Transcript Transplant-related Research Projects

Dissecting the Immunobiology of Post-Transplant
Skin Cancer :
The unholy trio of Sun Damage, Immunosuppression and Inflammation
A.M. VanBuskirk
Division of Surgical Oncology, OSU Department of Surgery
D.F. Kusewitt
OSU Department of Veterinary Biosciences
T.M. Oberyszyn
OSU Department of Pathology
Arthur G. James Comprehensive Cancer Center and R.J. Solove Research Institute
Outline
 Background/scope
of the problem
 Data in humans (almost all epidemiological,
NOT immunological)
 Data in animal models
 Where do we go from here?
The Former Problem in Transplantation
"The surgeon looks to the left, pivots to the right,
transplants the organ and ... whoa! Rejected!"
Immunosuppressive medication
Both the Blessing and Bane of Transplantation
Photo courtesy of Dr. Allan Kirk, NIH/NIDDK
Post-transplant Complications
 Chronic
Rejection
 Infectious Diseases
 Malignancies
– Post-Transplant Lymphoproliferative
Disorders (PTLD)
– Skin Cancer (particularly Squamous Cell
Carcinomas)
Cancer in Transplant Patients: factoids




Transplant patients are at increased risk for developing cancer (on
average, a 2- to 4-fold risk of developing any cancer compared to the
general population).
Non-melanoma skin cancer (NMSC) is the most common cancer after
transplantation, with a 50-250-fold increase compared to the general
population.
Risk factors for skin cancer in transplant recipients include older age at
time of transplantation, fair skin, history of sun exposure and length of
time since transplantation.
Transplant patients tend to develop multiple skin cancers that are
aggressive and can be life-threatening. SCC is reported as the cause of
death for 27% of Australian cardiac transplant recipients who’d survived
greater than 4 years. Also recently reported to be cause of death in a
significant number of Swedish transplant recipients. Data on SCC are
NOT routinely collected in North America.
Immunosuppressive medication
Both the Blessing and Bane of Transplantation
Photo courtesy of Dr. Allan Kirk, NIH/NIDDK
Warty-like lesions
Photo courtesy of Dr. Eggert Stockfleth, Charite, Berlin
Field Cancerization: Multiple Actinic Keratoses,
Squamous Cell Carcinomas
Photo courtesy of Dr. Eggert Stockfleth, Charite, Berlin
What other Immunosuppressed populations
exhibit increased Skin Cancer?
 HIV/AIDS
patients
 Cancer patients
 Autoimmune disease patients
What is a commonality among transplant
recipients and these other immunosuppressed
populations?
Exogenous/Therapeutic Immunosuppression
A reduced number of circulating CD4+ cells
The reduced number of CD4+ T cells is thought to impair immune
surveillance.
Approximately 23% of transplant patients have reduced numbers of CD4+ T cells
(Hutchinson, 2003)
Transplant patients with SCC have lower CD4+ T cell numbers than patients
without SCC (Ducloux, 1998)
However, the immunobiology of skin cancer in
the context of therapeutic immunosuppression
or CD4 leukopenia has not been systematically
investigated.
Animal models are effective pre-clinical tools.
Experimental Models of Skin Cancer




Chemically induced (SCC,
melanoma)
Ultraviolet radiation-induced
(SCC)
Transplantable skin tumors
– Human (SCC,melanoma)
– Murine (SCC, melanoma)
Tumors arising in transplanted skin
or skin cells
– Human (SCC,melanoma)
– Murine (SCC, melanoma)
D.F. Kusewitt
Mouse versus Human Skin
Human
Mouse
Epidermis
Papillary dermis
Reticular dermis
Arrector pili
Pilosebaceous unit
Eccrine gland
Apocrine gland
Panniculus carnosus
Subcutis/hypodermis
D.F. Kusewitt
How Mouse Skin Differs from Human Skin
The skin is thinner
 The skin lacks eccrine and apocrine glands
 Melanocyte location is restricted
 The mouse is fully haired
 No known papillomaviruses infect mouse
skin

D.F. Kusewitt
Our friend, the SKH/hairless mouse
Outbred
Functioning immune system
Develop SCC and SCC precursors upon
repeated exposure to UVB
Pros:
Accepted model of SCC
carcinogenesis, reflects outbred
population, excellent for prevention
studies
Cons:
Difficult to do immunological
experiments [Inbred SKH strain has
been offered to us, but must be rederived (currently in MHV+ facility)]
Also, currently breeding the hairless
gene onto FVB/n (6th generation)
The Importance of Inflammation to Carcinogenesis
Using pre-clinical models, the link between early inflammation and
the development of UV skin tumors is well established (Fischer,
Pentland and Oberyszyn groups). Early inflammation under
Mean units of MPO
(x 10-2)
conditions of immunosuppression needs further investigation
0.6
0.5
0.4
0.3
* *
0.2
0.1
0
Wilgus et al, 2000.
Ace
Celecoxib
UVB/Ace
UVB/
Celecoxib
Mean number of tumors per mouse
Reducing inflammation with Celecoxib
results in fewer skin tumors
20
15
UVB/Acetone
UVB/Celecoxib
10
* *
5
0
* *
* *
*
*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Weeks UV and treatment
Wilgus et al, 2003
*
What happens to UVB-induced
inflammation and carcinogenesis when
therapeutic immunosuppression is
present?
Experimental Scheme
Outbred
SKH/hairless
UVR 3x/week
1 week
Or 1 exposure
CD4 depletion
Experimental
immunosuppressant
Outbred
SKH/hairless
Skin Parameters
Skin thickness
MPO
Neutrophil Infiltration
p53+ basal layer cells
average # CD4+ cells
Background:
CD4+ cells infiltrate the epidermis in response to UVB
3.5
3
2.5
2
1.5
1
p=.003
0.5
0
CTRL
UV
UV
UV
Anti-CD4 Anti-CD8
**Have recently developed protocol to isolate 98% pure CD4+ CD3+ cells from the epidermis of UV-exposed mice.
Neutrophil
number
Ly6G+ cells
MPO
MPO units ( x 10-2)
CD4-depletion increases MPO and
neutrophil infiltration
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
CTRL
UV
UV
Anti-CD4
CTRL
UV
UV
Anti-CD4
20
15
10
5
0
avg # of p53+ cells/field
CD4-depletion increases the number of p53+
cells in the basal layer of the epidermis
6
5
4
3
2
1
0
CTRL
UV
UV
Anti-CD4
1 week of UVB exposures, harvest 24 hours after last UVB exposure
CD4-depletion increases skin production of PGE2
PGE2 pg/mg protein
35
P<.006
30
25
20
15
P<.002
10
5
0
IgG
UV/IgG
UV/anti-CD4
1 week Treatment
UV/anti-CD8
Nice, but what kind of CD4+ T cell is this and
how is it modulating UVB-induced inflammation?
 T-regulatory
(CD3+ CD4+ CD25+), TH-3
– MHC Class 2 restricted
– cell contact dependent or cytokines- IL10/TGF-b
 TH-2
(CD3+CD4+)
 CD4+
NKT (CD3+, NK+/-, TCR: Va14-Ja18+)
– MHC Class 2 restricted
– cytokines- IL4/IL5/IL10/IL13
– CD1 restricted
– direct killing, cytokines -IFN-g/IL4/IL10/IL13
Identifying Different CD4+ cell types
 Isolate
epidermal infiltrating CD4+ cells in
SKH mice and assess
– surface phenotype, fox-p3 protein, intracellular
cytokines
– TCR usage by PCR
– fox-p3 by PCR
 Use
NKT-deficient mice (Balb/c background)
Initial data: NKT cells can be detected in hairless mice
WT CD1d-/Marker
M1
M3
M4
K1
T1
Spleen Spleen
H2O
Va14Ja18
268 bp
B-actin
348 bp
Are NKT cells present in UVB-exposed skin?
Are NKT cells reduced/ depleted in anti-CD4 treated mice?
Are NKT-associated cytokines reduced in CD4-depleted mice?
If NKT cells modulate UVB inflammation, then NKTdeficient mice should have exacerbated inflammatory
responses to UVB.
1.6
Skin Thickness (mm)
1.4
1.2
1
No UV
0.8
UV
0.6
0.4
0.2
0
Balb/c
Ja-18 -/-
CD1d -/-
Mice were shaved and treated with hair remover 3 days prior to UVB exposure. Forty-eight hours after UVB
exposure, animals were sacrificed and edema (skin thickness) measured. Star indicates p<0.001 compared
to No UV control.
Average Fold Increase in MPO
NKT-deficient mice have exacerbated UVBinduced inflammatory responses
p<.015
25
20
p<.03
15
10
5
0
Balb/c
Ja18 -/-
CD1d -/-
Dorsal skin punches were taken from wild-type and NKT deficient mice 48 hours after UVB exposure.
Data are shown as the average fold increase in MPO over matched no UV controls.
Conclusions (1)
 CD4-depletion
increases neutrophil number and
activity
 CD4-depletion increases DNA damage, evidenced
indirectly as an increase in p53+ epidermal cells
 CD4-depletion results in increased PGE2 in the skin
 Preliminary data indicate that CD4+ NKT cells are
important regulators, as NKT deficient mice have
exacerbated inflammatory responses to UVB.
Importance of Inflammation even after chronic UVB exposure
Deplete CD4+ TUVB
cells
UVB
Exposure
Determine
Exposure
(inject Abs every 3
Assess MPO tumor number
weeks)
activity
10 wks 11 weeks
SKH-1 hairless
mouse
UVB
exposure 3
times
weekly
only
Continue
UVB
exposure
Trend toward increased MPO in
CD4-depleted mice at week 11
T.M. Oberyszyn
25 weeks
Tumor number per mouse
3x weekly
70
P<0.03
60
50
40
30
20
10
0
UV/IgG
UV/anti-CD4
25 week Treatment
Mutational analysis: anti-CD4 vs IgG
Group
Number of
Tumors
Number
with
mutations
Type of
Mutation
Codon
UVB/ IgG
21
3
C>T
C>T
C>T
R270C,
P275S
P275S
R270C,
P275S
UVB/ antiCD4
24
6
C>T
C>T
C>T
C>T
C>T
C>T
R270C,
R270C
P275S
R270C
R270C
R270C
R270C
Preliminary analysis of p53, exon 8 (S. Tanner)
Conclusions (2)
 CD4+
cells modulate inflammation after both
acute and chronic UVB.
 Celecoxib reduces inflammation after acute
and chronic UVB.
 CD4 depletion enhances tumor development
after chronic UVB.
 Tumors in anti-CD4 treated mice have more
detectable p53 mutations.
All that’s nice, but what happens when clinically relevant
immunosuppressants are used?
In the few published studies, immunosuppressants decreased the time to
tumor development and sometimes increased the number of tumors.
Kelly et al. 1987. Transplantation 44(3): 429-434.
Daynes et al. 1979. J. Natl. Cancer Institute 62:1075.
Reeve et al. 1985. Aus. J. Exp. Biol. Med. Sci. 63: 655.
However, the most commonly used immunosuppressants today were either
not tested, or were tested in non-therapeutic doses.
None of these studies looked at UVB-induced inflammation.
None of these studies used combination therapies.
Mean Stimulation Index
Effect of anti-CD4 and clinically relevant
immunosuppressants on Con A driven proliferation
2.5
2.0
p=.05
P<.011
1.5
P<.007
1.0
0.5
0
CTRL
IgG
Anti-CD4
PBS
CsA
TAC
CsA: 20mg/kg/day, ip
TAC: 2mg/kg/day, ip
p=.01
300
MPO units (x 10-4)
MPO units (x 10-4)
Effect of clinically relevant Immunosuppressants
on MPO activity at 48 hours post-UVB
250
200
150
100
50
0
PBS
CsA
No UV
TAC
PBS
CsA
UV
TAC
140
120
100
80
60
40
20
0
48 hours
No UV
controls
UV
IgG
UV
Anti-CD4
Conclusions
 Systemic
cyclosporine treatment reduces
the splenic MLR response, but increases
UVB-induced inflammation (MPO increased
4-8-fold).
 Systemic tacrolimus treatment reduces
the splenic MLR response, but does not
increase or decrease UVB-induced
inflammation (MPO activity similar to PBS
controls).
CsA vs Tac:
Why Different Responses?
 The
simple answer: We don’t know
 Possibilities:
– differential effects on neutrophil activity or
trafficking.
– Differential effects on T cell function.
– Differential effects on
monocyte/macrophage/dendritic cell
functions.
Fold increase in MPO
no UV vs UV
Effect of single and dual therapies on UVB-induced MPO activity:
48 hours after a single UVB exposure
**preliminary/new data**
5
4
3
2
1
0
PBS
CSA: 20mg/kg
TAC: 2mg/kg
MMF: 20mg/kg
SIR: 2 mg/kg
CSA
MMF
SIR
TAC
CSA
+
MMF
TAC CSA
+
+
MMF SIR
Mice treated for 1 week, then exposed to UVB. MPO activity measured at 48 hours after UVB.
Future Plans
Basic Research





Determine patterns of cellular infiltration and
whether these are altered by immunosuppression.
Assess mechanisms by which immunosuppressants alter
UVB-induced inflammation: effects on neutrophils,
keratinocytes, endothelial cells.
Assess the effects of clinically relevant
immunosuppressants on skin carcinogenesis.
Assess effectiveness of new topical treatments to
reduce inflammation and carcinogenesis.
Post-Transplant Research Group web site: funded by
Research on Research Grant, TELR (1 of 10 Universitywide)
Future Plans
Clinical Research




What is the scope of the problem in the OSU
transplant population?
Assess distribution of cytokine gene polymorphisms in
patients who develop skin cancer rapidly after
transplantation compared to those who do not.
Assess UVB-induced inflammatory responses in
transplant patients.
Assess new topical treatments to prevent skin cancer
in transplant patients.
Main difficulty is a lack of dermatology infrastructure linked to the
transplant program at OSU. So, currently we need outside collaborators:
ITSCC and SCOPE members have offered to help with samples.
“When I say “I”, I mean we,
when I say “we”, I mean they”
-Dr. Frank Fitch
Quote co-opted by Dr. Charles Orosz, and in turn, by me
Acknowledgements
Kusewitt Laboratory
Donna Kusewitt
Allison Parent
Erin Brannick
Brutkiewicz Laboratory
Randy Brutkiewicz
“Emily” Yin-Ling Lin
Oberyszyn Laboratory
Tatiana Oberyszyn
Jennifer Hatton
Kathy Tober
Brian Wulff
Stoner Laboratory
Gary D. Stoner
VanBuskirk Laboratory
Anne VanBuskirk
Sagal Ali
Tyler Hoppes
F Jason Duncan
Kelly Johnson Nye
Tanner Laboratory
Stephan Tanner
OSU Comprehensive Cancer Center- RJ Solove Research Institute
(MCC program and Immunology program)
National Institutes of Health- NCI
American Heart Association, Ohio Valley Affiliate
American Cancer Society, Ohio Division
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