Journal Club

Question 1

Describe Figure 1

Answer 1

• Cloning strategy
• Chemotactic assay
  
  • Results:
    
    • Lymphocytes from CCR7-/- mice did not migrate towards CCR7 ligand
• FACS analysis of lymphocytes in various locations
  
  • Peripheral blood, BM, Spleen, and various LNS
  • Results:
    
    • In CCR7 -/- mice:
      
      • Increased lymphocytes in blood and spleen
      • Decreased lymphocytes in LNs

Question 2

Describe Figure 2

Answer 2

• Immunohistochemistry
  
  • Looked at architecture of 2° lymphoid organs
  • Conj. abs against B220 and Thy antigens
  • Results:
    
    • T cells not in PALS, but instead in rim around B cell follicles in PP and in red pulp in spleen

Question 3

Describe Figure 3

Answer 3

• ELISA analysis of antibody in the serum of CCR7-/- mice
  
  • Capture Ab on the wells was specific for light chains
  • Detection Ab against the specific isotype
  • Was able to determine the relative amounts of the isotypes
  • Results:
    
    • Increased IgG2a, IgG2b, IgE, IgA
    • Decreased IgG3

Question 4

Describe Figure 4

Answer 4

• Looked at DC migration from skin into LNs
  
  • Skin painted with FITC
  • LNs from mice isolated and cells suspended and presence of FITC positive DCs analysed with flow cytometry
  • Results
    
    • In CCR7-/- mouse: no FITC positive DCs in LNs implies that DCs could not migrate from skin
    • In WT mice: population of FITC positive DCs found in the LNs

Question 5

Describe Figure 5

Answer 5

• Looked at whether the defect was intrinsic to the cells
• Adoptive transfer assays
  
  • Lymphocytes from WT or CCR7-/- mice transferred to CCR7-/- or WT mice
  • Transferred lymphocytes detected by AM-cystein expression (green fluorescence)
  • Results:
    
    • CCR7-/- lymphocytes transferred into WT mice still could not migrate into lymphoid organs
    • WT lymphocytes transferred into CCR7-/- mice could migrate into lymphoid organs (in fact, increased migration)

Question 6

Describe Figure 6

Answer 6

• Looked at B cell activity in spleen
• B and T cells isolated from CCR7-/- mice using MACS
• Immunohistochemistry
  
  • Abs against B220 and Thy
  • Transferred cells identified by their AM-cystein expression
  • Results
    
    • Transferred B cells not retained in outer PALS, but quickly move into B cell areas
    • Transferred T cells not enriched in the PALS, but rather found in the red pulp

Question 7

Describe Figure 7

Answer 7

• Looked at primary B and T cell responses
• T cell responses
  
  • Contact sensitivity assay
    
    • FITC applied to skin
  • Delayed type hypersensitivity assay
• B cell responses
  
  • KLH injected
  • Looked at titres of the various isotypes of Abs against KLH over time
  • Results:
    
    • Delayed isotype switching
    • Due to non-coordinated interactions between B and T cells during generation of a primary B cell response
    • CCR7 needed for B and T cells to migrate towards the boundary and for T cells to provide T cell help in this region