Vol.1, No.2, 15-26 (2011)
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJI/
Open Journal of Immunology
Molecular and cellular pathways involved in the
therapeutic functions of MHC molecules; a novel
approach for mitigation of chronic rejection
Thomas S. Skelton1, Malgorzata Kloc*, Rafik M. Ghobrial*
1Department of Surgery, Methodist Hospital and Methodist Hospital Research Institute, Houston, USA.
*Department of Surgery, Methodist Hospital, Fannin St., Houston, USA; RMGhobrial@tmhs.org; mkloc@tmhs.org
Received 24 June 2011; revised 20 July 2011; accepted 28 July 2011.
The mutated major histocompatibility complex
(MHC) class I that contains donor-type epitopes
displayed on recipient-type molecule was show-
n to inhibit acute and chronic rejection and in-
duce indefinite survival of heterotopic cardiac
allografts when administered in combination
with a sub-therapeutic dose of cyclosporine
(CsA) in a rat transplantation model. To eluci-
date the molecular pathways involved in the
immunosuppressive effects of the mut a ted MHC
molecule, we analyzed gene and protein ex-
pression profile during early and late phase
following post-transplantation. Cytoskeletal str-
ucture analysis and expression status of Rho
GTPase proteins, vacuolar transport and cy-
toskeleton regulatory pathways involved in im-
mune response in T and dendritic cells demon-
strated the nov el mechanism fo r the ab rogati on of
chronic rejection. Our studies confirm a new role
of Rho GTPase pathway in the modification of T
cell motility and infiltration of the graft. We dis-
cuss these results within the framework of the
most recent literature on MHC and molecular
machinery controlling T cell functions and den-
dritic cell an tigen p resentation.
Keywords: MHC; Transplantation; Allograft;
Chronic Reje cti on; Rat
The immune system plays a critical role in maintaining
an individual’s autonomy, and essential to its function is
the ability to differentiate self from potentially harmful
agents, such as microbial or viral proteins, foreign lipids
and polysaccharides, which are viewed as non-self and
are capable of eliciting an immune response. The trans-
plantation of genetically incongruous organs produces an
immunological response through allorecognition of non-
self histocompatibility antigens. These antigens are lo-
cated on cell surfaces and are capable of inducing an
immune response in genetically dissimilar (allogeneic)
recipients, resulting in graft injury and acute rejection of
tissue or cells bearing non-compatible antigens.
Solid organ transplantation had been unsuccessful for
many decades until the discovery in 1967 of the human
major histocompatibility complex (MHC), which led to
the introduction of human leukocyte antigens (HLA)-
matching method [1,2]. However, in spite of that dis-
covery, patient and graft survival remained poor. Not
until the 1980s, when the immunosuppressant cyc-
losporine became available for clinical use, did a sig-
nificant improvement in transplant success rates appear
[3,4]. Despite this discovery and the development of
other novel immunosuppressants, better organ preserva-
tion, refined surgical techniques, and post-operative care,
all of which help to eliminate acute rejection, chronic
rejection continues to plague the majority of allografts
and is a major obstacle for the long-term success of
transplants. For instance, the one-year kidney allograft
survival from cadaveric and living related donors has
increased from 50% to roughly 90% and 95%, respect-
tively, when compared with the allograft survival out-
comes of 20 years ago, but the 10-year graft survival
rates have fallen below 60%, due to chronic allograft
dysfunction [5,6].
The rejection response elicited by transplantation be-
tween members of the same species is regulated through
T and B cell recognition of tissues expressing genetically
encoded polymorphisms within the MHC and the pep-
tide fragments that they carry [6]. In humans, the MHC
region resides on the short arm of chromosome 6 and
contains more than 200 genes of which more than 40
known as the HLA genes, encode codominantly expressed
T. S. Skelton et al. / Open Journal of Immunology 1 (2011) 15-26
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cell surface proteins or components of the complement
system. These genes and their translational products
have been further grouped into three MHC classes,
which are termed I, II, and III, based on their tissue dis-
tribution, structure, and function [7-9]. An important
discovery in 1974 revealed that MHC molecules are
functionally responsible for presenting peptide antigens
to immune cells, thereby allowing the T cell receptor
(TCR) to interact specifically with peptide fragments of
a foreign protein bound within the peptide-binding
groove of a MHC molecule [10]. Each MHC molecule
consists of an extracellular peptide-binding cleft an-
chored to the cell by transmembrane and cytoplasmic
domains. There is a high degree of allelic variation asso-
ciated with the HLA system. For instance, there are 200
known variants of HLA-B, 500 variants of HLA-A, and
over 1000 variants of HLA-DR [11,12]. Polymorphisms
in the amino-acid sequence encoded by these alleles
typically reside within or adjacent to the peptide cleft
and facilitate antigen binding and presentation to T cells.
Because of the amino acid variability in this region, each
MHC binds an individual repertoire of protein antigens
that are recognized by distinctly different T cells. The
ability to potentially present a wide range of peptides on
MHC molecules is the primary cause of HLA polymor-
phism and may provide a balance between maximizing
the immunological response against invading antigens
while preventing self-recognition and autoimmunity [13].
MHC class I and class II molecules both use very similar
peptide binding domains, and peptide binding plays an
integral role in the proper folding of the final pep-
tide-MHC complex [14]. Peptide antigens are held in
place by non-covalent interactions between the peptide
and binding cleft, which determines peptide specificity,
and a network of hydrogen bonds that determine the
length of bound peptide [15]. Given the role of MHC
molecules in antigen presentation, in the case of trans-
plantation, allogeneic MHC molecules can act as both
antigen-presenting molecules and as foreign antigens
capable of eliciting an immune response [13].
Class I MHC is present on all nucleated cells and is
composed of a 45-kd α heavy chain encoded by genes of
the HLA-A, HLA-B, and HLA-C loci on chromosome 6
in association with a 12-kd protein, β2-microglobulin,
which is encoded by a gene on chromosome 15 [2,9].
Class II MHC is expressed by professional antigen pre-
senting cells (APC), namely, dendritic cells (DCs) and
by B lymphocytes, macrophages, endothelial cells, and
thymic epithelial cells [15]. Class II MHC is a het-
erodimer composed of non-covalently associated α and β
chains of approximately 230 amino acids, each encoded
by the HLA-DR, HLA-DP, and HLA-DQ genes’ loci [2].
Class III MHC genes encode components of the com-
plement system and will not be discussed in this review.
CD8+ cytotoxic T cells bind preferentially to class I
MHC molecules and CD4+ helper T cells bind to class II
MHC molecules.
Three unique, but not mutually exclusive, pathways of
antigen presentation, including the direct, indirect, and
semi-direct pathways, are used by MHC class I and class
II molecules for the presentation of peptide antigens to
CD8+ and CD4+ T cells, respectively, in the presence of
transplanted tissue (Figures 1-3).
Transplantation of a solid organ from a genetically
identical (syngeneic) individual does not lead to an im-
munological response against the graft. Disparities be-
tween self and non-self major histocompatibility com-
plex (MHC) molecules, as in the context of genetically
disparate (allogeneic) individuals, result in the activation
of the innate immune response, as well as the initiation
of an adaptive immune response, which subsequently
leads to graft rejection and possible patient death [6,15].
Adaptive immunity involves allorecognition of alloge-
neic or foreign MHC antigens. An alloresponse to the
graft occurs through the presentation of donor-specific
antigens to recipient T cells by engagement of a recep-
tor-ligand system between the T cell receptor (TCR) and
a foreign peptide-MHC complex. Following TCR liga-
tion, additional co-stimulatory signals lead to T cell ac-
tivation, proliferation of allospecific T cells, and re-
cruitment of effector leukocytes to the graft itself [13,16].
This specificity depends on the recognition of short pep-
tide antigens bound to polymorphic MHC-encoded gly-
coproteins on the cell surface of APCs and the antigenic
nature of the MHC molecules themselves [6,17]. Addi-
tionally, B lymphocytes participate in the humoral re-
sponse by presenting antigen to CD4+ T cells, which
subsequently help B cells differentiate into all- anti-
body-producing plasma cells.
The direct antigen-presentation pathway, or the en-
dogenous class I pathway (Figure 1(a)), is active in
nearly all cells and consists of mechanisms for display-
ing peptides produced within cells in the context of class
I MHC. This method of antigen presentation allows for
the internal proteome to be sampled and immunologi-
cally surveyed by cytolytic CD8+ T cells, which have the
ability to kill cells expressing viral proteins or tumor
antigens [15]. During class I presentation, the MHC class
I heavy chains assemble with the β2-microglobulin fol-
T. S. Skelton et al. / Open Journal of Immunology 1 (2011) 15-26
Copyright © 2011 SciRes. http://www.scirp. org/journal/OJI/
Donor derived APCs prime recipient CD4+ and CD8+ T cells by direct presen-
tation of intact donor peptide-MHC complexes (pMHC). CD4+ T cells then
provide helper signaling for cytotoxic CD8+ T cells primed by the same APC.
Recipient APCs phagocytose foreign MHC debris and present the processed
peptides on recipient MHC II to class II restricted CD4+ T cells.
(a) (b)
Intact donor class I or II MHC can be transferred to recipient APCs through cell-to-cell contact or exosomal
vesicles. These, now chimeric, APCs can directly present intact donor peptide-MHC I or II (pMHC) to recipi-
ent CD8+ and CD4+ T cells, respectively, as well as indirectly present processed donor MHC to recipient class
II restricted CD4+ T cells.
Figure 1. (a) Direct antigen presentation. (b) Indirect antigen presentation. (c) Semi-direct
antigen presentation.
lowed by assembly of the peptide loading complex within
the endoplasmic reticulum (ER) where endogenous pep-
tides generated through the action of peptidases are deliv-
ered by the heterodimeric transporter associated with anti-
gen processing (TAP). These peptide-MHC complexes are
then transported to the Golgi for final processing and fi-
nally delivered to the cell surface for presentation (Figur e
1(a); Figure 4) [18]. In the context of transplantation, di-
rect presentation relies on the allorecognition of passen-
ger leukocytes, specifically, the recognition of dendritic
cells expressing intact surface class I or class II MHC, by
recipient CD8+ and CD4+ T cells, respectively (Fi gures
1-2). This recognition primes CD8+ and CD4+ T cells.
Next, the CD4+ cells are able to provide additional stimu-
latory signals for the differentiation of allospecific effector
CD8+ cells that can wreak havoc upon the graft [16,19].
The presence of such donor APCs in the circulation fol-
lowing organ revascularization was demonstrated by
Lechler and Batchelor [20], who showed reduced failure
rates of rat renal allografts following transplantation of the
graft in an intermediately immunosuppressed recipient, thereby
depleting the graft of DCs before the final transplantation.
Openly accessible at
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Endogenous proteins are processed through proteasomal peptidases, and are transported via TAP into
the ER. MHC I heavy chains assemble with β2-microglobulin (β2 M) within the ER. Endoplasmic re-
ticulum aminopeptidase (ERAAP) mediates final peptide trimming before or after loading into the MHC
peptide-binding groove. Secretory vesicles transport peptide-MHC I complex to the Golgi apparatus for
final processing. Subsequently, the complex is transported to the cell surface via exosomes (secretory
vesicles) for presentation to CD8+ T cells. The red colored portion of the diagram represents stages in
antigen processing affected by allochimeric MHC treatment (see text).
Figure 2. Class I antigen processing.
Extracellular antigen is endocytosed and digested within endocytic vesicles and transferred to the en-
dosomal compartment where it is loaded into the protein-binding groove of class II MHC. MHC II was
previously assembled in the ER in combination with its invariant chain (II), which is partially cleaved in
the endosomal compartment leaving only the MHC class II-associated invariant-chain peptide (CLIP).
HLA-DM catalyzes the substitution of CLIP for the processed extracellular peptide antigen and the class
II pMHC complex is then transported to the Golgi and ultimately to the cell surface via exosomes (sec-
retory vesicles) for presentation to CD4+ T cells. The red colored portion of the diagram represents
stages affected by allochimeric MHC treatment (see text).
Figure 3. Class II antigen processing.
T. S. Skelton et al. / Open Journal of Immunology 1 (2011) 15-26
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(a) (b)
Figure 4. Actin cytoskeleton and formation of the immunological synapse (IS). (a) Actin cytoskeleton polymerization allows T cell
polarization and motility, bringing TCR and co-stimulatory receptor, CD28, into the proper spatial and temporal orientation to facili-
tate IS formation between TCR-pMHC and CD28-CD80/86. (b) Treatment with allochimeric MHC (see text) results in dysregulation
of actin cytoskeleton. This leads to failure in T cell polarization, IS formation, T cell motility and graft infiltration (see text).
During T cell development, thymic education ensures
the selective survival of those lymphocytes capable of
recognizing self MHCs. This process enables the mature
T cell repertoire to become restricted to the recognition
of foreign peptides bound to self MHCs, tolerant of self
peptides, and adept at mounting an immune response
toward foreign peptides through the process of positive
selection, while cells bearing self-reactive TCRs are de-
leted through the process of negative selection [21]. Ac-
cordingly, it is surprising that the T cell repertoire con-
tains such a high frequency of direct anti-donor reactive-
ity. It is unclear why, in the context of such MHC re-
striction, recipient T cells should ever recognize the al-
logeneic MHC molecules that are present in genetically
incongruous allografts or mixed lymphocyte reactions. It
has been shown that up to 10% of the T cell pool can
react with intact allogeneic class I or II MHC expressed
by passenger DCs that enter the circulation through the
transplantation of vascularized grafts [22-24]. This
paradox can only be explained by significant TCR cross-
reactivity between self-MHC and allogeneic MHC-pep-
tide complexes. Two of the models proposed to account
for this high frequency of alloreactive T cells in the di-
rect pathway are the “high determinant density” model
and the “multiple binary complex” model [25,26] in
which either alloreactive T cells directly recognize ex-
posed amino acid polymorphisms on intact allogeneic
MHC molecules or differences in the allo-MHC peptide
binding groove result in presentation of a set of peptides
different from those of the self-MHC homologue [27,28].
The direct pathway dominates the early postoperative
period and the acute rejection response but, despite the
high frequency of allo-MHC specific T cells, it gradually
declines with the depletion of donor APCs. The studies of
Pietra et al. [29] provide additional evidence for the role
of the direct pathway in allograft rejection. Both immu-
nodeficient mice with severe combined immunodefi-
ciency and recombination-activating-gene double nega-
tive (Rag-/-) mice, when reconstituted with singeneic
CD4+ T cells, were able to reject class II MHCs ex-
pressing cardiac allografts but had limited rejection of
grafts taken from MHC class II-deficient mice. Addi-
tionally, Rag-/- MHC II-/- mice receiving allogeneic
cardiac grafts were fully capable of rejecting such grafts
when reconstituted with CD4+ T cells [29]. While the
direct pathway can directly activate CD8+ T cells, the
importance of CD4+ T cells cannot be overstated, given
that these mice lacked CD8+ T cells and the capability to
generate an indirect response.
In contrast, the indirect pathway of antigen presenta-
tion, or the exosomal class II pathway, involves endocy-
tosis of extracellular antigens by recipient APCs. Akin to
conventional antigen presentation for nominal antigens,
following transplantation, host APCs migrate through
the graft, picking up soluble donor MHCs, apoptotic
cells and necrotic debris, all of which are internally
processed and presented to recipient CD4+ T cells as
peptide fragments within the peptide binding groove of
self-class II MHC molecules (Figure 1(b)) [20,24,30,31].
Class II MHC assembly occurs initially in the ER in
association with a transmembrane chaperone protein
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invariant chain. Within the endosomal compartment a
series of protease cleave the invariant chain, leaving an
MHC class II-associated invariant chain peptide (CLIP).
Subsequently, the intracellular protein HLA-DM cata-
lyzes the removal of CLIP and the loading of endocyto-
sed peptides, or the peptide exchange, before their trans-
port to the cell surface for immunologic presentation
(Figure 1(b); Figure 3) [11,17,32,33]. Studies in mice
suggested a role for the indirect antigen presentation
pathway in transplant rejection through the presentation
of allogeneic MHC by self-class II MHC H. DCs from
H-2Ab recipients, which lack the H-2E antigen, stained
positive for H-2E antigen after recipient injection with
H-2E expressing H-2k cells [34]. Furthermore, CD8+-
depleted or MHC class I-deficient mice receiving class II
MHC deficient skin grafts rejected their grafts through
the presentation of donor class I MHC on re- cipient
class II molecules [35]. The pathways for cellular pep-
tide antigen processing and their subsequent presen-
tation involve an elaborate multistep process consisting
of numerous cytosolic vesicles and organelles as well as
a number of catalytic and degradation enzymes that
function in peptide cleavage and loading into the protein
binding groove of the MHC [36]. The gradual decline in
direct alloreactivity, as donor APCs are depleted, is evi-
dent in grafts both with and without signs of chronic
rejection, indicating that direct presentation does not
play a major role in the development of chronic rejection.
In contrast, indirect antigen presentation, by virtue of
requiring the processing and presentation of endocytosed
proteins, is a slower process and persists for as long as
the graft is present, highly contributing to the develop-
ment of chronic rejection [37]. The indirect antigen
presentation pathway has been shown to be sufficient for
the development of chronic allograft vasculopathy and
arteriosclerosis in heart transplant models [38]. The re-
quirement of antigen processing and presentation by
recipient APCs in the context of self-class II MHC signi-
fies that the indirect pathway of antigen presentation is
largely dominated by CD4+ T cells [6]. However, some
overlap exists, as alloreactive CD4+ T cells can directly
respond to intact class II MHC, and alloreactive CD8+ T
cells can be indirectly activated through “cross priming”
which involves recipient APCs presenting donor anti-
gens shed by surrounding cells in the context of
self-class I MHC molecules [39]. The significance of the
role that these indirectly responding CD8+ T cells play in
facilitating chronic rejection remains unclear, but the
importance of the indirect pathway as a whole has been
well documented. While the majority of studies focus on
the interaction between APCs and responding T cells,
evidence also exist for the ability of recipient-derived
endothelial cells to utilize indirect antigen presentation
to promote further the rejection cascade. The endothe-
lium of a transplanted graft is gradually replaced with
recipient cells capable of presenting peptide in the con-
text of class II MHC [34]. Targeting of these replaced
cells by allo-reactive T cells provides further insight into
the role of the indirect pathway in the development of
the characteristic vascular lesions associated with
chronic rejection [40].
Recently, an additional mechanism of antigen presen-
tation, known as semi-direct allorecognition has been
uncovered (Figure 1(c)) [41]. The traditional model of
cross talk between CD4+ and CD8+ T cells involves a
linked “three cell” system in which the generation of
antigen-specific CD8+ effector T cells by APCs requires
additional stimulatory signaling from helper CD4+ T
cells activated by the same APC [42]. Transplantation
reveals limitations in this “three cell” model and sug-
gests the presence of an unlinked “four cell” model in
which crosstalk between directly activated CD8+ T cells
relies on amplifying signals from indirectly activated
CD4+ T cells stimulated by completely different APCs
[43,44]. Immunological cells have the capability to ex-
change surface molecules through either cell-cell con-
tact or through exosomes [45,46]. Semi-direct allorec-
ognition therefore resolves the “four cell” conundrum by
stipulating that recipient DCs can acquire and present
intact donor class I or II MHC directly to CD8+ or CD4+
T cells while maintaining the ability to internalize, proc-
ess and present donor MHC as peptides indirectly to
CD4+ T cells (Figure 3). In this manner, both direct and
indirect antigen presentation occurs in a three cell man-
ner involving the DC, CD4+ T cells, and CD8+ T cells
[6,41]. The transfer of intact donor MHC could be a
means of continual direct antigen presentation in the face
of diminishing donor APCs. Although no evidence has
been identified for the in vivo occurrence of this mode of
antigen presentation regarding allograft rejection, the
semi-direct pathway could provide an alternative expla-
nation for several scenarios that cannot be explained by
only considering direct and indirect pathways. One ex-
ample of these scenarios is the acute rejection of injected
embryonic thymic epithelium in the absence of indirect
presentation. Devoid of DCs, this response presumably
occurs through the acquisition of donor thymic antigens
presented directly to recipient CD8+ T cells [47].
Large-scale efforts in clinical research and basic sci-
ence have been devoted to unraveling the molecular ba-
sis of T-cell allorecognition, allograft rejection, and the
T. S. Skelton et al. / Open Journal of Immunology 1 (2011) 15-26
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/OJI/
development of allograft tolerance without the need for
chronic immune suppression. Allorecognition by any of
the previously mentioned pathways can ultimately lead
to the activation and recruitment of allospecific T cell
clones capable of producing aninflammatory response in
the graft. However, the outcome of allorecognition is not
as clear-cut as once thought. Allorecognition, in trans-
plantation is also capable of inducing a state of graft
acceptance, or tolerance. Allograft tolerance has been
observed in numerous allogeneic animal transplant mod-
els across class I/II MHC barriers as well as in humans,
especially following liver transplantation [48] in which
the liver has an inherent ability to resist rejection
through the expression of class I and II MHCs. This type
of protective allorecognition is also evident during
pregnancy where semi-allogeneic fetal tissue, consisting
of both paternal and maternal antigens, is present yet the
fetus is not rejected. These disparities allow a distinction
to be made between antigenicity and immunogenicity, or
the ability to recognize a foreign substrate and the ability
to produce an immune response to eliminate it.
The development of immune tolerance involves the
deletion of a large proportion of T cells with direct al-
lospecificity and the continual suppression of remaining
direct and indirect alloreactive cells that continue to be
primed for the life of the graft. Many cells with such
suppression or regulatory activity have been described in
both mice and humans. The most studied of them are the
CD4+ CD25+ T regulatory cells (Tregs), which express
high levels of the transcription factor Foxp3 [49]. The
development of Tregs occurs through the indirect path-
way after recipient dendritic cells have migrated to the
peripheral lymphoid tissue. The dependence on this
pathway has been recently shown in rats in which deple-
tion of host APCs caused the abrogation of tolerance in
Lewis liver allografts in Brown Norway recipients [50].
The role of indirect antigen presentation in antibody-
mediated rejection is also influenced by Tregs. The re-
jection of class I disparate cardiac allografts in rats has
been shown to be antibody-mediated and driven by indi-
rect T cell help, which was abrogated following toleriz-
ing protocols that induced specific CD4+ regulatory cells
Studies looking at the potential for MHC to serve as
immunomodulating agents have been underway for
years, sparked by the work of Billingham, Brent and
Medewar, who showed donor-specific tolerance to skin
grafts produced by exposing recipients to donor antigens
during fetal life [52]. Since then numerous studies inves-
tigating the therapeutic value of MHC molecules have
been performed. Immunization with allogeneic peptides,
eliciting only an indirect response is sufficient to propagate
transplant rejection. However, hyporesponsiveness of indi-
rectly allo-specific T cells can be achieved through intra-
thymic injection of MHC peptides [35,53]. Synthetic
peptides corresponding to specific HLA sequences, spe-
cifically those within the α1 helix of HLA-A have been
shown to inhibit cytotoxic T cell proliferation, produce
immunological tolerance and prolong survival in animal
transplant models. This peptide therapy was licensed
under the brand name Allotrap and showed inhibition of
cell-mediated immune responses to kidney allografts in
phase II trials [54,55]. Synthetic peptides based on the
α1 helix of HLA class II molecules similarly blocked T
cell proliferation [55]. Although all of these studies in-
dicate the potential therapeutic value of MHC molecules,
the mechanisms underlying their immunomodulatory
effects remain largely elusive.
We have previously produced donor-specific trans-
plantation tolerance in a rat heterotopic cardiac trans-
plant model through the pre- and peri-operative admini-
stration of an allochimeric class I MHC. Dominant
amino-acid epitopes identified on the α1 helix of the do-
nor MHC were inserted into the α1 helical region of the
recipient class I MHC, creating the allochimeric con-
struct [α1 l/u]-RT1.Aa [56]. By delivering this protein, in
combination with sub therapeutic doses of cyclosporine
(CsA) through the portal vein at the time of transplanta-
tion, we achieved donor specific tolerance, the attenua-
tion of acute and chronic rejection, and prolongation of
graft survival [56]. These observations have led our
laboratory to investigate the molecular and cellular
mechanism underlying the immunosuppressive effects of
soluble allochimeric class I MHC.
Heterotopic cardiac transplantation was performed
between donor Wistar Furth (WF) rats and ACI recipe-
ents. Recipient animals were either untreated, treated for
7 days with therapeutic CsA, or treated with 3 days of
sub-therapeutic CsA plus a single intraoperative dose of
allochimeric class I MHC molecule delivered through
the portal vein. Animals that received no immunosup-
pressant or received only sub-th e rap euti c CsA rejected
their grafts in 5.4 or 16.2 days, respectively, whereas
grafts from animals treated with combined low dose CsA
and allochimeric peptide survived indefinitely [56]. Mi-
croarray analysis of the gene expression profile of heart
allograft tissue and splenic T cells, demonstrated that
allochimeric class I MHC therapy caused increase in
expression of genes involved in the structural integrity of
the heart muscle, Nexilin and Myocardin, at day 1 and 3
T. S. Skelton et al. / Open Journal of Immunology 1 (2011) 15-26
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post-transplantation compared with acutely rejecting
controls, as well as downregulation in the
pro-inflammatory cytokine IL-1β within the cardiac al-
lograft [57]. There was also decreased expression of the
partitioning defective 6 homologue beta (PAR6) gene,
which functions in cell polarity and motility [57]. In
addition, genes that play a role in actin filament polym-
erization, including RhoA and Rac1, cell adhesion, in-
cluding Vcam, vacuolar transport, including RhoB and
the MAPK pathway, including Spred1, were shown to be
downregulated in splenic T cells isolated from animals
treated with allochimeric MHC compared with rejecting
controls [58]. Since all these genes play a role in cell
polarity and motility we proposed that the early immu-
nosuppressive effects attributed to allochimeric MHC
treatment was related to the impairment of T cell motility,
immunological synapse formation, and ability of T cells
to infiltrate the allograft (Figures 4-5) [57,58].
We further investigated the role of RhoA, a member
of GTPase protein family, in splenic T cells and its con-
tribution to the attenuation of chronic rejection. Using a
combination of RT-PCR and Western blot analysis, we
found that T cell expression of RhoA was significantly
reduced following allochimeric MHC treatment com-
pared with untreated and CsA-treated controls [59]. This
finding suggested that the early post-transplantation in-
hibition of RhoA played a role in diminishing the early
immune response against a foreign graft. In fact, immu-
nostaining for RhoA showed aberrant RhoA distribution
within splenic T cells in the allochimeric MHC-treated
animals isolated at day 1 and 3 post-transplantation
compared with CsA-treated controls [59]. Given the role
dysregulation.of RhoA in cytoskeleton organization and
actin filament polymerization, we further showed that
distribution of actin was highly disorganized in T cells
from the allochimeric MHC-treated cohort (Figure 6)
[59]. In addition, actin binding partner, protein Hip55
was partially dislocated from the actin in allochimeric
treated T cells (Figure 7) [59]. We suggested that dis-
ruption of the T cell cytoskeleton led to the impairment
of T cell migration toward its target, whether it is an
APC or foreign protein antigen (Figures 4-5) [59]. In
fact, histology of allografts from animals treated with
allochimeric MHC showed significantly reduced T cell
infiltration into allografts compared to rejecting controls
Another possible mechanism underlying the immu-
nosuppressive properties of soluble MHC is the way that
this protein molecule is processed by antigen-presenting
cells, such as the DCs. Through an elaborate multistep
process involving vesicular trafficking between the en-
doplasmic reticulum (ER) and the Golgi apparatus, pro-
tein antigens are displayed to peripheral T cells, and an
immune response is executed. However, nothing is
known about how allochimeric class I MHC is processed
Rho GTPase pathway related cell functions and events (marked in red)
potentially inhibited by allochimeric MHC I treatment in T cells and den-
dritic cells in rat cardiac allograft model system (see text).
Figur e 5 . Molecular and cellular targets of Rho pathway.
within the DC and presented to T cells. Looking at the
vesicular trafficking pathway and the morphological
appearance of the ER and Golgi apparatus, we discov-
ered stark differences in DCs isolated from allochimeric
MHC treated recipients compared with untreated and
CsA-treated controls [36]. We also found that there was
decreased expression and abnormal distribution of RhoB
in DCs from animals treated with allochimeric MHC [36].
Endocytic/intracellular membrane trafficking relies on
RhoB [60] and its decreased expression compared with
controls would indicate that the earliest step in antigen
processing has been dysregulated [36]. Furthermore,
looking at the ER and Golgi resident proteins KDEL and
GM130, we found distinctly abnormal morphology of
these two organelles in DCs from allochimeric MHC
treated animals. We found that the ER lost its vesicular
appearance and dissociated from its normal paranuclear
localization. The Golgi apparatus also lost much of its ve-
sicular appearance and became markedly reduced in size
compared to controls [36]. These changes suggest that in
our model system, the intracellular processing of soluble
antigen, such as allochimeric MHC, has been highly dis-
regulated (Figur es 2-3).
Our previous and recent studies indicate that in-
tra-operative treatment with allochimeric class I MHC
attenuates chronic rejection. Chronic rejection is charac-
terized by perivascular graft inflammation, graft vascular
intimal hyperplasia and progressive luminal narrowing,
as well as necrotizing arteritis. These lesions were shown
by Singer et al. [38] to be diminished in long-term
graft-surviving ACI recipients of WF grafts compared with
T. S. Skelton et al. / Open Journal of Immunology 1 (2011) 15-26
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/OJI/
(A-C, D1, D3, E-K) Rhodamine-phalloidin staining (red) of actin in T cells
isolated from spleen. (D2, D3, I-K) T cell nuclei are counterstained with
Hoechst (blue). (D3, I-K) Merged images of actin (red) and nuclear (blue)
Hoechst staining. (A) T cells from control,untreated animals at day 1 and (I)
at day 3 post-transplantation. (A, I) Uniform layer of cortical actin is visible
underneath the cellular membrane. (B) T cells from animals treatedwith a
sub-therapeutic dose of CsA at day 1 and (J) at day 3 post-transplantation.
(B, J) Actin distribution becomes patchy; small aggregates of actin underlie
the cellular membrane. (D1-H) T cells from animals treated with a
sub-therapeutic dose of CsA in conjunction with allochimeric molecule at
day 1 and (J) at day 3 post-transplantation. Actin forms large aggregates
unevenly distributed below the cellular membrane. Bar is equal to 10 μm.
(Figure originally published in Skelton et al. Transplant Immunology 23
(2010) 185-193; with the permission from Elsevier).
Figure 6. Allochimeric molecule treatment induces changes in
cortical actin distribution in T cells.
untreated and CsA-treated controls. In addition, the
adoptive transfer of T cells conditioned with allochi-
meric class I MHC into secondary recipients also re-
sulted in decreased evidence of chronic rejection, which
suggests the role of antigen-specific T regulatory cells in
attenuating detrimental graft injury [61].
Although further studies are necessary to fully under-
stand the role of MHCs in the attenuation of chronic
rejection, our studies indicate that the development of
tolerance is multifactorial. We believe that periopera-
tive exposure to donor-specific epitopes of class I MHC
alters the recipient T cell and DC subcellular and Rho
pathway related events (Figure 5). Thus, the effector
function of recipient T cells and DCs is reduced, leading
(A1, B1, C1 and D1) Immunostaining of T cells with anti-Hip55 antibody
and FITC-conjugated secondary antibody (green) at day 1 post-transplanta-
tion. (A2, B2, C2, D2) Rhodamine-phalloidin staining (red) of actin in T
cells. (A3, B3, C3, D3) Merged images of actin (red) and Hip55 (green)
staining. (A1-A3) T cells from control, untreated animals. A uniform layer
of cortical actin is visible underneath the cellular membrane and colocalizes
with the actin-binding protein, Hip55. (B1-B3) T cells from animals treated
with a sub-therapeutic dose of CsA. Actin and Hip55 distribution becomes
patchy; small aggregates (on average 21 aggregates per cell), containing
colocalized actin and Hip55, underlie the cellular membrane. (C1-D3) T
cells from animals treated with a sub-therapeutic dose of CsA in conjunc-
tion with allochimeric molecule. Actin and Hip55 form large aggregates
unevenly distributed below the cellular membrane. Some of these aggre-
gates show colocalization of actin and Hip55 (thick arrows), and some
Hip55-positive aggregates (on average 2.3 aggregates per cell) do not con-
tain actin (thin arrows), which indicates that actin partially dissociates from
its binding partner, Hip55. Bar is equal to 10 μm. (Figure originally pub-
lished in Skelton et al.. Transplant Immunology 23 (2010) 185-193; with
the permission from Elsevier).
Figure 7. Dissociation of actin-binding adaptor protein Hip55
from actin in T cells from allochimeric molecule-treated rats.
to the suppression of the recipient’s immune response
and the maintenance of immunological tolerance.
We acknowledge NIH Grant RO1 AI49945 support to R. M. Gho-
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