PowerPoint Template

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The effect of TLR on transplantation and
tolerance of the cornea
Mee Kum Kim M.D1, Jinho Jeong M.D 1, Won Ryang
Wee M.D 1, Jin Hak Lee M.D 2   1Department of
ophthalmology, Seoul National University
Hospital, Seoul, Korea 2Department of
ophthalmology, Seoul National University Bundang
Hospital, Bundang, Korea
The authors have no financial interest in the
subject matter of this poster.
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Purpose

• To investigate the role of toll-like receptor
  (TLR)
• On the MHC mismatched allograft rejection in
  corneal transplantation
• On the immune tolerance of the cornea

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Materials and Methods

• Mouse cell culture
• Group
• Group K keratocytes only 1x104 cells
• Group D dendritic cells only (DC2.4) 1x104
  cells
• Group M macrophages only (Raw264.7) 1x104
  cells
• Group KD keratocytes dendritic cells
  0.5x104 0.5x104 cells
• Group KM keratocytes macrophages 0.5x104
  0.5x104 cells

Mouse primary corneal keratocyte DMEM/F12 (11)
with 10 FBS and 1PS
Mouse DC cell line DC2.4 RPMI1640 with 10FBS
and 1PS
Mouse macrophage cell line Raw264.7 DMEM with
10FBS and 1PS
Kindly provided by Kim TJ
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• TLR stimulation with agonist
• LPS (TLR4 agonist)
• Pam3csk4 (TLR2 agonist)
• Change in TLR expression and its products of TLR
  signaling pathway
• TLR2 and TLR4 expression
• RT-PCR
• Stimulation for 6 and 24 hours with LPS or
  Pam3csk4
• Phospho-IkappaB-alpha (p-I?B-a)
• Western blot
• Stimulation for 2, 6, and 24 hours with LPS or
  Pam3csk4
• Cytokine assay
• ELISA
• Candidate cytokines IL-6, TNF-a, TGF-ß, IL-10
• Stimulation for 24 hours with various
  concentration
• LPS 1, 10, 100, 1000, 10000 ng/ml
• Pam3csk4 1, 5, 10, 50, 100 ng/ml

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• Corneal transplantation

• Group (Donor ? Recipeint)
• C57BL/6 (B6) ? B6 Autograft (n10)
• C3H/HeN (C3H) ? B6 MHC mismatched allograft
  (n11)
• C3H ? TLR4 knockout (TLR4KO) (n13)
• Transplantation procedure
• Donor /recipient 3.0 / 2.0 mm trephine
• 8 to 10 interrupted 10-0 Nylon sutures
• Clinical evaluation
• Analysis of graft survival using Slit-lamp
  biomicroscopy
• Definition of rejection (end point) 100 graft
  edema
• Kaplan-Meier survival analysis (Log rank
  (Mantel-Cox) test)
• Immune cell assay
• Cervical lymph node and spleen
• 12 days 4 weeks after transplantation
• Flow cytometry (FACSCalibur) and FlowJo software
• CD11c, CD80/CD86 CD4/8, CD44/69 CD4/8, IFN?

TLR4 knock-out mice on a C57BL6 (B6 background)
were bred at a SPF animal facility and were
kindly provided by Dr. S-Y Seong at the
Department of Microbiology and Immunology, Seoul
National University College of Medicine
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Results

• TLR expression after agonist stimulation
• P-I?B-a expression after agonist stimulation

? TLR4 in M, D, and K
? TLR2 in D and K
? P-I?B-a in M, D, and K
? P-I?B-a in M,and D
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• Cytokine secretion after agonist stimulation
• After LPS stimulation
• Coculture K D
• Coculture K M

Markedly increased anti-inflammatory cytokine
IL-10 and TGF-ß1 in coculture condition
K B6 Keratocyte D Mouse dendritic cell line
(DC 2.4) M Mouse macrophage cell line (Raw
264.7)
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• After Pam3csk4 stimulation
• Coculture K D
• Coculture K M

Markedly increased anti-inflammatory cytokine
IL-10 and TGF-ß1 in coculture condition
K B6 Keratocyte D Mouse dendritic cell line
(DC 2.4) M Mouse macrophage cell line (Raw
264.7)
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• Corneal allograft survival and immunologic
  profiling
• Median survival time
• C3H ? B6 gt C3H ? TLR4KO

• IFN-? secreting CD8 T cell
• C3H ? B6 lt C3H ? TLR4KO

CD8 IFN-? in L/N at 12 days
Plt0.05, Mann Whitney U test
Plt0.05, Log Rank test
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At 12 days
At 4 weeks
1.90
3.53
1.15
5.49
CD86
CD 11c
Lymph nodes
CD 11c
2.70
10.64
1.81
2.84
CD80
2.77
1.75
1.57
2.27
CD80
CD 11c
Spleens
CD 11c
3.48
4.13
3.17
1.95
CD86
C3H ? B6
C3H ? TLR4KO
C3H ? B6
C3H ? TLR4KO
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0.27
0.31
0.74
0.29
0.60
0.29
0.34
0.52
0.93
C3H ? B6
C3H ? TLR4KO
C3H ? B6
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Conclusions

• TLR4 is likely to be involved in immune
  modulation of MHC mismatched corneal allograft
  rejection
• Fibroblast seems to be involved in immune
  modulation through TLR signaling