Ghép Da: Chỉ Định, Ứng Dụng Và Các Nghiên Cứu Hiện Tại (2011)

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skin area and in the most immunoactive zone on the histological slides. The details of this
visual scoring system are shown in (Table 1).

Grade 0
No rejection
Normal epidermal and dermal appearance without evidence of
rejection
Grade 1
Indeterminate/
supposed
rejection
Very early visual signs of rejection
Focal (25% of limb or DSSG surface) inconspicuous changes in the
skin color (pink/pinkish or slight red/reddish spots) and/or in the
texture of the skin (dryness, fine scaling of epidermis)
Grade 2
Mild rejection
Clear visual signs of rejection
Diffuse (25-50 % of DSSG or limb skin surface) clear changes in color
and texture of skin, sometimes with slight limb edema, thickening of
the skin
Grade 3
Moderate
rejection
Extensive (more than 50% of DSSG or limb skin surface) and
advances alteration of skin quality – desquamation, scabs, skin crusts
Grade 4
Severe rejection
Destruction of epidermis with intact dermis or complete destruction
of the skin
Table 1. Visual grading system for assessment of rejection
Three animals (20%) showed early rejection and salvage therapy was successful in all cases.
One of rats developed a second rejection episode, successfully treated with salvage therapy.
In two of these three rats skin damage after resolution of rejection prevented assessment of
grades 1 and 2 rejection do to incomplete or deficient healing. The remaining 12 rats (80%)
were free of rejection during the treatment period.
The average time for the onset of grade 1 rejection in limb skin after immunosuppression
ceased was 6.75 ± 1.42 days (median day 6.5) and 8.75 ± 2.38 days (median day 8) for grade
2. The mean interval between the onset of grade 1 and the development of grade 2 rejection
in limb skin was 2 ± 1.35 days for rats free of early rejection (n= 12).
Visual rejection episodes in group 2 (rats with limb transplant and DSSG)
During the first 6 weeks 28 rats (62%) were rejection free. Seventeen rats (38%) developed
early rejection. Thirteen rats (29%) were excluded from the study due to the poor quality of
skin doe to incomplete or deficient healing after salvage therapy. In total, 32 rats, 28 without
early rejection episodes and 4 rats with one episode of very early or early rejection,
successfully reversed with salvage therapy, were evaluated after termination of im-
munosuppressive therapy (Fig 2).
The average onset grade 1 rejection was 5.06 days (median day 5) for DSSG, and 6.41 days
(median day 6) for limb skin. The average interval between grade 1 rejection of the skin graft
and grade 1 rejection of the limb skin was 1.35 days. Rats evaluated in group 2 displayed a
strong statistic difference in the onset of grade 1 rejection of the skin graft and the limb skin
(p < 0.0005).
The average onset grade 2 rejection was 8.28 days (median day 8) for DSSG, and day 9.21 7
(median day 9) for limb skin. The interval between grade 2 rejection of the skin graft and
grade 2 rejection of the limb skin was 0.91 days. There was a small but statistically

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343
significant difference between the first day with visual signs of grade 2 rejection of the skin
graft and the limb skin (p < 0.05).







Fig. 2. Clinical (visual) aspect of rejection on the limb skin and on the DSSG in the earlier
stages of rejection A, B,C Grade 0 (No rejection) - Normal epidermal and dermal appearance
without evidence of rejection D, E, F Grade 1 (Inconspicuous / indeterminate / supposed rejection)
- Very early visual signs of rejection; Focal (25% of limb or DSSG surface) inconspicuous
changes in the skin color (pink/pinkish or slight red/reddish spots) and/or in the texture of
the skin (dryness, fine scaling of epidermis) G, H, I Grade 2 (Mild rejection) - Clear visual
signs of rejection; Diffuse (25-50 % of DSSG or limb skin surface) clear changes in color and
texture of skin, sometimes with slight limb edema, thickening of the skin
The time difference between the onset of grade 1 rejection and the development of grade 2
rejection was on average 3.4 days for DSSG and 2.9 days for limb skin. There was no
significant difference between grades 1 or 2 rejection of the limb skin of control animals and
rats with both a limb transplant and sentinel skin graft. As a result, we conclude that appli-
cation of a skin allograft does not influence the rejection of a transplanted limb.
Histologic rejection results
161 biopsies from 45 animals were evaluated histologically for signs of rejection. Specimens
were taken for suspected rejection or when the visual grade appeared to alter. Samples were

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taken concurrently, from both the sentinel skin graft and limb allograft. Rejection was
assigned histologically, based on changes in the most immunoactive zone on the histological
slides. All slides were analyzed in a blinded fashion and scored based on a new histologic
grading system. The new grading system was introduced to describe the very early changes
in skin pathology observed during onset of rejection. The criteria used for histologic
assessment of skin rejection are shown in (Table 2, Fig 3).





Grade 0
Normal epidermal and dermal appearance without evidence of
rejection
Dermal mononuclear cell infiltration < 10 cells / 500X optic field
Grade 1
Indeterminate
rejection
Dermal mononuclear cell infiltration 10-20 cells / 500X optic field
Epidermal (especially on epidermo-dermal junction) mononuclear
cells infiltration 1-3 cells / 200X optic field
Grade 2
Mild rejection
Diffuse intercellular edema (spongiosis) on basal layer of epidermis
with normal continuity of epidermo-dermal junction
Dermal mononuclear cell infiltration 10-20 cells / 500X optic field
Epidermal (especially on epidermo-dermal junction) mononuclear
cells infiltration > 3 cells / 200X optic field
Grade 3
Moderate
rejection
Discontinuity of epidermo-dermal junction
Focal basal cells vacuolization
Dermal mononuclear cell infiltration more than 20 cells / 500X optic
field
Epidermal (especially on epidermo-dermal junction) mononuclear
cells infiltration > 3 cells / 200X optic field
Intercellular edema on basal layer of epidermis (spongiosis)
Dyskeratosis of squamous cells in the epidermis or hair follicle
epithelium
Grade 4
Severe rejection
Complete separation at the epidermo dermal junction
Complete epidermal degeneration and necrosis
Dense inflammatory infiltration in the dermis > 20 cells / 500X optic
field





Table 2. Histology grading and scoring system for assessment of rejection

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Fig. 3. Histological aspects of rejection in the earlier stages of skin rejection
A, B Grade 0 (H&E; 200x and 500x) Normal epidermal and dermal appearance without evidence of
rejection - Dermal mononuclear cell infiltration < 10 cells / 500X optic field C, D Grade 1
(H&E; 200x and 500x) Indeterminate rejection - Dermal mononuclear cell infiltration 10-20
cells / 500X optic field; Epidermal (especially on epidermo-dermal junction) mononuclear
cells infiltration 1-3 cells / 200X optic field E, G. Grade 2 (H&E; 200x and 500x) Mild
rejection - Diffuse intercellular edema (spongiosis) on basal layer of epidermis with normal
continuity of epidermo-dermal junction; Dermal mononuclear cell infiltration 10-20 cells /
500X optic field; Epidermal (especially on epidermo-dermal junction) mononuclear cells
infiltration > 3 cells / 200X optic field
The average grade of limb rejection noted in 35 samples harvested from 13 control rats was
1.57. In group 2, 126 samples from 32 rats were analyzed. Analyzing the early stages of re-
jection (grade 0 - 2), for corresponding skin graft and limb skin samples, the average grade
of rejection observed for limb skin was 1.08 compared with 1.46 for the skin grafts. The
difference in severity of rejection was found to be significant using a paired t test at p <0.005.
The higher average grade of rejection seen in sentinel skin infers that rejection develops
sooner at this site. This finding confirms the hypothesis that a distant sentinel skin graft
rejects before limb skin.
There was no significant difference using an unpaired t-test (p value = 0.86) between the
onset of limb skin rejection in groups 1 and 2. The mean rejection grade was 1.57 for group 1
and 1.42 for group 2. This result confirmed visual analysis, and demonstrated that the

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sentinel skin graft did not influence the rejection of limb allografts when transplanted
simultaneously.
4. Conclusions
In conclusion, these findings confirm a small, but significant delay in rejection of limb skin
compared to a DSSG. Skin grafts transplanted simultaneously with a hind limb are thus a
useful marker of rejection by providing advanced waning of an impending episode. This
allows earlier intervention with salvage therapy and the consequent rapid initiation of
therapy that is of benefit in a clinical setting. The DSSG can be placed on a site usually
covered by clothing and may obviate the need for biopsies of the transplanted limb, with
their associated scarring, functional and aesthetic consequences. The skin DSSG did not
appear to significantly alter the incidence of severity of rejection and appear to be safe in
this animal model. Modifications of the current visual and histopathological criteria for
diagnosis of rejection were introduced to allow early detection.
The concept of DSSG is already applied in clinical settings. A series of three hand
transplants performed where patients received an additional simultaneous full-thickness
donor skin graft transplanted to the left hip area. This skin was used as a source for skin
biopsies and as an additional area for monitoring rejection (distant sentinel skin graft,
DSSG). The DSSG used in all three patients proved to be of benefit, allowing prediction of
rejection of the hand by as much as 7 days. This allowed appropriate treatment to be started
immediately. In one patient, salvage therapy (systemic and topical) was delayed until the
hand showed the same clinical and histological signs of rejection. The skin changes resolved
completely within 5 days at both locations. In the other two patients salvage therapy was
started immediately DSSG showed the onset of rejection and rejection of the hand was not
observed. However, the DSSG was of value only in the early postoperative phases. With
time DSSG cells were replaced by "creeping substitution" of recipient skin and monitoring
value was lost.
In our study, we found that the DSSG showed the first signs of rejection (grade 1), with a
delay of 1.35 days before the limb skin detection. Sensitivity was reduced to 0.91 days for
grade 2 rejection. The 7 days interval noted in Italian clinical cases, may be explain by a
gradual taper of immunosuppressive therapy, rather than the complete withdrawal used in
this study.
In the case of the first face transplantation a sentinel radial forearm flap harvested from
the donor’s left upper limb was transferred to the recipient’s left submammary fold and
suture end-to-end to the thoracodorsal vessels. This vascularized composite tissue flap,
hidden under the hanging breast, was used to monitor indirectly the immunological
behavior of the graft, aiming to avoid damage to the reconstructed face by repeated skin
biopsies.
5. References
Andrew Lee W. P., Yaremchuk M J, Pan YC, Randolph MA, Tan CM, Weiland AJ. Relative
Antigenicity of Components of a Vascularized Limb Allograft. Plast Reconstr
Surgery, March 1991, Volume 87, Issue 3

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Askenasy N Localized Bone Marrow Transplantation Leads to Skin Allograft Acceptance in
Nonmyeloablated Recipients: Comparison of Intra-Bone Marrow and Isolated Limb
Perfusion. Stem Cells 2002; 20: 86-93
Black KS., Hewitt CW., Aniel M., Grisham GR., Caiozzo VJ., Achauer BM.
Neuromuscular
capabilities
in
long-term
composite
tissue
allografts.
Transplant Proc 1988;20:369
Bos JD, Das PK, Kapsenberg ML. The skin immune system (SIS), 2nd Ed. Boca Raton: CRC
Press, 1997
Buttemeyer R., Jones NJ., Min Z., Rao U. Rejection of the Component Tissues of Limb
Allografts in Rats Immunosuppressed with FK506 and Cyclosporine. Plast Reconst
Surg 1996;97:139
Doi K. Homotransplantation of limbs in rats. A preliminary report on an experimental study
with nonspecific immunosuppressive drugs. Plast Reconstr Surgery 1979
Nov;64(5):613-21
Guzman-Stein A, Shons AR. Functional recovery in the rat limb transplant model: a
preliminary study. Transplant Proc. 1987 Feb;19(1 Pt 2):1115-7.
Hewitt CW, Black KS, Fraser LA, Howard EB, Martin DC, Achauer BM, Furnas DW.
Composite tissue (limb) allografts in rats. I. Dose-dependent increase in survival
with cyclosporine. Transplantation. 1985 Apr; 39(4):360-4.
Hewitt CW, Black KS, Fraser LA, Howard EB, Martin DC, Achauer BM, Furnas DW.
Composite tissue (limb) allografts in rats. I. Dose-dependent increase in survival
with cyclosporine. Transplantation. 1985 Apr; 39(4):365-8.
Hewitt CW., Black KS., Aguinaldo AMA., Lin SS., Howard EB. Pathologic alterations in the
skin component of composite tissue and skin allografts treated with Cyclosporine.
Transplant Proc 1988;20:1003
Jones NF., Heberbraud D., Buttemeyer R., Zhao M., Benhaim P., Rao U. Comparison of
Long-Term Immunosuppression for Limb Transplantation Using Cyclosporine,
Tacrolimus and Mycophenolate Mofetil: Implications for Clinical Composite Tissue
Transplantation. Plast Reconstr Surg 2001;107:777
Kim SK, Aziz S, Oyer P, Hentz VR. Use of cyclosporine-A in allotransplantation of rat
limbs. Ann Plast Surg 1984; 12: 249-255
Lanzetta M, Dubernard JM. Hand Transplantation. Springer Milan 2007, 263-268
Lanzetta M, Dubernard JM. Hand Transplantation. Springer Milan 2007, 425-433
Llull R. An open proposal for clinical composite tissue allotransplantation. Transplant Proc
Volume 30, Issue 6, September 1998, 2692-2696
Press BHJ, Sibley RK, Shons AR. Limb allotransplantation in the rat: extended survival and
return
of
nerve
function
with
continuous
cyclosporine/prednisone
immunosuppression. Ann Plast Surg 1986 1986 Apr;16(4):313-21.
Scott A. Gruber, Mansour V. Shirbacheh, Jon W. Jones, John H. Barker, Warren C.
Breidenbach. Local drug delivery to composite tissue allografts (p 407-411).
Microsurgery Volume 20 Issue 8 , 357 - 469 (2000)
Shapiro RI, Cerra FB. A model for reimplantation and transplantation of a complex organ:
the rat hind limb. J Surg Res 1978;24:501–506

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Zamfirescu DG, Owen E, Lascar I, Molitor M, Zegrea I, Popescu M, Bishop GA, Lauer CA,
Simionescu M, Climov M, Lanzetta M., Sentinel skin allograft-a reliable marker for
monitoring of composite tissue transplant rejection. Transplant Proc. 2009
Mar;41(2):503-8.
Zdichavsky M, Jones J, Ustuner TE, Ren X, Edelstein J, Maldonado C, Breidenbach WC,
Gruber SA, Ray M, Barker JH. Scoring of skin rejection in a swine composite tissue
allograft model. J Surg Res 1999;85:1–8

25
Immunological Considerations for
Inducing Skin Graft Tolerance
Ethel J. Gordon
North Carolina Agricultural & Technical State University
United States of America
1. Introduction
While the first uses of skin transplantation may be found in anecdotal images involving the
use of foreign tissue to cover burns and damaged skin, the modern era of skin grafting
attempts to understand the phenomenon scientifically. One well documented case describes
Joseph E. Murray’s surgical procedure to use skin from a recently deceased person to cover
the skin of a pilot who suffered disfiguring burns in 1944 (Murray, 1992) The transplanted
skin survived for more than thirty days, despite any predictions of rejection that might have
destroyed the tissue in two weeks or less. Murray hypothesized that the procedure was
successful because of the weakened state of the recipient’s immune system. Murray
continued his interest in transplantation immunology, and ten years later his team was the
first to engraft an organ from a living donor, (Murray, 1992).
In 1944 Medawar described rejection of allografts, that is, organs or tissues exchanged
between individuals of the same species (Medawar, 1944). His pioneering work described
the consequences of allograft and autograft transplantation in exquisite detail. He used the
rabbit skin model to show differences in acceptance of autografts, using the same individual
or same strain as the tissue source, vs. allografts that exchanged skin between different
animals of the same species. He further used meticulously measured tissue size as a
surrogate marker for skin antigen dose and documented the process of healing for
autographs opposed to the “actively acquired immune reactions” that resulted in the
necrosis of allografts. R. E. Billingham (1959) reviewed the fate of engrafted tissue that is
rejected by the host in light of the then recently discovered major histocompatibility
complex in the mouse (MHC). The MHC genes encode cell surface proteins that allow the
immune system to discriminate between its own cells and foreign cells. Billingham
referenced the use of isogenic mouse strains wherein each animal of the strain has identical
MHC genes, and these animal models facilitated the understanding of graft versus host
disease, rejection mediated by immunocompetent cells within a graft (Billingham, 1959).
Billingham, Brent, and Medawar (1953) described immunological tolerance that is actively
acquired following prenatal exposure to foreign tissue in a mouse model. This phenomenon
described the survival of engrafted tissue, whereas the same tissue generated immunity
resulting in graft destruction when transplanted in the adult. After eight weeks, the tolerant
mice were challenged with adult skin of the same donor origin as the primary transplant, in
order to evaluate the persistence of tolerance. These experiments documented the utility of

Skin Grafts – Indications, Applications and Current Research
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mouse models as a practical means to help us understand immunological processes. Five
years later, Billingham and Silvers (1958) induced specific tolerance to the male chromosome
associated Y antigen in neonatal female mice, again using the skin graft model. Researchers
continue to use these basic methods to uncover the underlying mechanism of tolerance and
immunity.
Skin grafts, with their enhanced antigenicity and abundant supplies of antigen presenting
cells, remain a vigorous test of any tolerance induction system (Murray, 1971, Nasir et al.,
2009, Tobin et al., 2009, Akdis, 2010, van den Berg et al., 2011, Kaplan, 2010, del Rio et al.,
2010). Some of the skin associated antigens include autoantigens found on keratinocytes and
appear to induce T lymphocyte reactivity that results in skin eruptions (Jackman et al.,
2002). Antigenicity of skin has remained a concern for those promoting translational
medicine, including those who promote the use of composite tissue allografts (Swearingen
et al., 2008). The grafting of composite tissue is often used in limb replacement surgery.
These grafts consist of complex combinations of multiple tissue types, including skin,
adipose tissue, bone and others (Siemionow et al., 2009). The complexity of these composite
tissue allografts is such that a new classification system was devised in order to accurately
describe their attributes. Tissue antigenicity is one of the criteria on which the classification
system is based (Gordon et al., 2009). These historical experiments helped to usher in the era
of modern skin transplantation.
2. Generalized Immunosuppression to facilitate skin graft acceptance
Medical science has pursued a multiplicity of scientific directions in order to utilize solid
organ transplantation as a potential cure for life-threatening diseases and as a treatment for
sever disfigurement. Organ transplantation has been referred to as “One of the most
remarkable achievements of medicine during the 20th century” (Goldstein, 2011). Scientific
advances gleaned from the use of skin grafts and other tissue and organ grafts have restored
tissue function, extended the life, and promoted the general well-being of the graft host. The
spoken or unspoken hypothesis behind the use of generalized immunosuppression may be
stated as follows: If the immune system in an otherwise healthy individual is capable of
recognizing foreign tissue and mounting an attack against it, then significantly reducing all
such recognition may allow for engrafted tissue to remain intact and functional. Many of
these procedures required protracted or life-long treatments with unanticipated or negative
sequelae (Ingvar et al., 2010, Berardinelli et al., 2009). The goal of these broad-based
protocols was to protect the viability of the graft in order to protect the life of the host. Some
notable examples of the use of generalized immunosuppressive agents are described below.
2.1 Three categories of drugs targeting lymphocytes have been described
Information gleaned from reviews in major journals describes three general categories of
immunosuppressive agents (Halloran, 2004, Lindenfeld et al., 2004, Gonzalez Posada, 2006,
1970). These include antibodies directed against lymphocytes, steroids, and agents that
interfere with metabolism or that have toxic effects on cells. The first category is often
referred to as antilymphocyte or antithymocyte globulin (ALG or ATG). ALG in
combination with an intrathymic dose of donor spleen cells was shown to induce graft
survival in a rat model (Shen et al., 1996). Lewis-Brown Norway cardiac allografts showed
extensive survival in Lewis rats; whereas skin grafts from the same donor strain were

Immunological Considerations for Inducing Skin Graft Tolerance
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rejected at a similar rate as third party grafts, that is, grafts bearing transplantation antigens
different from those of the donor or host. These experiments demonstrated further the
increased antigenicity of skin in relation to other tissues. The author hypothesizes that
additional non-MHC skin antigens may be responsible for the rejection. Additional aspects
of skin antigenicity are described above (Murray, 1971, Nasir et al., 2009, Tobin et al., 2009,
Akdis, 2010, van den Berg et al., 2011, Kaplan, 2010, del Rio et al., 2010).
Adding ALG to a protocol that includes azathioprine and prednisolone may reduce the toxic
effects of ALG. Corticosteroids such as prednisolone reduce the inflammatory response,
interfere with protein synthesis and are cytotoxic for lymphocytes. Despite the lymphocyte
toxicity, these reagents were not found to prolong skin graft survival. The cytotoxic and
antimetabolite drugs appeared to target DNA function, activity, or synthesis.
Cyclophosphamide is a strong DNA cross-linker and alkylating agent. Methotrexate is a
folic acid inhibitor that blocks DNA synthesis, and therefore interferes with cell division in
lymphocytes. Azathioprine and 6-mercaptopurine are two related purine analogs that have
been show to delay graft rejection. The effect of all of these drugs was variable depending
on the species treated and the regimen used. Optimum regimen included using the drugs in
combination with the specific antigen in order to have a more prolonged graft-protecting
effect rather than a temporary dampening of the body’s ability to mount an immune
response.
2.2 Protection of rabbit skin by phenothiazine derivatives
Eyal and associates (1965) used phenothiazine related products to suppress immune
responses to rabbit skin allografts. The team focused their attention on the action of three
different compounds: chlorpromazine, perphenazine, or promethazine. They chose these
reagents because of reports that these compounds could minimize cell death and tissue
necrosis, albeit a protective effect was not witnessed with the engraftment of guinea pig
skin. Eyal and colleagues hypothesized that an optimal dose and specific compound
combination would be protective. These compounds produced different degrees of graft
protection in controlled experiments where they were tested against normal saline in
otherwise untreated animals. Both treatment and control groups of rabbits were given ear
skin allografts of approximately equal sizes. They found that the most beneficial compound
was promethazine, followed by perphenazine; and that the least protective was
chlorpromazine. They attribute the extended skin graft survival of treated animals to a
membrane protective effect of the drugs on graft recipient cells and to a reduced loss of
donor antigen from the graft itself.
2.3 Prolongation of graft survival mediated by methylhydrazine derivatives
Floersheim (1967) used methylhydrazine derivatives to induce tolerance in MHC disparate
adult mice and reported significant improvement in graft prolongation when the treatment
was combined with an infusion of donor specific cells derived from spleen or from kidneys
and liver. Graft survival was increased to at least twenty percent when this combined
therapeutic approach was used. According to the US Environmental Protection Agency
(EPA) these drugs may have a negative impact on liver and kidney primarily, and on blood
and spleen secondarily (EPA, 2007). Acute but not chronic effects have been reported for
humans (EPA, 2007). Thus despite some benefits in terms of graft survival, potentially
harmful side effects limited more widespread use.

Skin Grafts – Indications, Applications and Current Research
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2.4 The use of urethane in combination with X-irradiation to protect skin grafts
Cole and Davis (1962) used a reagent known as a DNA antagonist and an inhibitor of
mitosis in order to prolong survival of skin grafts in sub-lethally X-irradiated mice. They
were aware of studies that demonstrating that high doses of radiation could protect a
graft by suppressing an immune response. Cole and Davis hypothesized that a lower
radiation dose in combination with urethane treatment would be immunoprotective and
showed that sixty percent of the mice had significant skin graft survival. Mean graft
survival in the treated animals was 40 days vs. 18 days for untreated mice of the same
strain. Their study included an early attempt to develop irradiation bone marrow
chimeric mice. A subsequent study induced skin graft tolerance that endured for more
than 130 days and was specific for transplantation antigens of the bone marrow donor, as
third party grafts were rejected (Davis and Cole, 1963). In such experiments, the native
bone marrow is ablated, in whole or in part, by radiation, and specific donor derive bone
marrow is used to replace the ablated cells of the host. Additional examples of irradiation
bone marrow chimerism appear below.
More recently, urethane or polyurethane has been used as a dressing to promote wound
healing of split thickness skin graft donors (Cigna et al., 2009), and as a component of
negative pressure dressings for young burn patients who receive skin grafts (Psoinos et al.,
2009).
2.5 Calcineurin inhibitors
Calcineurin is a protein phosphatase found in eukaryotic cells whose activity is significantly
blocked by cyclosporine A and Fk506, now referred to as tacrolimus (Rusnak and Mertz,
2000). The once-popular calcineurin inhibitors (CNI) fell out of favor due to their observed
nephrotoxicity (Groetzner et al., 2004). Additional complications of the use of CNI drugs
include associated malignancies. Doesch et al. (2010) studied the development of neoplasias
in cardiac patients treated with immunosuppressive drugs. They found a strong correlation
between the use of CNIs or the drug azathioprine and the development of malignancies.
These drug-associated malignancies include multiple forms of skin cancer, including
squamous cell carcinoma (Wu et al., 2010), but exclusive of melanoma (Signorell et al., 2010).
In contrast, the rate of malignancies was reduced in patients treated with non-CNI based
agents such as mammalian target of rapamycin (m-TOR) inhibitors (Doesch et al., 2010).
3. Pre-engraftment reduction of tissue antigenicity
3.1 Cultured tissues and antibodies
Jacobs and Uphoff (1974) reviewed a variety of methodologies used to reduce the potential
immune reaction between the donated tissue or organ and the graft recipient and reported
that phenotypic changes including reduced antigenicity may be observed in cultured cells.
These changes vary with species. The advantage of using these techniques may be a reduced
dependence on the use of immunosuppressive agents. Antibodies may be used to treat the
graft donor or the tissue itself. They summarized the experiments of Hellman and Duke
(1967) who used a skin allograft model to demonstrate the reversal of tolerance to syngeneic
skin when the tissue was incubated with skin of a MHC disparate donor. Unpredictably,
pre-incubating donor and recipient skin together did not result in prolonged skin allograft
survival.

Immunological Considerations for Inducing Skin Graft Tolerance
353
3.2 Preconditioning with allogeneic RNA or DNA
In agreement with this data, the benefits of altering donor skin antigenicity was further
demonstrated when Guttman et al. (1964) prevented acceptance of syngeneic skin grafts by
pretreating mouse skin in vitro with allogeneic RNA. In contrast to these results, Lemperle
et al. (1968) demonstrated that pre-treating donor tissue with RNA or DNA facilitated
extended skin graft survival in MHC disparate donor and host combinations.
4. The use of chimerism to protect engrafted tissues
4.1 Bone marrow chimerism induced by whole body Irradiation
An early model of irradiation induced bone marrow chimerism was developed by two
scientist working at the National Institute of Allergy and Infectious Disease (Liacopoulos
and Goode, 1964). Liacopoulos and Goode were aware that overwhelming the immune
system of potential tissue donors with a bolus of strong antigens from different sources may
block reactivity to a specific unrelated antigen. This procedure is known as protein
overloading and is directed towards the graft donor. The group tested the hypothesis that
spleen cells, bone marrow or skin from animal models given a large dose of antigen would
be tolerated in another animal. The graft recipients were irradiated and pretreated with
donor strain derived cells prior to skin grafting. Rabbit gamma globulin or Limulus
hemocyanin served as the strong antigens. The authors used both a mouse allograft model
and a concordant rat-to-mouse xenograft model (Liacopoulos and Goode, 1964).
Owen et al. (1945) had observed earlier that naturally occurring tolerance could develop
between dizygotic calves in utero. Since these calves each had a different immunological
makeup, the prenatal exposure to each other’s disparate antigenic makeup was
undoubtedly the source of the immunological unresponsiveness. Stone et al. (1965)
expanded the initial findings by engrafting skin between dizygotic twins known to be
chimeric at the erythrocyte level. The discovery of chimerism in these calves provided the
scientific basis for adapting this concept to the development of irradiation bone marrow
chimerism as a model for the induction of transplantation tolerance.
Following these discoveries, a series of studies tested the hypothesis that the phenomenon
of chimerism could be experimentally induced in order to prolong allograft survival (Matheʹ
et al., 1963, Seller, 1967, Lubaroff and Silvers, 1973, Buckley, 1975). Mathe‘ et al. (1965)
applied this concept in order to protect a patient from leukemia. The patient was given
whole body irradiation followed by a bone marrow transplant comprised of tissues from six
different male and female donors, all of whom were related to the host. The bone marrow
graft restored the myeloid and erythroid compartments. A skin graft from one of the male
bone marrow donors remained intact and viable for more than seven months, while skin
grafts from other donors were rejected. These results implied that the donor and the host
were more histocompatible. More recent applications of irradiation bone marrow chimerism
are discussed below.
Tolerance to allografts following whole body irradiation has been reviewed (Strober et al.,
1979, Slavin et al., 1985, Sprent et al., 1993, Monaco, 2004). A potential mechanism to explain
the success of these grafts was suggested by Okada and Strober (1982)who used a mixed
lymphocyte reaction (MLR) to demonstrate that spleen cells from animals given whole body
irradiation induced the proliferation of large numbers of cells directed against specific
antigenic targets. This work extended earlier studies that identified two distinct populations
of suppressor cells induced by these methods. One population responded specifically to

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bovine serum albumin (BSA) as an antigen, while the other responded non-specifically
(Slavin and Strober, 1979).
4.2 The induction of chimerism without myeloablation
Several investigators have reported the induction of allogeneic bone marrow chimerism
without myeloablation (Sykes, 1996, Pan et al., 2003, Fuchimoto et al., 2000, Shapira et al.,
2003, Matthews et al., 2004, Ciurea and Andersson, 2009, Przepiorka et al., 1999).
Myeloablation refers to the massive radiation induced destruction of the host’s bone
marrow cells, especially the T cell lineages, and the use of allogeneic bone marrow to replace
the destroyed cells. Because of the radiation, with or without other treatments, the host is
unable to reject the donated bone marrow cells. The major side effects associated with lethal
whole body irradiation, have caused some investigators to develop non-myeloablative
procedures (Sykes, 1996).
4.2.1 Chimerism to induce tolerance to allografts and xenografts
Sykes used low dose (3 Gy) whole body irradiation in combination with a higher dose (7
Gy) irradiation of the thymus and antibodies that deplete CD4+ and CD8+ T cells to generate
specific tolerance to the cells and tissues of the donor (Sykes, 1996). Sykes used this protocol
to show tolerance to allografts in the mouse, and tolerance of rat skin in murine hosts. She
used the swine–to–mouse model to show that the protocol could be expanded to a
discordant model, that is, one involving hyperacute reactions between widely disparate host
and donor species. She depleted T cells and natural killer cells (NK) in thymectomized mice,
then engrafted the thymus of a fetal pig. These mice were shown to accept a skin graft from
the same donor strain as the thymus graft. Despite being described as “non-myeloablative,
the procedure combines low dose total body irradiation with focused high level irradiation
targeted to the thymus.
4.2.2 Irradiation-free chimerism in a murine allograft model
Pan et al. (2003) used a combination of costimulation blockade and a metabolism antagonist
to reduce the donor T cell population in donor bone marrow to induce chimerism in mice.
Depletion of donor T cells reduced the risk of graft vs. host disease (GVDH) wherein donor
T cells initiate an immune response against the new host. Pan and colleagues used a
combination
of
fludarabine,
described
below,
the
immunosuppressive
drug
cyclophosphamide, and interruption of the CD40/CD154 T cell activation pathway to
facilitate donor bone marrow engraftment. The protocol resulted in mixed chimerism in the
host and allowed donor origin skin grafts to survive.
4.2.3 Irradiation-free chimerism in a swine animal model
Fuchimoto and colleagues (2000) developed a technique to create a hematopoietic system
composed of both donor origin and host origin cells, referred to as “mixed chimerism” to
distinguish the results from full chimerism that often occurs when the host is given lethal
irradiation followed by an allogeneic bone marrow transplant. They used high doses of stem
cells separated from peripheral blood to induce mixed chimerism. The chimeric host
accepted skin grafts from a donor whose transplantation antigens (swine leukocyte antigens,
SLA) matched those of the bone marrow, but did not accept skin grafts from a third party
donor, one that matched neither the bone marrow donor nor the new host.

Immunological Considerations for Inducing Skin Graft Tolerance
355
4.2.4 Irradiation-free chimerism in humans
Shapira et al. (2003) reported an attempt to induce chimerism with no radiation in high risk
patients who were not able to tolerate whole body radiation. The use of the drugs
fludarabine and busulfan had a myeloablative effect on the host bone marrow cells.
Fludarabine has been reported to minimize the development of neoplasias as well as to
suppress metabolism (Matthews et al., 2004). Busulfan has been described as a
myeloablation agonist that has been used to engraft bone marrow and stem cells (Ciurea
and Andersson, 2009, Przepiorka et al., 1999). Eight percent of the patients developed full
chimerism as evidence by donor origin hematopoietic cells. Regrettably, graft-versus-host
disease (GVHD) was identified in most patients despite a brief low dose treatment of
cyclosporine-A, and mortality was high.


5. Costimulation blockade to interrupt the process of T cell activation
If the events reported by Stone’s group represent naturally occurring prenatal tolerance
(Stone et al., 1965), how then may we induce tolerance in an adult? One answer may be
found by dissecting the pathway for the development of immunity. These experiments
tested the hypothesis that interference with one of the key steps leading to an
immunological response would prevent the downstream events from occurring. This type of
tolerance is referred to as peripheral tolerance as it leaves the host with the ability to
respond to other immunological perturbans. Exposure of untreated animal models to a
source of foreign antigen initiates the process of immunity and may provide the stimulus to
recruit potentially alloreactive or xenoreactive cells from their histological sites. Subsequent
interference with the reaction between CD40 and CD40L (CD154) or the reaction between B7
molecules and cytotoxic T lymphocyte-associated antigen (CTLA)-4 provides a scenario
wherein cells that recognize the foreign antigen receive the initiating signal that recruits
them to the site of antigen deposit, but not the required secondary signals. Costimulation
blockade may be mediated by monoclonal antibodies directed against specific cell surface
molecules, by the use of agonistic monoclonal antibodies to prevent reactivity induced by
donor origin cells within the graft, referred to as graft-versus-host disease, (Albert et al.,
2005, Yu et al., 2003, Yu et al., 2000) by the use of anti-sense RNA to facilitate apoptosis (Yu
et al., 2004), or by deletion of cellular components expressing costimulation molecules. In
systems where CD4 cells are important, graft survival may be accompanied by the
expansion of CD4+ CD25 Foxp3 regulatory cells.
When the elements of one’s own immune system come into contact with particles, agents,
cells or tissues not belonging to the host, those foreign agents are recognized as non-self.
Immunity is the complex process of activating the body’s adaptive immune defenses in
order to protect the body from these specific invading elements. T cells are components of
the adaptive immune system, and as such may be geared up to protect the body from
foreign invaders, including pathogenic organism and transplanted cell and tissue grafts.
They are increasingly protective upon re-exposure of the organism to the same antigen
(Janeway C.A. et al., 2001).
An important feature of immunity is the activation of thymus derived lymphocytes or T
cells. Grakoui et al. (1999) provided a detailed study of the environment and the molecular
interactions required for T lymphocyte activation. In a host with an intact immune system,
immunity would result in the destruction of engrafted tissue or organs from another
individual who is not an identical twin. T cell activation is an essential feature of immunity,

Skin Grafts – Indications, Applications and Current Research
356
and thus a significant player in the acceptance or rejection of a graft. A hypothesis utilizing
this information might be stated as follows: If T cell activation is important for immunity
and graft rejection, then interruption of the T cell activation cascade will prevent or delay
graft rejection. Preventing an immune reaction between graft and host would provide an
environment conducive to transplantation tolerance. In particular, preventing an immune
response to the specific graft-associated antigens, often described as anergy, would be more
beneficial than a generalized immunosuppressive response because the host immune
system would still be capable of protecting the body from other invaders.
Modern transplantation immunology owes a great debt to the pioneering work of Jenkins
and Schwartz (1987) who demonstrated that antigen presentation devoid of a MHC context
failed to stimulate T cell clones in vitro or T cells in vivo. They used a chemical crosslinking
agent to affix pigeon cytochrome peptides to the surface of spleen cells. If properly
presented with the cell’s MHC surface molecules, the process would have resulted in T cell
proliferation, IL-2 production (Jenkins et al., 1987) and antigen recognition (Jenkins and
Schwartz, 1987). The inappropriate antigen presentation resulted in T cells unresponsive to
their cognate (recognizable) antigen; and thus the authors concluded that the mechanism
involved was the deletion of antigen-specific T cells (Jenkins and Schwartz, 2009). Their in
vitro blockade of T cell activation was reproduced in the mouse model.
In 1993, Boussiotis et al. (1993) published their research implicating the costimulatory
molecule B7 as a contributor to allograft immunity. Using a transfection model, the authors
showed that interrupting the B7:CD28/CTLA4 pathway was consistent with an anergy
model of immune unresponsiveness to one specific human derived transplantation antigen,
HLA-DR7. In contrast, blocking the reaction between the intercellular adhesion molecule 1
(ICAM1) and lymphocyte function antigen 1 (LFA1) appeared to be associated with
immunosuppression. Important contributors to T cell activation have been reviewed by
Wingren et al. (1995) and include the work of Ford and Larsen (2009). The work of Jenkins,
Schwartz, and colleagues has shown antigen specific T cell unresponsiveness (Jenkins et al.,
1987, Jenkins and Schwartz, 1987, Jenkins and Schwartz, 2009). Further research led to
clarification of the mechanisms of T cell signaling and activation. One set of stimulatory
reactions was a consequence of the binding of B7 (CD80 or CD86) with CD28; while a
different cascade of events followed the binding of CD40 with CD154 (CD40L). Many
features of these pathways were elaborated by Bluestone et al. (1995) and have been
reviewed by Lenschow et al. (1996). The three main pathways of T cell activation have been
described by ML Ford and CP Larsen (2009).
Larsen et al. (1996) showed the importance of interrupting CD40/CD28 binding in skin and
cardiac allografts, although mechanistic details remained for future investigations. Some
mechanisms may include the induction of anergy, as mentioned above, the reduction of
alloreactive T cell populations (Iwakoshi et al., 2000), the induction of regulatory T cells and
the interactions of other cells with regulatory functions (Gordon and Kelkar, 2009), as
discussed below, or diverting the proliferation of a specific T cell type (Li et al., 2009). Pree
et al. have been advocates of the translational aspect of costimulation blockade. In a clinical
setting, these protocols may facilitate mixed chimerism and prevent a GVH reaction that
may otherwise ensue (Pree et al., 2009, Pan et al., 2003).
5.1 The B7/CD28 pathway and skin grafts
Preventing binding of B7 molecules on B cells to their ligands, CD28 on T cells has been
shown to be effective for interrupting T cell activation. Shiao et al. (2007) used a humanized

Immunological Considerations for Inducing Skin Graft Tolerance
357
antibody directed against the T cell CD28 molecule to prevent the expansion of alloreactive
T cells. The experiments were done in beige/SCID mice bearing human skin grafts and
infused with peripheral blood cells expressing different human transplantation antigens
(HLA) than those of the skin donor. Liu et al. (2007) used a gene therapy model to infuse
antisense B7.1 in order to suppress alloreactivity to rat spleen cells. This model was not
associated with GVHD as measured by examination of intestines, liver, skin, or other
tissues(Liu et al., 2007). Rulifson et al. (2002) demonstrated that Langerhans cells resident in
the skin of the donor were such effective antigen presenting cells (APCs) that they could
prime T cells for activation independently of either the B7 or CD40 activation pathways. In
their hands, the strength of this direct antigen presentation reaction limited the success of
tolerance induction mediated through the interruption of either B7-CD28 or CD40-CD154
pathway. These findings are consistent with the work of Tao et al. (1997) who concluded
that weak T cell receptor (TCR) signaling primed CD4+cells in a CD28/B7 dependent
manner. Early experiments using the skin allograft model were important for demonstrating
that additional costimulatory molecules must be important for completing the T cell
activation cascade (Kawai et al., 1996). Their report demonstrated suppressed proliferation
and cytokine production by CD28 depleted T cells, nonetheless, these cells were capable of
initiating skin allograft rejection.
5.2 Interrupting CD40 and B7 ligation
The combined effect of blocking the CD28 and CTLA4 ligation reactions was tested in a
murine skin graft model (Li et al., 2006). The combined treatment was delivered via a
replication incompetent adenovirus vector. Significant skin graft survival was
demonstrated in the treated mice in contrast to rapid rejection of skin grafts in mice that
were untreated or given non-expressing vectors. A detailed study outlining the efficacy of
CTLA4Ig produced by different sources as an efficient tool for interrupting costimulatory
signals produced by the ligation of B7/CD28 molecules was completed (Najafian and
Sayegh, 2000). CTLA4Ig’s higher affinity for B7 competitively inhibits B7/CD28 ligation,
therefore interfering with the delivery of costimulatory signals. Other investigators found
that CTLA4 signaling facilitated allograft survival of skin and islet grafts in mice treated
with an infusion of donor derived antigen plus ant-CD154 monoclonal antibody (mAb)
(Zheng et al., 1999). Tung et al. (2008) showed the combined effect of blockading both the
B7 and CD40 pathways in a series of limb graft experiments. Balb/c male mice were
donors of limbs engrafted onto C57Bl/6 female mice heterotopically. Costimulation
blockade was mediated by anti-CD154 antibody with or without CTLA4-Ig. Anti-CD154
alone resulted in limb survival of 75 days, but graft survival was extended to 120 days by
the combined therapy. Larsen et al. (1996) demonstrated the advantage of blocking both
of these costimulatory pathways in order to facilitate the engraftment of skin and cardiac
tissue. Both in vivo and in vitro confirmation of the effect was discussed.
Protracted survival of murine skin allografts in thymectomized mice treated with an
infusion of donor derived spleen cells and anti-CD154 mAb was achieved only in mice
expressing CD4+ T cells, gamma interferon, and CTLA4 (Markees et al., 1998). In contrast,
the studies by Gordon et al. (1998) using the concordant rat-to-mouse xenotransplantation
model, showed no requirement of CD4+ T cells or for gamma interferon for prolonging the
survival of rat skin and islets in mice. The role of B7/CD28 ligation in providing
costimulatory signals to effect T cell activation was confirmed by the studies of Onodera et

Skin Grafts – Indications, Applications and Current Research
358
al. (1997). They pre-sensitized Lewis rats with Brown-Norway skin grafts and evaluated the
survival of cardiac grafts when recipients were treated with CTLA4Ig with or without an
infusion of donor antigen. They hypothesized that graft survival was mediated by either
clonal anergy or the deletion of alloreactive CD8+ cells.
5.3 Interfering with CD40/CD154 ligation
Although the first study of the CD40/CD154 pathway to promote graft survival utilized a
concordant rat-to-mouse islet xenograft model (Markees et al., 1996), the first use of this
system to facilitate skin graft acceptance was reported by Larsen at al. (1996), and
described aspects of the costimulatory process related to both CD28-B7 ligation and CD40-
gp39 ligations. The “gp39” designation was later changed to CD154 in keeping with the
system for naming cell surface molecules. In their hands, each set of costimulatory
molecules provides necessary but not identical signals resulting in fully activated T
lymphocytes. Interrupting both of these pathways provided conditions for prolonged
survival of heart tissue and skin allografts.
Interrupting the reaction between CD40 and CD154 has been confirmed as an effective
method for limiting T cell costimulation. This pathway is required for the generation of T
cells that are fully activated against alloantigen (Yamada and Sayegh, 2002), and
interruption of this pathway promotes graft survival or tolerance. Moodycliffe et al. showed
that the binding of these molecules encouraged the migration of dendritic cells from the skin
to the lymph nodes for effective antigen presentation (Moodycliffe et al., 2000). Their model
assessed dendritic cell (DC) function in wild type C57Bl/6 mice or those devoid of CD154
molecules. A combined therapy consisting of a bolus infusion of donor derived spleen cells
and a brief course of treatment with anti-CD154 mAb resulted in prolonged survival of rat
skin grafts in mice. Graft survival was enhanced significantly in mice devoid of CD4+ T
cells (Gordon et al., 2001).
Nikolic et al. used a skin graft model to differentiate between models that blocked immunity
to alloantigens vs. those that interrupted autoimmunity (Nikolic et al., 2010). They used
normoglycemic non obese diabetic mice (NOD) that received whole body irradiation, T cell
depletion, and anti-CD154 mAbs with or without bone marrow cells from C57Bl/6 mice.
The treatment delayed or prevented hypoglycemia, but only prevented isletitis in mice that
received bone marrow cells as well as the other conditioning treatments.
Xu et al. (2010) studied the role of minor histocompatibility antigens in bone marrow grafts.
They found that when they used B10.BR skin to sensitize AKR mice to alloantigens, donor
origin cells were cleared more rapidly than in unsensitized mice of the same strain.
Recipient mice treated with antibodies to CD154 did not produce antibodies to minor
histocompatibility antigens of the donor. These studies suggest that blockade of CD40-
CD154 ligation may facilitate bone marrow transplantation and reduce the risk of reactivity
to minor histocompatibility antigens.
Although blockade of CD40 and CD28 activation pathways did not produce the
vasculopathy shown with other methods, translating this method into a clinical protocol
was unexpectedly problematic because of the development of thromboembolisms
(Yamada and Sayegh, 2002). Scientists have since analyzed the cause for the
pathophysiological problems identified clinically. Their reports help to identify platelets
as a source for soluble CD154 or CD40 ligand and demonstrate a correlation between
increased circulating levels of this molecule and increased thrombus formation (Yacoub et

Immunological Considerations for Inducing Skin Graft Tolerance
359
al., 2010, Yuan et al., 2010). These studies show further that CD154’s concentration is
increased in hypertensive patients included non-diabetic and pre-diabetic persons with
metabolic syndrome (Unek et al., 2010).
In an effort to overcome these complications, Gilson et al. (2009) compared two different
isotypes of anti-CD40 monoclonal antibodies. Isotypes are any one of the five main
structurally and functionally distinct categories of antibodies (Janeway C.A. et al., 2001).
Gilson et al. (2009) found that the IgG2b, but not the IgG1 antibody had a synergistic effect
on graft survival when combined with CTLA-4-Ig, and hypothesized that the use of
alternative antibody isotypes may encourage the development of new reagents that are as
effective as anti-CD154 antibodies, but without harmful clinical effects.
6. The induction of regulatory T cells (Tregs)
In 1975, Gelfand and Paul (1975) used an anti-thy1.2 antibody to facilitate C57Bl/6 skin
grafts on Balb/c mice. The antibody, which was directed against thymocytes, resulted in
prolonged skin graft survival in comparison to untreated mice. The study demonstrated
infectious tolerance as the effect could be transferred to new hosts given cells of the treated
mice. The authors thus hypothesized that the treatment generated a population of
suppressor cells that regulated the processes responsible for graft survival. In the same year,
Gershon hypothesized the existence of suppressor T cells that down modulated immune
responses and detailed predominant requirements for their functionality (Gershon, 1975).
These concepts were controversial at the time that they were advanced.
During the 1980’s, the concept began to take hold that there existed a specific population of
regulatory T lymphocytes with the capacity to specifically suppress immune responses.
Maki et al. showed that B6AF1 mice pretreated with ALS and given both BMC and skin
grafts from CH3 donor mice had suppressed reactivity against donor antigen as
demonstrated by in vitro assays (Maki et al., 1981a, Maki et al., 1981b). Streilein and
Niederkorn (1985) described the induction of a suppressor T cell population that helps to
explain the phenomenon of the anterior chamber of the eye functioning as an
immunologically privileged site. The suppressor activity was associated with improved skin
graft survival and other host responses. The suppressor cells were described as Thy1.2 and
L3T4 expressing CD4+ T lymphocytes.
Subsequent studies by Subba and Grogan (1986) using rat skin allografts implanted in the
anterior eye chamber resulted in minimal alloreactivity as measured in vitro. Treatment
with cyclophosphamide or removal of the allograft reversed the immunosuppression seen
in mixed lymphocyte reactions (MLR), thus implicating the involvement of suppressor
cells.
Based on a wealth of more recently generated data, the scientific community no longer
needs to be convinced that a regulatory cell population exists or can be induced. Regulatory
T cells have been identified or induced by some of the methods shown in Table 1 below. We
now accept the description of the predominant category of regulatory cells responsible for
allograft survival, the generation of chimerism and mixed chimerism, protection from
autoimmunity and reduced risk of graft vs. host disease (GVHD) and host vs. graft disease
(HVG) as being CD4 + CD25 +Foxp3 + regulatory T lymphocytes. These Tregs may be
naturally occurring in the thymus or adaptively induced in response to foreign antigen and
costimulation blockade (Guo et al., 2008, Schwartz, 2005, Sakaguchi, 2005, Coenen et al.,

Skin Grafts – Indications, Applications and Current Research
360
2005, von Boehmer, 2005). The Tregs express the forkhead transcription factor Foxp3
(Fontenot and Rudensky, 2005), and there is some evidence that the expression level of this
transcription factor may be a key indicator of the level of immunosuppression potential
(Chauhan et al., 2009) The immunosuppressive effect of these cells has been shown by in
vivo and in vitro experiments.


Graft or Target
Authors
Method of Induction
Skin grafts
Autoimmunity
(Oderup et al., 2006)
Il2Rbeta-/- recipients

Heart
(Oderup et al., 2006)
Rag 1 -/- given anti-154 and anti-B7

Chimerism
(Iudaev et al., 1975)
Sublethal WBI and BMC
Hy antigen and HVG
(Weng et al., 2007)
Sublethal WBI
Chimerism
Skin allografts
(Yamazaki et al., 2007)
BMC, DST and anti-CD154
Skin graft
(Chai et al., 2005)
Foxp3 transduced expression in naïve
CD4+ CD25_
Skin graft
(Banuelos et al., 2004)
DST and anti-CD154 in CD4, CD8, or
CD25 depleted skin and islet graft
recipients
Skin graft
(Sanchez-Fueyo et al.,
2007)
Wt or Class II – mice

Skin graft
Mixed chimerism
(Pilat et al., 2010)
Rapamyacin and costimulation
blockade;
Skin graft
(Kim et al., 2011)
Anti-CD154, Non cytolytic anti-CD4
Composite tissue
allograft
(Bozulic et al., 2011)
Anti-TCR, WBI

Legend: Table 1 describes some milestones in the history of regulatory T cell recognition. Abbreviations:
WBI, whole body irradiation, BMC, bone marrow cells; HVG, host vs. graft reaction; Hy, male specific
antigen; DST, donor specific antigen; wt, wild type; TCR, T cell Receptor
Table 1. The Induction of CD4 + CD25 + Regulatory T Cells

Immunological Considerations for Inducing Skin Graft Tolerance
361
7. Other cells with regulatory functions
7.1 Double negative T cells
Double negative T cells, described as CD3+ T lymphocytes bearing typical αβT cell receptors,
but expressing no CD4, CD8, or NK1.1 surface molecules. These cells use the process of
trogocytosis to gain access to the alloantigen, xenoantigen, or self peptides that are then
expressed on their cells surfaces (Ford McIntyre et al., 2008). Trogocytosis is the process of
utilizing broken pieces of plasma membrane as an antigen capture mechanism (Hudrisier et
al., 2007). B and T cell receptors as well as some costimulatory co-receptors appear to be
involved in this process.
7.2 CD8+ T cells
CD8+ T cells have been shown to promote skin allograft survival in fully mismatch donor-
host combinations. Graft survival was initiated by rapamycin treatment of graft hosts (El
Essawy et al., 2011). El Essawy and colleagues found that C57Bl/6 mice devoid of CD4
molecules handily rejected DBA mouse skin allografts, but showed prolonged (median
survival, >100 days) survival of these grafts following rapamycin treatment of the grafts
hosts. These regulatory/suppressor T cells were CD28 + but showed low levels of IFN-γ, IL-
2, and IL-10.
7.3 Natural Killer T (NKT) cells
Natural Killer T (NKT) cells are lymphocytes bearing TCR T cell receptors. A regulatory
role for these cells was described in mice devoid of the Jα18 segment of the TCR and in
mice lacking the CD1 restricting element associated with NKT cells following
costimulation blockade mediated by a transfusion of donor antigen and a short course of
anti-CD154 mAb (Gordon and Kelkar, 2009). Graft survival was abbreviated in the Jα18
and in the CD1 deficient mice as compared to wild type mice of the same strain (Gordon
and Kelkar, 2009).
7.4 Induction of tolerogenic dendritic cells
Adorini and Penna (2009) report on the use of vitamin D receptor (VDR) agonist as a means
of inducing tolerogenic dendritic cells (DCs). These DC are associated with an
immunosuppressive effect. The use of secosteroid hormones that function as VDR agonist
help to induce DCs that then facilitate the induction of CD4(+) CD25(+) Foxp3(+) regulatory
T cells.
8. The humanized mouse as a model for the study of the human immune
system
The humanized mouse model takes advantage of the existence of basic cellular
deficiencies in non-obese diabetic (NOD) and CB-17 scid/scid strains of mice. Some strains
are crosses between the two and bear the deficiencies of each parental type. The severe
combined immunodeficient substrain lacking a functional IL-2 receptor gamma gene has
become a more advanced mouse model. These mice may be infused with human
hematopoietic stem cells that reconstitute a functional human immune system in the
murine environment. These genetically altered mice provide important tools for studying

Skin Grafts – Indications, Applications and Current Research
362
autoimmunity, cellular and molecular interactions of the human immune system, solid
tumors and other forms of cancer, and may provide models for studying human
retroviruses or other infections where no appropriate animal model exists. Many of these
strains have been developed by Lenny Schultz and others at Jackson Laboratories (Bar
Harbor, ME). Some phenotypic characteristics of the most recent strain include the lack of
T cells, B cells, and NK cells. There is no IL-2R γ chain, both the innate and adaptive
compartments of the immune system are defective, and most antibody responses are very
low. Salient features of these mice including timelines for their development have been
reviewed recently (Van Duyne et al., 2009, Ishikawa et al., 2008, Hill et al., 1991). In the
section below, we review the use of this important model for investigation in several areas
of biomedical research.
8.1 Humanized mouse system for the study of skin grafts and wound healing
Racki et al. (2010) compared usage of NOD-scid IL2rgamma(null) mouse (NSG) vs. the
CB17-scid bg (SCID.bg) mouse to study skin allograft survival vs. rejection. They found
that components of the human immune successfully engrafted into the NSG mouse, but
that skin graft survival was poor. In contrast, the SCID.bg mouse was less successful in
engrafting human cells, but showed improved skin graft survival over the NSG model.
Their results showed that infiltration of transplanted human skin by glucocorticoid
receptor isoform 1 (GR-1+) cells impaired graft survival. GR-1+ cells are immature myeloid
cells that may differentiate into dendritic cells, have been identified in human bone
marrow recipient, and are characterized as having an immunosuppressive effect (Li et al.,
2004). When Racki’s team treated NSG mice with antibodies to GR1, the cellular infiltrate
decreased and skin graft survival was improved. Additional studies that impact skin graft
survival and wound healing used an alternative model, the nude mouse (Escamez et al.,
2004). This mouse lacks a thymus, and therefore has no mature T lymphocytes. They
found that treating laboratory engineered “skin equivalent” with keratinocyte growth
factor improved wound healing.
Gilet et al. (2009) used SCID mice reconstituted with human PBLs and engrafted with
human skin to study T cell subset recruitment mediated by the cytokine CCL17. Erdage and
Morgan (2004) used the same animal model (huPBMC-SCID) to study the differences in allo-
and xenoreacivity to bioengineered skin modified with or without keratinocyte growth
factor. The team used the same model to study the survival of human fetal vs. neonatal skin
grafts. They found that the lower MHC Class I and Class II expression in fetal skin was
associated with longer graft survival than that of neonatal skin (Erdag and Morgan, 2002).
Issa et al. (2010) separated regulatory T cells from human PBLs and used them to control
immune responses to skin allografts in a humanized mouse model.
8.2 The humanized mouse system and autoimmunity
The humanized mouse model has become an important research tool for the study of type 1
diabetes and psoriasis. Pearson et al. (Pearson et al., 2008) described the NOD-Rag1null
Prf1null Ins2Akita spontaneously hyperglycemic mouse model. This model has no
autoimmune defect, but may be used to study type 1 diabetes in an environment that avoids
any toxicity associated with chemically induced diabetes models. King et al. (2008) describe
the engraftment of peripheral blood mononuclear cells (PBMC) vs. human stem cells. They

Immunological Considerations for Inducing Skin Graft Tolerance
363
induced diabetes and showed that an infusion of allo-PBMCs leads to islet graft rejection,
thus they proposed the use of this model to study mechanisms of human islet allograft
rejection. Other aspects involving the use of these animal models to study diabetes have
been reviewed (King et al., 2008, Shultz et al., 2007).
Humanized mouse models of psoriasis have been developed that use bioengineered skin
derived from biopsies and from patients with psoriasis (Guerrero-Aspizua et al., 2010). A
psoriasis pathology was duplicated by injecting cultivated T cell populations along with IL-
17and IL-22, and depriving the skin of its stratum corneum. This model will be used to
assess the pathophysiology of the disease and to suggest alternative therapeutic targets, and
to test new hypothesis related to patient outcomes in pre-clinical investigations.
Bhagavathula et al. (2005) used the severe combined immunodeficient (SCID) mouse to
demonstrate the effectiveness of humanized neutralizing mAbs directed against transduced
amphiregulin as a therapeutic agent reversing some phenotypic characteristics of psoriasis
in transplanted skin. The mAb decreased epidermal thickness in vivo and reduced
keratinocyte growth in vitro.
Because TNFα antagonists have been used to counter the effects of psoriasis, Gordon et al.
(2005) used a humanized mAb directed against TNFα to dissect the mechanism of action of
these reagents. They injected the reagent into otherwise unaffected skin engrafted onto the
SCID-HU mouse model and they injected highly activated T cells in order to simulate an
acute psoriasis phenotype. The concentration of epidermal Langerhans cells (LCs) in the
plaques began to increase within one week of the mAb treatment, and thus the loss of LCs
was associated with plaque development. These studies help to clarify the method of action
of the TNFα antagonist and suggest a method for reversing the course of this disease in
patients.
8.3 The humanized mouse system and immunity: Graft vs. Host Disease (GVHD)
This model has been used to study GVHD. Soluble FasL was used to treat graft recipients
in order to prevent alloreactivity of immunocompetent donor T cells that recognize the
host antigens (Bohana-Kashtan et al., 2009). Pinot et al. (2010) describe the development
of the NSG mouse model and its use in studying xeno-GVHD. Vlad et al. (2009) used this
mode to demonstrate the prevention of GVHD through the use of an immunoglobulin
component, the immunoglobulin-like transcript 3-Fc protein, that induces CD8+
suppressor cells.
8.4 The humanized mouse system as a source for induced pluripotent stem cells
Hanna et al. (2007) used a humanized mouse model of sickle cell anemia to describe the
benefits of using induced pluripotent stem cells (IPS) derive from adult fibroblast cells. This
“knock-in” model replaced mouse globin genes with mutated human Aγ and βS globin
genes. The homozygous mutant developed many phenotypic characteristics of the sickle cell
disease. Tail snips from these mice were used as a source of fibroblast cultures. Retroviral
vectors were used to transduce expression of the four transcription factors needed to
reprogram the adult mouse cells or to express selectable markers. The investigators used
homologous recombination to repair the genetic defect responsible for sickle cell disease.
The investigators anticipate future studies that eliminate the use of retroviral vectors and
oncogenes in applications of IPS for human use.

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9. Conclusions
The modern world of skin grafting, arising from its historical roots to new depths of
scientific understanding, will help to advance translational science and medicine. Skin grafts
allow us to understand specific immunological functions, help dissect the mechanisms
responsible for tissue and organ specific tolerance, identify additional regulatory cells or
functions required for transplantation tolerance, test the development of mouse models of
the human immune system and help answer fundamental questions related to the use of
stem cells as curative agents for human disease.
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