Visualizing Dendritic Cell Networks In vivo

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Visualizing Dendritic Cell Networks In vivo
Lindquist RA et al., Nat Imm. 5,12 (2004).
Mike Leibowitz Exp Basis of Immunology January
10th, 2007
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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.

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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

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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

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Experimental System Transgenic Mouse
C57BL/6
EYFP
11c
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Experimental System Flourophores

• 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

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Experimental System Two Photon Microscopy

• 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

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Experimental System Two Photon Microscopy
E2
Pulsed infrared lasers
E2.1
E1
E1.1
E0
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 ordered (most biologics)
• Used to image collagen fibers, microtubules

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Experimental System 2 photon vs Confocal
http//depts.washington.edu/keck/intro.htm Accesse
d 1-9-07
Helmchen et al. Nat Meth. 2 (6) 2005.
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Experimental System Two Photon Microscopy

• Advantages of 2 photon microscopy vs confocal
  microscopy
• Reduced damage to cells due to low energy high
  wavelength light
• Increased resolution b/c less light scattered
  81/?
• Infrared light allows for deep tissue penetration
  (up to 800um)
• Absence of absorption in out of focus planes
  limits photobleaching to focal plane

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Experimental Plan

• Created transgenic mouse with DCs expressing EYFP
• 2 photon microscopy to image steady-state DCs in
  lymph node
• Label T and B cells with EGFP to study
  interaction of DCs and lymphocytes in lymph node
• Study interactions of LPS activated mature ECFP
  expressing DCs with lymphocytes as compared with
  steady-state immature EYFP expressing DCs in
  adoptive cell transfer experiments