, Cancer regression in patients after transfer of genetically engineered lymphocytes.ppt

Visualizing Dendritic Cell Networks In vivo Previous work 2 photon microscopy was used to investigate cellular contacts involved in T cell development1 and the dynamics of CD8+ T cell activation by mature DCs in the lymph node2 The dynamics of DC mediated CD4+ T cell activation in the lymph node using 2 photon microscopy3 1Bousso P et al. Science. 296 (5574) 2002. 2Bousso P et al. Nat Imm. 4 (6) 2003. 3Miller MJ et al. J Ex Med. 200 2004. Hypothesis ??? Aim- Interested in studying the activity of steady-state and mature dendritic cells (DCs) in lymph nodes of live mice using 2 photon microscopy Dendritic Cells APCs DCs in periphery Capture Ag and induce tolerance or home to lymph node DCs in Lymph node Immature DCs establish T cell tolerance Mature DCs induced effector T cell fxns Experimental System: Transgenic Mouse Experimental System: Flourophores Experimental System: Two Photon Microscopy Experimental System: Two Photon Microscopy Experimental System: 2 photon vs Confocal Experimental System: Two Photon Microscopy Experimental Plan * Lindquist RA et al., Nat Imm. 5,12 (2004). Mike Leibowitz Exp Basis of Immunology January 10th, 2007 EYFP 11c C57BL/6 EYFP 11c EYFP? enhanced yellow fluorescent protein Label CD11c+ DCs ECFP? enhanced cyan fluorescent protein C57/BL6 mice expressing ECFP on B-actin promoter Label DCs with different flourophore EGFP? enhanced green fluorescent protein Label T and B cells Eo hv hv E1 E2 E3 Light Pulsed infrared lasers Principle: Used for high resolution imaging of thick tissue sections A laser emits pulses within femtoseconds The energy from both photons excites the flourophore to a high energy state Upon relaxation, the flourophore emits light energy E0 hv hv E1 E2 Pulsed infrared lasers E1.1 E2.1 E0.1 Second Harmonic Generation Two or more photons are scattered generating a single photon of exactly twice the incoming quantum energy Requires that molecules are inversion asymmetric and spatially ord