Technologies
  3. 3. Cancer Immunoediting Strongly immunogenic tumors would fail to develop to the point that they become clinically significant. In this way, the immune system may exert a selective pressure for cancercausing mutations (such as ras point mutation, complete loss of p53 or Rb) that are largely immunologically silent – a process that is termed immunoediting. . In other words, immunoediting is a process ranging from immune surveillance to immune escape. It’s an immune selection. . -3- Sample & Assay Technologies
  4. 4. Cancer Immunoediting – 3E Theory . . Phase 1: Recognition, infiltrating, recruiting Phase 2: Induce tumor death Elimination . Phase 3: Promote more tumor death Phase 4: Destroy all tumor cells Equilibrium: Tumor cells which have survived the elimination phase enter the equilibrium phase. . Escape: Tumor cells which have acquired resistance to equilibrium enter the escape phase. . -4- Sample & Assay Technologies
  5. 5. Cancer Immunoediting -5- Sample & Assay Technologies
  6. 6. Tumor Escape Mechanisms – Immunosuppression . Tumor-Derived Soluble Factors Tumor cells produce immunosuppressive factors: . IL-10, TGF-β, VEGF, Prostaglandins, etc. . . Cellular Components: Regulatory T cells (Tregs), Myeloid-derived suppressor cells (MDSCs) and invariant Natural Killer T cells (iNKTs). . -6- Sample & Assay Technologies
  7. 7. How do Cancer Cells Become Resistant to Elimination? Paper 1: We demonstrate that the strong immunogenicity of an unedited tumor can be ascribed to expression of highly antigenic mutant proteins. Outgrowth of tumor cells that lack these strong antigens through a T-cell-dependent immunoselection process represents one mechanism of cancer immunoediting (Nature, Feb 2012). . . -7- Sample & Assay Technologies
  8. 8. How do Cancer Cells Become Resistant to Elimination? Paper 2: By comparing the development of tumors in immunecompetent mice to that in mice with broad immunodeficiency or specific antigenic tolerance, we show that recognition of tumor-specific-antigens by lymphocytes is critical for immunoediting. Furthermore, primary sarcomas were edited to become less immunogenic through the selective outgrowth of cells that were able to escape T cell attack. Loss of tumor antigen expression or presentation on MHC (Major Histocompatibility Complex) class I was necessary and sufficient for this immunoediting process to occur. These results highlight the importance of tumor-specific-antigen expression in immune surveillance, and potentially, immunotherapy (Nature, Feb 2012). . . -8- Sample & Assay Technologies
  9. 9. Conclusion T cell recognition of tumor antigens is key to cancer immunoediting. The Clinical Case of Cancer Immunoediting: Most glioblastoma at relapse are negative for EGFRvIII expression, a relevant and direct example of cancer immunoediting (Expert Rev Anticancer Ther, Nov 2011). . . . -9- Sample & Assay Technologies
  10. 10. The Induction of Anti-Cancer Immunity 1. Tumor cells express tumor-associated antigens on MHC class I. . 2. Cytotoxic T cells recognize tumor-associated antigens and kill tumor cells. . 3. Dendritic cells (DCs), which are important antigen presenting cells (APCs), take up and process antigen from dead or dying tumor cells, present the tumor-associated antigens on MHC class I and II and stimulate T cells and other immune cells. . - 10 - Sample & Assay Technologies
  11. 11. Anti-Cancer Immunotherapies . 1. Nonspecific immune stimulation: * Stimulate T cells IL-2 . IFNα * Activate dendritic cells . TLR7 agonist imiquimod anti-CD25 antibody * Inhibit / deplete Treg cells . low dose cyclophosphamide - 11 - Sample & Assay Technologies
  12. 12. Anti-Cancer Immunotherapies 2. Immune Checkpoint Blockade Block CTLA4-B7 and PD1-PDL1 interactions – The CTLA4-blocking antibody ipilimumab (Bristol-Myers Squibb) has been approved by FDA for treating melanoma in March 2011. - 12 - Sample & Assay Technologies
  13. 13. Anti-Cancer Immunotherapies . 3. Adoptive Cell Transfer Two main approaches are being explored: . * T cells that reside in the tumor are cultured and expanded ex vivo in the presence of IL-2. When enough of these polyclonal T cells are obtained, they are re-infused into the patient. . * Isolated peripheral blood T cells are transfected to express tumorantigen-specific TCRs (T Cell Receptors) and then re-administered to the patient. This strategy has the advantage that enough T cells can be obtained for infusion in all patients, but a potential drawback is that the TCRs that are transfected into the T cells have a limited antigen-specificity repertoire. . - 13 - Sample & Assay Technologies
  14. 14. Anti-Cancer Immunotherapies . 4. Cellular Vaccines * Tumor cell or tumor antigen vaccination Extracted, irradiated tumor cells are re-administered to the patient. Adjuvants such as TLR agonists or GM-CSF are required. . . * APC vaccination APCs are extracted from a patient’s bloodstream, cultured and activated with cytokines or adjuvants, loaded with tumor antigen ex vivo, and re-administered to the patient. Sipuleucel-T (made by Dendreon) has been approved by FDA for prostate cancer treatment in April 2010. . . * DC vaccination DCs are cultured from peripheral blood monocytes in the presence of IL-4 and GM-CSF, and activated and loaded with tumor antigen ex vivo, and readministered to the patient. . . - 14 - Sample & Assay Technologies
  15. 15. Inflammasomes and Anti-Cancer Immunity Inflammasomes in carcinogenesis and anticancer immune responses . Nature Immunology 13, 343-351, 2012 (March 18, 2012) . The inhibition of inflammasomes or neutralization of their products, mainly interleukin 1β (IL1β) and IL-18, has profound effects on carcinogenesis and tumor progression. . --------------------------------------------------------------------------------------------------------------------------- . Inflammasomes PCR Arrays . . - 15 - Sample & Assay Technologies
  16. 16. Profiling Gene Expressions – Using RT2 Profiler PCR Array 84 Pathway-Specific Genes of Interest 5 Housekeeping Genes Genomic DNA Contamination Control Reverse Transcription Controls (RTC) n=3 Positive PCR Controls (PPC) n=3 - 16 - Sample & Assay Technologies
  17. 17. PCR Array Work Flow cDNA Synthesis (kit) 45 minutes Load Plates (Use 8-Channel Pipettors) 2 minutes Run 40 cycle qPCR Program 2 to 2.5 hours compatible with all major real-time PCR instruments Upload and Analyze Data 15 minutes - 17 - Sample & Assay Technologies
  18. 18. T Cell and B Cell Activation PCR Array . T-Cell Activation: Regulators of T-Cell Activation: CD2, CD276, CD47, DPP4, CD3D, CD3E, CD3G, CD4, CD7, CD80, CD86, CD8A, CD8B, FOXP3, ICOSLG, IRF4, LAG3, LCK, MAP3K7 (TAK1), MICB, NCK1, TNFSF14, VAV1. T-Cell Proliferation: CD28, CD3E, ICOSLG, IL1B, IL10, IL12B, IL18, NCK1, RIPK2, TNFSF14. T-Cell Differentiation: ADA, APC, BCL2, BLM, CD1D, CD2, CD27 (TNFRSF7), CD4, CD80, CD86, EGR1, IL12B, IL15, IL2, IRF4, NOS2 (iNOS), PTPRC, SOCS1. T-Cell Polarization: CCL3, CCR1, CCR2, CCR3, CCR4, CCR5, CD274, CD28, CD4, CD40LG (TNFSF5), CSF2 (GM-CSF), CXCR3, CXCR4, IFNG, IL12A, IL12RB1, IL12RB2, IL18R1, IL2, IL4, IL4R, IL5, TGFB1. Regulators of Th1 and Th2 Development: CD2, CD40 (TNFRSF5), CD5, CD7, CSF2 (GM-CSF), IFNG, IL10, IL12A, IL13, IL3, IL4, IL5, TLR2, TLR4, TLR9. Th1 & Th2 Differentiation: CD28, CD40 (TNFRSF5), CD40LG (TNFSF5), CD86, IFNG, IL12A, IL12B, IL12RB1, IL12RB2, IL18, IL18R1, IL2, IL2RA, IL4, IL4R, IL6. . B-Cell Activation: Antigen Dependent B-cell Activation: CD28, CD4, CD40 (TNFRSF5), CD40LG (TNFSF5), CD80, FAS (TNFRSF6), FASLG (TNFSF6), IL10, IL2, IL4. Other Genes involved in B-Cell Activation: ADA, CXCR5, ICOSLG, IL6, IL7, MS4A1, TGFB1. B-Cell Proliferation: BCL2, CD27 (TNFRSF7), CD40 (TNFRSF5), CD81, IL10, IL7, PTPRC. B-Cell Differentiation: ADA, AICDA, BLNK, CD27 (TNFRSF7), IL10, IL11, IL4, RAG1. . Other Immune Cell Activation: Macrophage Activation: IL13, IL4, TLR1, TLR4, TLR6. Neutrophil Activation: IL8. Natural Killer Cell Activation: CD2, IL12A, IL12B, IL2. Leukocyte Activation: CX3CL1. . . - 18 - Sample & Assay Technologies
  19. 19. Th1-Th2 Responses PCR Array . Th1 Genes: Th1 Markers: CCR5, CXCR3, IFNG, STAT4, TBX21. Th1 Immune Response: CCR2, CD80, EBI3, IL12B, IL18, IL1RL1 (ST2), IL27, IL27RA, IL4R (CD124), SLC11A1, SOCS5, TLR4, TLR6, TNFSF4, VEGFA. Other Th1-Related Genes: CD28, CD40LG (TNFSF5), CSF2 (GM-CSF), HAVCR2, IL12RB2, IL18R1, IL2, IL2RA, IRF1, SOCS1, STAT1, TNF. Cell-Mediated Immunity: BCL6, CD27 (TNFRSF7), CD28, CD40LG (TNFSF5), IFNG, IL10, IL12B , IL2, IL27RA, IL4, IL7R, LTA (TNFB), PTPRC, SLC11A1, STAT6, TBX21, TLR4, TNF, TNFSF4. Th2 Genes: Th2 Markers: CCR4, GATA3, IL13, IL25 (IL17E), IL2RA (CD25), IL4, IL4R (CD124), IL5, STAT6. Th2 Immune Response: BCL6, CCR2, CD86, IL10, IL18, IL27RA, IL6, SOCS5, TNFSF4. Other Th2-Related Genes: CCL11 (Eotaxin), CCL5 (RANTES), CCL7 (MCP-3), CCR3, CEBPB, GFI1, GPR44, ICOS, IL13RA1, IL1R1, IL9, IRF4, JAK1, MAF, NFATC1, NFATC2, PCGF2 (RNF110). Humoral Immunity: CD28, EBI3, IFNG, IL6, IL7, LTA (TNFB), TNF. CD4+ T Cell Markers: CD27 (TNFRSF7), CD4, CD40LG (TNFSF5), CD80, CD86, CREBBP, CTLA4, FASLG (TNFSF6), IL15, IL6, IL6R, IL7, JAK2, LAG3, LAT, MAPK8 (JNK1), PTPRC, TGFB3, TNFRSF8, TNFRSF9, TNFSF4 (OX40L), TYK2, YY1. Other Genes Involved in Inflammatory Response: JAK2, LTA (TNFB), SPP1 (Osteopontin). Other Genes Involved in Immune Response: IL3, SFTPD. . . . . . . . . . . ----------------------------------------------------------------------------------------------------------------------------------------------Nakata Y, Brignier AC, Jin S, Shen Y, Rudnick SI, Sugita M, Gewirtz AM. c-Myb, Menin, GATA-3, and MLL form a dynamic transcription complex that plays a pivotal role in human T helper type 2 cell development. Blood. 2010 Aug 26;116(8):1280-90. . . . - 19 - Sample & Assay Technologies
  20. 20. Th17 PCR Array . Surface Molecules: CD28, CD34, CD4, CD8A, ICAM1, ICOS, TLR4. . Chemokines: CCL1 (I-309), CCL2 (MCP-1), CCL20 (MIP-3A), CCL22 (MDC), CCL7 (MCP-3), CX3CL1, CXCL1, CXCL12 (SDF1), CXCL2, CXCL5 (ENA78/LIX), CXCL6 (GCP-2), IL8, MMP3, MMP9. . Cytokines: CSF2 (GM-CSF), CSF3 (GCSF), IFNG, IL10, IL12B, IL13, IL15, IL17A, IL17C, IL17D, IL17F, IL18, IL1B, IL2, IL21, IL22, IL23A, IL25 (IL17E), IL27, IL3, IL4, IL5, IL6, IL9, TGFB1, TNF. . Cytokine Receptors: CCR2, CCR4, CCR6, IL1R1, IL12RB1, IL12RB2, IL17RA, IL17RB, IL17RC, IL17RE, IL23R, IL6R, IL7R. . Signaling Pathway Molecules and Transcriptional Factors: CD40LG (TNFSF5), CEBPB, CLEC7A (Dectin-1), FOXP3, GATA3, IRF4, ISG20, JAK1, JAK2, NFATC2, NFKB1, RORA, RORC, RUNX1 (AML1), S1PR1, SOCS1, SOCS3, STAT3, STAT4, STAT5A, STAT6, SYK, TBX21, TRAF6. --------------------------------------------------------------------------------------------------------------------------------------------Kobayashi T, Matsuoka K, Sheikh SZ, Elloumi HZ, Kamada N, Hisamatsu T, Hansen JJ, Doty KR, Pope SD, Smale ST, Hibi T, Rothman PB, Kashiwada M, Plevy SE . NFIL3 is a regulator of IL-12 p40 in macrophages and mucosal immunity. J Immunol. 2011 Apr 15;186(8):4649-55. . . . . - 20 - Sample & Assay Technologies
  21. 21. Cytokines and Chemokines PCR Arrays . Chemokines: CCL1, CCL11, CCL13, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL24, CCL3, CCL5, CCL7, CCL8, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, CXCL2, CXCL5, CXCL9, PF4, PPBP, XCL1. . Interleukins: IL10, IL11, IL12A, IL12B, IL13, IL15, IL16, IL17A, IL17F, IL18, IL1A, IL1B, IL1RN, IL2, IL21, IL22, IL23A, IL24, IL27, IL3, IL4, IL5, IL6, IL7, IL8, IL9. . Interferons: IFNA2, IFNG. . Growth Factors: BMP2, BMP4, BMP6, BMP7, CNTF, CSF1, CSF2, CSF3, GPI, LIF, MSTN, NODAL, OSM, THPO, VEGFA. . TNF Superfamily: CD40LG, FASLG, LTA, LTB, TNF, TNFRSF11B, TNFSF10, TNFSF11, TNFSF13B. . Other Cytokines: ADIPOQ, MIF, SPP1, TGFB2. . Anti-Inflammatory Cytokines: CCL18, CCL19, CCL21, IL10, IL11, IL12A, IL12B, IL13, IL18, IL2, IL22, IL23A, IL24, IL4, IL6, TGFB2. . - 21 - Sample & Assay Technologies
  22. 22. T Helper Cell Differentiation PCR Arrays . Cytokines & Receptors: CCL5 (RANTES), CCL7 (MCP-3), CCR3, CCR4, CCR5, CCR6, IL12B, IL12RB2, IL13, IL13RA1, IL17A, IL17RE, IL18, IL18R1, IL18RAP, IL1R1, IL1R2, IL1RL1, IL2, IL21, IL2RA, IL4, IL4R, IL5, IL9, TNF. . . T Helper 1 Subtype Markers: CCR5, HAVCR2, IGSF6, IL12B, IL18, IRF1, SOCS1, SOCS5, TLR4, TLR6, TNF. . . T Helper 2 Subtype Markers: CCL5 (RANTES), CCL7 (MCP-3), CCR3, CCR4, CEBPB, GFI1, GPR44, ICOS, IL13RA1, IL4R, JAK1, NFATC1, NFATC2. . . Transcription Factors: CEBPB, FOSL (FRA-1)1, FOXP3, GATA3, GATA4, HOXA10, HOXA3, ID2, IRF4, IRF8, MAF, NFATC1, NFATC2, NR4A1, NR4A3, POU2F2, REL, RELB, RORA, RORC, RUNX1 (AML1), RUNX3, STAT1, STAT4, STAT6, TOX, ZBTB7B. . . Epigenetically Regulated Genes: Th1 Cells: EOMES, IFNG, IL12RB2, IL18R1, IL18RAP, FASLG (TNFSF6), TBX21. Th2 Cells: ASB2, GATA3, IL13, IL1RL1, IL4, IL5, PPARG. Th17 Cells: IL17A, IL17RE, IL1R1, IL21, RORA, RORC. Inducible and Natural Regulatory T (iTreg and nTreg) Cells: CCR6, FOSL1 (FRA-1), FOXP3, IKZF2, IL9, IRF4, IRF8, MYB, NR4A1, NR4A3, POU2F2, REL, RELB, TGIF1, TNFSF11. Conventional Versus Regulatory T Cells: CACNA1F, CHD7, FOXP3, GATA4, HOPX, HOXA10, HOXA3, ID2, IKZF2, IL1R2, IL2RA, KIF2C, LRRC32, PERP, PKD2, TNFRSF9, TP53INP1, UTS2. . - 22 - Sample & Assay Technologies
  23. 23. T Cell Anergy & Immune Tolerance PCR Array . T-Cell Regulators: BTLA, CBLB, CD27, CD28, FAS, FOXP3, IL15, IL2, IL2RA, IL4, LAT, TGFB1, TNFRSF14, TNFSF14. B-Cell Regulators: BTLA, CD27, CD40, CD40LG, FOXP1, FOXP3, HDAC9, IL4. Cytokines, Cytokine Receptors and their related Proteins: CCL3L1, CCR4, CD40LG, CD70, CSF1, CSF2, FASLG, IFNG, IL10, IL10RA, IL13, IL15, IL17A, IL1A, IL2, IL2RA, IL2RB, IL31, IL4, IL5, IL6, IL7R, LEP, LTA, PRF1, PTGER2, PTGS2, TGFB1, TNFSF10, TNFSF14, TNFSF8. TNF Superfamily Members and their Receptors: CD40LG, CD70, FAS, FASLG, LTA, TNFRSF10A, TNFRSF4, TNFRSF18, TNFRSF4, TNFRSF8, TNFRSF9, TNFSF10, TNFSF14, TNFSF8. Transcriptional Regulators: CDK2, CDK4, EGR2, EGR3, EOMES, FOS, FOXP1, FOXP2, FOXP3, GATA3, HDAC9, IFNG, ING4, IRF4, JAK3, JUN, MEF2A, NFATC1, NFATC2, NFATC3, NFKB1, NHLH2, NOTCH1, STAT3, STAT6, TBX21, TGFB1. Other Genes involved in T-cell Anergy: CMA1, CTLA4, DGKA, DGKZ, GZMB, ICAM1, ICOS, ITCH, ITGA1, JAK1, LGALS3, PDCD1, PRKCG, RNF128, SELL. ----------------------------------------------------------------------------------------------------------------------------------------------. . . . . . . . . . . . . Gorczynski RM, Chen Z, He W, Khatri I, Sun Y, Yu K, Boudakov I Expression of a CD200 transgene is necessary for induction but not maintenance of tolerance to cardiac and skin allografts. J Immunol. 2009 Aug 1;183(3):1560-8. . . - 23 - Sample & Assay Technologies
  24. 24. Dendritic & Antigen Presenting Cells (APCs) PCR Array . . . . . . . . . . . . . . . . Antigen Uptake: CD44, CDC42, ICAM1, ICAM2, RAC1, TAP2 (ABCB3). Antigen Presentation: CCL19, CD1A, CD1B, CD1C, CD1D, CD209, CD28, CD4, CD40 (TNFRSF5), CD40LG (TNFSF5), CD74, CD80, CD86, CD8A, HLA-A, HLA-DMA, HLA-DPA1, TAPBP, THBS1. Dendritic Cell Chemotaxis: CCL19, CCL5 (RANTES), CCR1, CCR2, CCR5, CXCR1 (IL8RA), CXCR4. Dendritic Cell Differentiation: CSF2 (GM-CSF), LYN, RELB, TGFB1. Cytokines: CCL11 (Eotaxin), CCL13 (MCP-4), CCL16 (HCC4), CCL19, CCL2 (MCP-1), CCL3 (MIP-1A), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CSF2 (GM-CSF), CXCL1, CXCL10 (INP10), CXCL12 (SDF1), CXCL2, FLT3LG, IFNG, IL10, IL12A, IL12B, IL16, IL2, IL6, IL8, MIF, TNF, TNFSF11. Cytokine Receptors: CCR1, CCR2, CCR3, CCR5, CSF1R, CXCR1 (IL8RA), CXCR4, FLT3, ERBB2 (HER2). Other Cell Surface Receptors: CD2, CD40 (TNFRSF5), FCER1A, FCER2, FCGR1A, LRP1, TLR1, TLR2, TLR7, TLR9, VCAM1. Signal Transduction: CDKN1A (p21CIP1/WAF1), CEBPA, CLEC4C, FAS (TNFRSF6), IRF7, IRF8, ITGAM, ITGB2, LYN, NFKB1, PTPRC, RELA, RELB, STAT3, TGFB1. - 24 - Sample & Assay Technologies
  25. 25. Innate & Adaptive Immune Response PCR Array . Innate Immunity: Pattern Recognition Receptors: DDX58 (RIG-I), NLRP3, NOD1 (CARD4), NOD2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9. Cytokines: CCL2 (MCP-1), CCL5 (RANTES), CSF2 (GM-CSF), CXCL10, IFNA1, IFNB1, IL18, IL1A, IL1B, IL2, IL8, TNF. Other Genes: APCS, C3, CASP1 (ICE), CD14, CD4, CD40 (TNFRSF5), CD40LG (TNFSF5), CD8A, CRP, HLA-A, HLA-E, IL1R1, IRAK1, IRF3, IRF7, ITGAM, LY96 (MD-2), LYZ, MAPK1 (ERK2), MAPK8 (JNK1), MBL2, MPO, MX1, MYD88, NFKB1, NFKBIA (IkBa/Mad3), STAT1, TICAM1 (TRIF), TRAF6. . Adaptive Immunity: Th1 Markers/Immune Response: CCR5, CD80, CXCR3, IFNG, IL18, IL23A, SLC11A1, STAT4, TBX21, TLR4, TLR6. Th2 Markers/Immune Response: CCR4, CCR8, CD86, GATA3, IFNB1, IL10, IL13, IL18, IL4, IL5, IL6, NOD2, STAT6. Th17 Markers: CCR6, IL17A, RORC, STAT3. Treg Markers: CCR4, CCR8, FOXP3, IL10. T Cell Activation: CD80, CD86, ICAM1, IFNG, IL23A, IL6, SLC11A1. Cytokines: CCL2 (MCP-1), CCL5 (RANTES), CSF2 (GM-CSF), CXCL10 (INP10), IFNA1, IFNG, IL10, IL13, IL17A, IL18, IL2, IL23A, IL4, IL5, IL6, IL8, TNF. Other Genes: CD4, CD40 (TNFRSF5), CD40LG (TNFSF5), CD8A, CRP, FASLG (TNFSF6), HLA-A, IFNAR1, IFNGR1, IL1B, IL1R1, IRF3, IRF7, ITGAM, JAK2, MAPK8 (JNK1), MBL2, MX1, NFKB1, RAG1, STAT1. . . . Humoral Immunity: C3, CCL2 (MCP-1), CCR6, CRP, IFNB1, IFNG, IL6, MBL2, NOD2, TNF. Inflammatory Response: APCS, C3, CCL5 (RANTES), CRP, FOXP3, IL1A, IL1B, IL4, IL6, MBL2, STAT3, TNF. Defense Response to Bacteria: IFNB1, IFNG, IL23A, IL6, LYZ, MBL2, MYD88, NOD1 (CARD4), NOD2, SLC11A1, TLR1, TLR3, TLR4, TLR6, TLR9, TNF. . Defense Response to Viruses: CD4, CD40 (TNFRSF5), CD86, CD8A, CXCL10 (INP10), DDX58 (RIG-I), HLA-A, IFNAR1, IFNB1, IL23A, IL6, IRF3, NLRP3, TICAM1 (TRIF), TLR3, TLR7, TLR8, TYK2. . ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- . . . McMahon L, Schwartz K, Yilmaz O, Brown E, Ryan LK, Diamond G. Vitamin D-mediated induction of innate immunity in gingival epithelial cells. Infect Immun. 2011 Jun;79(6):2250-6. - 25 - Sample & Assay Technologies
  26. 26. Toll-Like Receptors (TLRs) PCR Array . Toll-Like Receptors: CD180 (LY64), SIGIRR, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10. Pathogen-Specific Responses: Bacterial: CCL2 (MCP-1), CD14, CD180 (LY64), FOS, HRAS, IL10, IL12A, IL1B, IL6, IL8, IRAK1, HMGB1, HSPA1A (HSP70 1A), JUN, LTA (TNFB), LY86 (MD-1), LY96, NFKBIA (IKBA/MAD3), PTGS2 (COX2), RELA, RIPK2, TLR2, TLR4, TLR6, TNFRSF1A, TICAM1 (TRIF). Viral: EIF2AK2 (PRKR), IFNB1, IFNG, IL12A, IL6, IRF3, PRKRA, RELA, TBK1, TLR3, TLR7, TLR8, TNF, TICAM1 (TRIF). Fungal/Parasitic: CLEC4E, HRAS, HSPA1A (HSP70 1A), IL8, TLR2, TIRAP. TLR Signaling: Negative Regulation:SARM1, SIGIRR, TOLLIP. TICAM1 (TRIF)-Dependent (MYD88-Independent): IRF3, MAP3K7 (TAK1), TAB1, NR2C2, PELI1, TBK1, TICAM2, TLR3, TLR4, TRAF6, TICAM1 (TRIF). MYD88-Dependent: IRAK1, IRAK2, MAP3K7 (TAK1), TAB1, MYD88, NR2C2, TIRAP, TLR1, TLR10, TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TRAF6. Downstream Pathways and Target Genes: NFkB Pathway:BTK, CASP8, CHUK (IKKa), ECSIT (SITPEC), FADD, IKBKB, IL10, IL1B, IRAK1, IRAK2, IRF3, LY96, MAP3K1 (MEKK), MAP3K7, MAP4K4, NFKB1, NFKB2, NFKBIA (IKBA/MAD3), NFKBIL1, NFRKB, PPARA, REL, RELA, TNF, TNFRSF1A, UBE2N. JNK/p38 Pathway:ELK1, FOS, IL1B, JUN, MAP2K3 (MEK3), MAP2K4 (JNKK1), MAP3K1 (MEKK), MAP3K7, MAPK8 (JNK1), MAPK8IP3, TNF. JAK/STAT Pathway: CCL2 (MCP-1), CSF2 (GM-CSF), IFNG, IL12A, IL2, IL6. Interferon Regulatory Factor (IRF) Pathway:CXCL10 (INP10), IFNA1, IFNB1, IFNG, IRF1, IRF3, TBK1. Cytokine-Mediated Signaling Pathway: CCL2 (MCP-1), CSF3 (GCSF), IL1A, IL1B, IL6, IRAK1, IRAK2, RELA, SIGIRR, TNF, TNFRSF1A. Regulation of Adaptive Immunity:CD80, CD86, HSPD1, IFNG, IL10, IL12A, IL1B, IL2, MAP3K7, TRAF6. Adaptors & TLR Interacting Proteins:BTK, CD14, HMGB1, HRAS, HSPA1A (HSP70 1A), HSPD1, LY86 (MD-1), LY96 (MD-2), MAPK8IP3, MYD88, PELI1, RIPK2, SARM1, TICAM1 (TRIF), TICAM2 (TRAM), TIRAP, TOLLIP. Effectors:CASP8 (FLICE), EIF2AK2 (PRKR), FADD, IRAK1, IRAK2, IRAK4, MAP3K7 (TAK1), TAB1, NR2C2, PPARA, PRKRA, ECSIT (SITPEC), TRAF6, UBE2N. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------. . . . . . . . . Schluter A, Espinosa L, Fourcade S, Galino J, Lopez E, Ilieva E, Morato L, Asheuer M, Cook T, McLaren A, Reid J, Kelly F, Bates S, Aubourg P, Galea E, Pujol . Functional genomic analysis unravels a metabolic-inflammatory interplay in adrenoleukodystrophy. Hum Mol Genet. 2012 Mar 1;21(5):1062-77. Chiesa S, Morbelli S, Morando S, Massollo M, Marini C, Bertoni A, Frassoni F, Bartolome ST, Sambuceti G, Traggiai E, Uccelli A A Mesenchymal stem cells impair in vivo T-cell priming by dendritic cells. Proc Natl Acad Sci U S A. 2011 Oct 18;108(42):17384-9. . . . . - 26 - Sample & Assay Technologies
  27. 27. Next Generation Sequencing (NGS) Why spend time sequencing the entire genome when your research is focused on a few genes? QIAGEN now offers GeneRead DNAseq Gene Panels, with which you can analyze genetic variants among specific genes of your interest or pre-selected, cancer-focused gene panels. These panels, each with a small number of thoroughly researched genes, enable: • Deep sequencing • Identification of low-frequency genetic variants • Savings in time, money and sample usage GeneRead DNAseq Gene Panels use multiplex PCR target enrichment technology based on sophisticated primer design and pooling algorithms. The PCR primer sets cover the protein-coding regions and untranslated regions (UTRs) of all human genes. Each panel includes integrated controls to identify poor-quality samples / libraries before sequencing. - 27 - Sample & Assay Technologies
  28. 28. Next Generation Sequencing (NGS) • Comprehensive Cancer • Breast Cancer • Colon Cancer • Prostate Cancer • Lung Cancer • Liver Cancer • Ovarian Cancer • Gastric Cancer • Leukemia - 28 - Sample & Assay Technologies
  29. 29. Next Generation Sequencing - Breast Cancer Gene Panel AKT1 CDH1 FBXW7 KRAS APC CDKN2A FGFR1 NRAS BRAF CTNNB1 FGFR2 PIK3CA BRCA1 EGFR HRAS PTEN BRCA2 ERBB2 KIT TP53 - 29 - Sample & Assay Technologies
  30. 30. We Provide Service – Send Samples to Us & Receive Results! Whole Genome Illumina Gene Expression Profiling Illumina Genotyping . Pathway / Focused Panel Mutation Profiling Methylation PCR Array miRNA PCR Array . . . . . Individual Gene / Locus Mutation Detection Methylation qPCR . . . . Sample Preparation – DNA, RNA Extraction and Purification Cells, Tissue or Biofluids Fixed Tissue Small Sample . . . . . - 30 - Sample & Assay Technologies
  31. 31. Contact Information . . . . . Jesse Liang Email: Technical Support: 1-888-503-3187 Email: Check Webinar Calendar: Next Generation Sequencing (NGS): December 18 (Tuesday) Mutation PCR Arrays: December 20 (Thursday) . . . - 31 - Sample & Assay Technologies