Molecular and cellular pathways involved in the therapeutic functions of MHC molecules; a novel approach for mitigation of chronic rejection

doi:10.4236/oji.2011.12003

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Vol.1, No.2, 15-26 (2011)

doi:10.4236/oji.2011.12003

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.

ABSTRACT

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

1. INTRODUCTION

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].

2. MHC AND THE IMMUNE RESPONSE

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

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.

3. MHC AND ANTIGEN PRESENTATION

PATHWAYS

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.

4. DIRECT PATHWAY

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

1717

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.

(c)

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

T. S. Skelton et al. / Open Journal of Immunology 1 (2011) 15-26

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

1919

(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.

5. INDIRECT PATHWAY

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

T. S. Skelton et al. / Open Journal of Immunology 1 (2011) 15-26

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].

6. SEMI-DIRECT PATHWAY

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].

7. ALLOGRAFT TOLERANCE

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

2121

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

[51].

8. MHC AS THERAPEUTIC AGENTS

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.

9. MOLECULAR AND CELLULAR

PATHWAYS INVOLVED IN INHIBITION

OF CHRONIC REJECTION BY MHC

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

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

[59].

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

2323

(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.

10. ACKNOWLEDGMENTS

We acknowledge NIH Grant RO1 AI49945 support to R. M. Gho-

brial.

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