Do heat shock proteins play a role in transplant immunity?

Do Heat Shock Proteins Transplant Immunity?

Play

a Role in

Rem!J. Duquesnoy, Kaihong Liu, Ricardo A. Moliterno, and Derrick At#eld

c

ellular rejection of allografts is mediated by donor-specific T lymphocytes that, via direct allorecognition, exert cytotoxic effects on donor tissues and through lymphokine release and recruit and activate other inflammatory cells that cause graft injury. Limiting dilution analysis has shown rather low frequencies of donor-specific T cells in rejecting allografts. In other words, the majority of graftinfiltrating lymphocytes do not function through direct allorecognition and must react through other specificity mechanisms. Recent studies have shown that the intragraft immune responses include indirect allorecognition whereby donor tissue-derived alloantigens are processed and presented by recipient antigen-presenting cells (ARC) to alloresponsive self-restricted T cells.‘-7 Donor-specific, major histocompatibility complex (MHC) allopeptides have been identified and this indirect allorecognition affects transplant outcome. Another specificity mechanism deals with the T-cell reactivity to minor histocompatibility antigens and the so-called tissue-specific antigens. We have forwarded the concept that, during rejection, the callograft undergoes a stress response that leads to increased expression of heat shock proteins (hsp) and triggers the infiltration and activation of hsp-responsive lymphocytes.s*g During recent years, an increasing number of investigators have begun to study the role of hsp in transplantation, including ischemia/reperfusion injury. At the last Basic Sciences Symposium of the Transplantation Society, a plenary session dealt with this topic for the first time (for progress report see DuquesnoytO). In this review, we discuss our views on the role of

From Ihe Dioirion of Tranrplanlakw~ burgh Medkal Center, Pi&burgh, PA.

Palhology,

Utkrri~

of P&s-

Suppor!ed ly NWD Granl No. AI-23567 and 4 the Palhology Educalion and Rereanh Foundation. DA. ir alro supp&ed by a GLAXO ruearch fellowship awardjmn rhe South AJiican Ga&wn!erology So&y and by the Soulh A&con Surgical Ruearch So&y. Addnw reptin reque.& IO Rena J. Duquemoy, PhD, Diction of Tranrplonlalion Palho&, Uniumily oJA’l/.sbutgh Medical Cenler, Biomedical Science Tower, Room Wl.552, Pillsburgh, PA 15261. ComkhlO I996by W.B. Saunders Company 0955470X/96/ 1003~0005%5.OOlO

Transplantation

Reviews,

hsp in transplant immunity and what data have been obtained so far. Topics include the expression of hsp in transplanted tissues and the role of hsp in humoral and cellular aspects of transplant immunity. We have also included some background information about hsp and their relevance in the immunology ofcertain disease conditions.

Hsp in Autoimmune Tumor Immunity

Disease and in

Also called stress proteins, hsp are common constituents of all types of prokaryotic and eukaryotic cells and they exhibit a high level ofevolutionaryconservation. Originally identified in cells exposed to sudden elevations in temperature,“J* hsp mediate responses to a variety of stressful stimuli, including ischemia and infIammation.i3J4 They play a major role in various cellular compartments by functioning as molecular chaperones involved in the assembly, folding, and translocation of intracellular polypeptides to interact with various receptors and, in stressed cells, to restore the functional activity of denatured proteins.‘“‘” Increased intracellular hsp levels increase the cellular resistance to injury. Many stress proteins have been classified as hsp according to molecular weight in kilodaltons. For instance, hsp60 operates primarily in mitochondria, whereas the cytosolic compartment contains the stress-inducible hsp72 and the constitutively expressed hsp73 (also called hsc73). Other stress proteins are referred to as glucose-regulated proteins (grp) because they were originally discovered in culture systems deprived of glucose.rg Grp78 (also called BiP*O) and grp94 are examples that operate primarily in the endoplasmic reticulum,*’ whereas grp75 is a mitochondrial chaperone. Many stress proteins, especially the members of the hsp70 family, have a common structure consisting of an amino-terminal domain with ATPase activity and an carboxy-terminal domain with structural similarities to the peptide-binding region of MHC molecules.22~23Stress proteins participate in the biogenesis of class I and class II molecules and in antigen processing and presentation.*“*’ The medical importance of stress proteins is

Vol IO, No3

(Jub),

199Gpp 175186

175

176

Duquesng cl al

apparentfrom numerousstudieson infection, inflammation, autoimmune disease,and tumor immunity.2s During infection, humoral and cellular immune responses are often directed to microbial hsp.2g Injurious stimuli to tissuesinduce an increasedproduction of hspthat may activate the immune system. In many experimental modelsand clinical situations, hspresponsivelymphocyteshave beenshownto participate in the pathogenesisof several autoimmune diseases.**HspGO-reactiveT cells are present in synovial fluids from arthritis patientssoand hsp70reactive T cellsappear in spinalfluids and demyelinated lesionsof multiple sclerosispatients.3’*32 Peripheral T-cell reactivity against hsp60 and increased serum levels of hsp60-specificantibodies have been reported for diabetes, atherosclerosis,and many other diseases.2s Hsp have alsobeenimplicated in the pathogenesis of experimentally induced autoimmune disease.For example, Van Eden’s group has shown that T cells specific for the 180-188sequenceof mycobacterial hsp65 confer adjuvant arthritis in rats33and that vaccination with the 256270 peptide providesprotections4In a murine model of insulin-dependentdiabetes, Cohen’s groups5has identified T cells that are specific for an epitope corresponding to an amino acid sequence shared between mycobacterial and mammalian hsp60. Administration of this peptide prevented diabetesin this model. Stressproteins are alsoinvolved in tumor immunity. Srivastava has shown that grp94 (or gp96), hsp70,and hsp90isolatedfrom methylcholanthreneinduced murine sarcomaselicit immune responses causing a specific regression of transplanted tumors.3637These hsp are associatedwith antigenic peptidesthat are chaperonedwithin and outside the endoplasmic reticulum during antigen processing and presentation by MHC molecules.3s*3g Other groupshave alsoshowna role for hsp70in antitumor resistance.40$41 Eberlein’s group postulated that tumor tissuemay undergo a stressresponseand that a portion of tumor-infiltrating lymphocytesmay recognize stress proteins.42Tumor-derived CD4 T-cell linescan react with heat-stressedB-cell linesthrough hsp70recognition. Others have shownhsp60-specific T cells with antitumor activity against a murine sarcoma43 and that injection of mice with tumor cells transfected with mycobacterial hsp65 induces immune protection against the original tumor.& Srivastava45hasproposedthat the involvement of hsp in the immune responsecan be categorized into four paradigms. First, hsp from foreign organisms

may act as classicalforeign antigens and they elicit immune responsesto nonconseIved hsp epitopes. Second,the host respondsto self-hsp,to which there is no central or peripheral tolerance. Becausehspare highly conserved self-molecules,one would expect that hspreactive T cellswould be negatively selected during thymus development. However, thymic tolerance might be prevented under certain situations such a tissue-restricted hsp expression, including conditions of stress and the specific intracellular localization of certain hspsuchashsp60in mitochondria. Third, cross-reactivity between hsp and other proteins, including bacterial hsp, may elicit immune responses.This would be related to the molecular mimicry concept of pathogenesisof autoimmune disease.Insufficient evidence is available for the concept that hsp, as antigens, provide a direct link between infection and autoimmunity.* The fourth paradigm dealswith the concept that hsp function as carrier moleculesof antigenic peptides that are then presented by APC to responding T cells. In other words, the hsp-antigencomplex elicits the immune responseto the antigen but not to the hsp. This concept wasprompted by observationsthat immunization with complexes of hsp with tumor antigen generates protective immunity to a transplanted specific sarcoma but not to other antigenically distinct sarcomas.36,47dg Moreover, in vivo cross-priming with gp96 complexed with antigenic peptides can elicit antigen-specificcytotoxic T-cell responses.3gp50 Another mechanismof immune lysis of tumor cells seemsrelated to the surface expressionof hsp70.4’a1 In conclusion,several concepts have emerged to explain the mechanism(s)behind how stressproteins play a role in tumor immunity and the pathogenesis of autoimmune disease.It seemslikely that similar concepts may evolve regarding the participation of stressproteins in transplant immunity.

Expression Transplanted

of Stress Proteins Tissues

in

The first report on stressproteins in transplantation waspublishedin 1987by Currie et al,52who reported an increased expression of hsp72 in rejecting rat cardiac allografts. Perdrizet et als3*#observed that the increased hsp expression associatedwith mild ischemia/reperfusioninjury may mediate a cytoprotective effect that promotes graft survival. In a rat kidney syngraft model, he hasshownthat heat shock of donor animals(whole body ischemia,at 42.5% for 15 minutes), followed by recovery before kidney

StressProleins in Transplant Immuni&

177

removal and cold storage (at 4°C for 48 hours), will ing the inflammatory stage of rejection, there are preserve renal function.55Whereas cold-stored kidseveral lower molecular weight bands that might ney grafts never functioned, the prior treatment of represent degradation products of hsp60,hsp72,and donor rats with heat shock and a 6- to 8-hour grp78. Other investigators have also reported the recovery period lead to a successfulengraftment of increased allograft expressionof hsp72 in this rat such kidneys. Thus, induction of a heat shock recardiac transplant mode1.5g Cyclosporine treatment sponsemay protect rat renal grafts from cold storage of allograft recipients inhibits this heat shock reinjury. This protection is associatedwith an ensponseto acute allograft rejection.60 hancedexpressionof hsp72. Other stressproteins alsoshowincreasedexpresWe have used a rat model of heterotopic heart sionduring allograft rejection. Mohanakumar’sgroup transplants (ACERTl” into LEW-RTl’) to study the has reported higher levels of hsp40 and hsp60 in kinetics of hsp expression during allograft rejecrejected human kidney transplants.61With the rat tion.j6 This wasperformed by immunoblot analysisof heart allograft model, we have studied the heme graft stromal tissueswith monoclonalantibodies to oxygenase isoenzymesHO-l and HO-2, which devarioushsp (from StressGen,Victoria, British Columgrade the hememoiety of senescenthemoglobinand bia, Canada). Three types of stressful stimuli apreact with a variety of intracellular proteins, such as peared to increase the hsp expression in the allomyoglobin, tissuehemoproteins,nitric oxide synthegraft, namely ischemia/reperfusion injury, the tase, and guanylate cyclase.6265 The expression of appearanceof graft-infiltrating lymphoid cells, and, HO-l, also known as hsp32,is inducible and reprefinally, the inflammatory stage of the rejection prosentsan indicator of oxidative stress.HO-2 is consticess.56During the first few days after transplantatutively expressedin most tissues.Immunoblotting tion, both allografts and syngrafts exhibit increased studies have shown a transient increase in HO-l expressionof hsp60, hsp72,grp75, and grp78. Alloexpression in syngrafts, but a progressiveincreasein grafts showedfurther increasesin grp78 and grp94 allografts. 66 These results suggestthat hsp32is inexpression on day 3 after transplantation, when duced after ischemia/reperfusion injury and during lymphoid infiltrates appear (Fig 1).As chaperonesin rejection. On the other hand, the expressionofHOthe endoplasmic reticulum, grp78 and grp94 are is similar in grafted and nongrafted hearts. involved in the biosynthesisof immunoglobulinchains, Rat heart allograft rejection, especiallyduring the MI-K molecules, and T-cell receptor chains and later stages,is associatedwith a higher expressionof participate in antigen presentation.‘7*s0~27~3s57~58 Durthe KDEL sequence-containing protein disulfide isomerase(PDI) and calreticulin.@jBoth function as molecular chaperonesin the endoplasmicreticulum ( _.... during the biosynthesisand transport of various -SW secretory proteins. Several reports have described - glp78 the presenceof autoantibodiesto PDI and calreticulin in sera from patients with autoimmune dis-s&Da ease.67-70 Our immunoblot data with the rat heart transplant model are summarized in Table 1. These results and the findings by other investigators provide further documentation that the stressresponse of an allograft during the various stagesof rejection Figure 1. Immunoblotsof cardiacstromal tissueextracts testedwith monoclonalantibodySPA-827(Stress- involvesa wide spectrum of hsp.An important aspect Gen) against the KSEKDEL sequenceof grp78. This is that increasedhspexpressionconveysa cytoprotecmonoclonalantibodycrossreacts with grp94and an undetive effect from inflammatory injury. For instance, fined50-kDbandthat may representa degradationprodhsp70 protects cells from injury causedby reactive uct of grp78or grp94.Syngrafts(lanesSl throughS5)and allografts(lanesAl through A5) were testedon days I oxygen species,including nitric oxide, and cytokines through5 after transplantation.LanesLEW andAC1are such as tumor necrosis factor-o (TNF-u).~’ The proteinsamples from normalrecipientand normaldonor immunological consequencesof the increased hsp hearts.LanesSRHandARH representheartsfrom 5-day expressionin the allograft might occur at humoral LEW recipientsof syngeneicor allogeneiccardiactransand cellular levels.Most information generatedsofar plants.(Reprintedwith permission from Qian et al.56)

178

Table

Duquem y el al

1. Stress Protein Expression in Stromal Tissues OfTransplanted

Rat Hearts Allo,Fraf

syngrafl (d l-5)

hsp60 hsp72 hsc73 w75 e-p78 grp% Hemoxygenase-I (Hsp32) Hemoxygenase-2 Protein disulfide isomerase Caheticulin Note: llwse immunoblot studies Syngeneic Lew into Lew transplants with normal AC1 and Lcw hearts increase; ++, marked increase. degraded forms orhsp molecules.

Ischemial Remion InjuIy (d 1-2)

+/+ 0 + + -I-/+ 0 + +

Immunity

Rejeclion-AmSated InJammation (d 4-5)

+ + +/+ ++ + + 0 + +

+D +/- D -k/-D +/+D 0 ++ 0 ++ ++

were performed on AC1 into Lcw hctcrotopic heart kwq~lants; their graft survival is 6 to 7 days. were used as controls. hsp-specific antibodies were obtained rrom StrcssGen. Comparisons wcrc made and the lcvcls were expressed as hollows: 0, no increase; +/-, modest or variable increase; +, definite D indicates kc presence or crossreacting lower molecular weight bands presumably representing

with various transplant models deaf with immune responses involving the hsp60 and hsp70 families.

Humoral Proteins

+ + 0 + + + + 0 + +

Appearance of Cellular Infiltrate (d 2-3)

to Stress

The literature contains many reports describing the appearance of hsp-specific antibodies associated with various infections and autoimmune diseases.2s Most commonly, these antibodies react with hsp60, but there are also many examples of humoral immunity to hsp70 and other hsp. Two groups of investigators have published transplant-related data on hspspecific antibodies. Rose et ali have found an association of humoral immune responses to stress proteins with transplant outcome. Western blot studies on cardiac tissue extracts and pretransplant sera have shown that the presence of high-titer antibodies to hsp60 and hsp70 correlates with a higher rejection rate in heart transplant patients. Goral et a173 have observed elevated serum levels of antibodies reactive with hsp70 during the development of acute graftversus-host disease (GVHD) in an Fl hybrid rat model. These antibodies reacted with mammalian hsp72/hsc73 preparation and a 70-kD protein in an autologous lymphoid cell lysate. Because the antibody levels correlated with the disease process, the investigators suggested that anti-hsp70 antibodies may contribute to the pathology of acute GVHJI.

Hsp-Induced Propagation of Lymphocytes From Human Transplant Tissues Our initial studies on stress proteins and cellular rejection were performed with endomyocardial biopsies from heart transplant patients.” These studies were based on the experience that the in vitro culturing of biopsies transplant tissues with interleukin-2 (L2) permits the outgrowth of graft-infiltrating lymphocytes and that the frequency of biopsy growth correlates with the degree of cellular rejection.74 We have used similar culture conditions whereby heart transplant biopsies were incubated with soluble Mycobachium tubercuhwis extract (MIX), a source of hsp.s In assays on 299 biopsies from 89 heart transplant patients, we found that h4TE can induce lymphocyte outgrowth and this correlated with the histological rejection grade. Similar observations were made with biopsy cultures incubated with recombinant mycobacterial hsp65.* These data provided first evidence that hsp-reactive T lymphocytes may infiltrate transplanted hearts during rejection. Similar tindings have recently been obtained by Trieb et al,75 who studied human T-cell lines propagated from rejected kidney allografts. These cells exhibited proliferative responses to recombinant human hsp72 in combination with autologous peripheral blood mononuclear cells as AF’C, but not with allogeneic APC. Our studies on the flow cytometric analysis of

Sh~s Pm!ein~ in Tran.@anl

MTE- and hsp65-propagated lymphocyte cultures showed higher frequencies of CD8+ cells for early posttransplantation biopsies, whereas later on, most cultures were predominantly CD4+ cells.* We noted also increasing proportions of TCR yS cells. During the first year after transplantation, about one-fourth of biopsy-derived lymphocyte cultures contained significant numbers of TCR yS T cells (> 10%) regardless of propagation stimulus. On the other hand, more than one-half of long-term transplant biopsies yielded yS T cells, especially when cultured with MTE and hsp65. The TCR -yZicell frequencies did not correlate with the histological rejection grade. MTE appears to preferentially stimulate the proliferation of the y9-positive, 62-positive subset of circulating TCR y6 T lymphocytes.‘” However, the MTE and hsp-propagated graft-infiltrating TCR ~8 cell population consisted almost exclusively of y9negative, 61-positive cells. Others have also reported that heart transplant biopsy-derived TCR -y6 cells are primarily of the 6 I subset.” TCR ~8 cells may react with hsp65 from mycobacteria and stressed mammalian tissue, and such cells may participate in autoimmune reactions.3”~7s*7g Relevant to our data are the findings by Wick et al”” on the upregulated hsp65 expression and the presence of hsp65-reactive TCR yS cells in arteriosclerotic lesions in an experimentally induced rabbit model. Several years ago, we reported the propagation of CD4-CD8TCR y6 cells from coronary arteries from long-term heart transplant patientswith chronic rejection.“’ Would such TCR ~8 cells react with hsp generated during the stress response associated with chronic rejection?

Hsp Reactivity of Lymphocytes Isolated From Heterotopic Rat Cardiac Allografts During Acute Rejection The rat heart transplant model has permitted more detailed studies of the effect of hsp on graftinfiltrating lymphocytes.” Incubation with mycobacterial hsp65 and especially hsp70 causes a marked increase of the proliferative response of allograftderived cells to irradiated donor spleen cells as allogeneic APC. Very little proliferation takes place with hsp70 and syngeneic APC unless a small amount of IL2 has been added to the culture. These findings suggest that IL-2 must be important for the hsp reactivity of intragraft lymphocytes. During cellular

179

Immuni~

rejection, donor-specific alloreactive lymphocytes would be a source of IL2. In this rat heart model, hsp reactivity of allograftinfiltrating cells is first seen about 3 days after transplantation and this is accompanied by the appearance of alloreactive T cells (Fig 2). In contrast, lymphocytes isolated from syngrafts or allografts from tacrolimus-treated recipients exhibit no or very little proliferation in the presence of hsp and selfAK.9

Hsp71 Dependency of Autoreactive T-Cell Clones Cultured From Allograft-Infiltrating Cells Allograft-derived lymphocyte clones are readily estab lished by culturing with self-APC and recombinant Mycobacterium tuberculosis hsp71 (Mtub71) in the presence of IL-2. Two groups of self-APC reactive T-cell clones have been identified; both of them are CD3+, CD4+, CD8-.s2 One group requires Mtub71 for self-APC-induced proliferation and E-2 release and they are referred to as hsp71-dependent autoreactive T cells. These clones proliferate in the presence of self-APC, whereas allogeneic or third-party APC are ineffective. Mtub71 does not appear to function as a conventional antigen because exposure to trypsin, ATP, or polymyxin B abrogates its stimulatory activity. s2 Rather, the hsp71 effect appears mediated by structurally intact hsp70 molecules that will induce self-APC to become stimulatory to this group of autoreactive T cells.

-l.EwAPc-

Figure 2. Proliferative responses of graft-infiltrating lymphocytes isolated from cardiac allografts during the first 5 days after transplantation. The assays were performed with irradiated donor spleen cells (ACI-APC) or with irradiated syngeneic spleen cells (IXW-APC) and recombinant Mycobacterum tuberculosis hsp71 (Mtub71) with a small quantity of IL2 (0.4 U/mL). (Adapted and reprinted with permission from Moliterno et al.‘)

180

Duqumqv et al

The second group of autoreactive T-cell clones respondreadily to self-ARCin the absenceofMtub71. These clones are referred to as hsp714ndependent autoreactive lymphocytes, although their proliferative responsesto self-ARCare frequently augmented by Mtub7 1.Becausethis augmentingeffect ofMtub7 1 is largely unaffected by trypsin, ATP or polymyxin B, it seemslikely that its mechanismis different from the mechanism of hsp7I-dependent autoreactive T-cell stimulation by self-ARC.This difference might reflect the stimulation of hsp71-independentautoreactive clones by self-ARC through a self-restricted presentation of antigens in the Mtub71 preparation. At present,we have not seena significant Mtub71 effect on alloreactive T-cell cloneseven after culturing with donor ARC plusMtub7 1. Therefore, hsp7Idependent and hsp714ndependent,autoreactive T cells may represent distinct componentsof cellular rejection mediated by alloreactive T cells. Table 2 summarizesthe proliferative responses of the autoreactive and alloreactive T-cell clones to self-ARC or auo-m. All theseclonesareCD4+ and monoclonalantibodies to MI-K classII, but not classI, inhibit their ARC-induced proliferation. Incubation with an antirat TCR-c@ monoclonal antibody (R73) has produced contrasting resultss2R73 inhibited the proliferative responsesof the alloreactive clonesand the hsp71-independentautoreactive clones, but an increasedproliferation was seenfor the hsp7ldependent autoreactive clones. This antibody has been reported to induce proliferation of normal spleno cytesand its stimulator-yeffect isdirected at T cellsa In severaltransplantation models,R73 can induce a considerableprolongation of graft survival.84~ffi An immunosuppressiveeffect seemsalsoapparent from our in vitro findings that R73 inhibits the proliferative responsesof graft-derived alloreactive hsp71independent autoreactive T-cell clones.

On the other hand, this antibody augments the proliferation of hps7ldependent T cells induced by Mtub7 1 treated self-ARC.Moreover, incubation with R73 enablestheseclonesto respondto self-ARC (and even allo-ARC) in the absenceof Mtub7 1.Studiesare currently in progress to determine how hsp71dependent and hsp71-independentSelf-APC-reactive T cells can be further distinguished by their responsepatterns to anti-TCRcl8 antibody R73. Although their functional significance in transplant immunity remains to be established,we propose that hsp7ldependent, autoreactive T cells reflect cellular immune mechanismassociatedwith a stressresponseto the rejection processand tissue inflammation.

Effect of Murine Hsp71-Dependent

grp78 on T Cells

The autoreactive T-cell dependencyis not limited to mycobacterial hsp71,becauseincubation with mammalian (ie, murine) grp78, a stressprotein generally found in the endoplasmicreticulum, will alsolead to proliferation of hsp7l-dependent T-cell clones (Fig 3):” This T-cell reactivity with grp78 becomeseven more interesting becauserejecting allografts have higher grp78 levels (Fig 1). Grp78 is constitutively expressedin mammalian cells and can be upregulated under various stress conditions.86Hightower and Guidona have reported the rapid release of grp78 and other hsp by cultured fetal rat cells respondingto stressfulstimuli. Mammalian grp78 and mycobacterial hsp71 are molecular chaperoneswith a considerable amino acid sequencehomology; also, their binding of pep tides is very similar.** The structure of hsp70 indicates a highly conserved N-terminal ATPase domains9and a lessconservedC-terminal domain with a peptide-binding site with some similarities to the

Table 2. Proliferation Characteristics of Autoreactive and Alloreactive T-Cell Clones Cultured From Cellular Infiltrates ofHeterotopic Rat Heart Allografts Undergoing Rejection Hs~7LDe~t, Autoreactive Ch Self-APC Self-APC Allo-APC Allo-APC Self-APC Self-APC Allo-APC

+ Mtub7 1 + Mtub7 I + anti-TCRcx@ + Mtub71 + anti-TCRcxp + anti-TCRc$

Adapted and reprinted with permission from Liu et aI.=

0 + 0 0 + ++ +

Hs~71-Indepndent, Autoreactive Clam + +or++ 0 0 0

Donor-S@$c, Alloreactive Clones 0 0 + + 0 0

Strss Pmlknr in Tram-piano Immunip

ACI-APC AC,-APC AWAPC

Figure 3. Stimulatory effectof grp78on the self-AKinducedproliferativeresponses of an allograftderiveclhsp7ldependent, autoreactive Tcell clone3I .5. The proliferation of an alloreactiveclone 5.5.isnot influencedbygrp78 nor Mtub7I. (Adaotedandreprintedwith permissionfrom Qian et als6)

ACI-APC

alone + Mlep66

+Mtub,l

+Mudna

Grp78

Allo,asc”ve

Clone

LEW-APC

LW-APC

LEW-APC

+Mlap66 +Mt”b7l

+Mudno

ACI-APC ACI-APC +Mudne

6

akIna

LW-APC

0~78

ACI-APC

AC&APC

181

aIma +Mlep66

+Mtub71 0~78

peptide-binding cleft in MHC molecules.22~23~90 Pep tide binding by hsp70 stimulates ATPase activity, and, in the presenceof ATP, the energy releasedby ATP hydrolysiscauses the dissociationof the hsp70peptide complex.glBecausethe data with trypsin- or polymyxin-treated Mtub71 suggestedthat the hsp71 dependencyof autoreactive T cellsrequires structurally intact hsp70,z* it is possiblethat this effect is mediated by an ATPdependent process.Indeed, we have found that Mtub71 passagedthrough an ATP column looses its stimulatory effect on allograftderived T cells.R*

Recipient sera have antibody reactivity to bacterial hsp and to heart extract protein bandscorresponding to hsp.Graft-infiltrating cellsreact with mycobacterial hsp65and lymphocyte lines have been generated with hsp71- and murine grp78-dependent reactivity to self APC, but not donor or third-party APC. This T-cell reactivity seemsto require intact hsp70 molecules, because treatment with trypsin, polymyxin, or ATP column chromatography abrogates the hsp71effect. These findings suggestthat hspdependent immune mechanismsmay play a role in the pathogenesisof chronic rejection.

Hsp Immunity in a Rat Allograft Model of Chronic Rejection

A Mechanistic Model for the Role of Hsp in Transplant Immunity

Murase et alg2has shown that heterotopic heart transplants from Lewis (RTl’) rats into Brown Norway (RTI”) recipients pretreated 100 days before with Lewis bone marrow cells and a brief course of tacrolimus exhibit long-term graft survival without immunosuppression.However, all these grafts develop chronic rejection as evidenced by a chronic obliterative arteriopathy and the presenceof endocardial infiltrates resembling Quilty lesionsa In contrast, pretreatment with a liver allograft prevents chronic rejection of the transplanted heart. Because graft-infiltrating lymphocytes exhibit low levels of donor-specificalloreactivity, we haveconsideredother immunologic mechanismsin this chronic rejection model. The involvement of hspimmunity hasbeenshown in this model.g3Immunoblot analysishas shown an increasedexpressionof hsp32,hsp60,protein disulfide isomerase,and calreticulin in stromal tissues from cardiac allografts undergoing chronic rejection.

Current experimental evidencesupportsthe concept that, during cellular rejection, graft-infiltrating cells induce a stress responsewithin the allograft that increases the expression of hsp and triggers the recruitment and activation of hspdependent lymph& cytes. Whereas a variety of stressproteins exhibit higher tissue levels during the different phasesof allograft rejection, our lymphocyte studieshave dealt, so far, primarily with the hsp70 family, namely mycobacterial hsp71and murine grp78. Hsp70doesnot appear to stimulate graft-infiltrating Tcells asa conventionalantigen. Rather, structurally intact hsp70 molecules seem to interact with self-APC, which then stimulate certain types of autoreactive CD4+ T cells to undergo proliferation. This hsp effect implies a previously unrecognized mechanism of transplant immunity. We postulate that a stressresponseto an injurious stimulus leads to increasedintragraft levels of hsp, someof which would function as intercellular chaperonesinvolved

182

Duqwmy

in signalling self-APC to become stimulatory towards graft-infiltrating T cells. This mechanism might be based on the peptide-binding properties of these stress proteins. The members of the hsp70 family function as molecular chaperones that have a common structure consisting of a C-terminal peptidebinding domain and a N-terminal ATPase domain that influences peptide binding.” For instance, grp78 can bind peptides of minimally 7 to 8 residues,gj and the peptide-binding region contains four major pockets that can accomodate large hydrophobic residues.s6 The peptide-binding properties of grp78 are more similar to those of class II molecules than those of class I molecules.“6 Recent studies have implicated that grp78 can interact with MHC gene products during their biogenesis to become functional antigenpresenting molecules.j7~g7 Our data indicate that hsp-dependent T cells probably do not actually “see” hsp70 on self-APC. It seems more likely that the reactivity of these T cells is because of recognition of peptides that have been transported by hsp70 into self-APC, which then would present them presumably in context with MHC molecules. This concept is analogous to Srivastava’s “fourth paradigm” that hsp function as carrier molecules of antigenic peptides that elicit specific tumor immunity.“j Because the grp78 and mycobacterial hsp7 1 dependency has been observed for CD4+ T cells only, we must consider other types of stress protein-dependent T cells in the allograft. An important consideration is that such stress proteins must play a role in antigen presentation. There are several candidates, including calnexin, PBP72/74 (or grp75), and grp94. We prefer grp94 (also called gp96) because of the convincing demonstrations by Srivastava’s group that grp94 can transport immunogenic tumor-specific peptides to APC that then induce protective immunity to transplantable tumors and the generation of tumor-specific cytotoxic CD8+ T cells.gs*99 Grp94 can cross-prime cytotoxic T-cell responses to minor histocompatibility antigens4550 and interferon-y upregulates grp94 expression. ‘O” Our data show that the allograft expression of grp94 is increased during rejection (Fig 2), and we have preliminary indications that CD8+ lymphocytes can respond to dendritic cells that have been pulsed with grp94-containing protein fractions prepared from allogeneic tissues. The inclusion of grp94 permits the development of a mechanistic model for the activation of hsp dependent T cells and their functional significance in transplant immunity (Fig 4). This model considers the intragraft release of stress proteins that function

et al

Figure 4. A proposed model how a stress response in an allograft may induce two pathways of hspdependent, self--MHC-restricted T-cell stimulation. The stress response of the allograft leads to the externalisation 01 peptideqarrying hsp70 and grp94 stress proteins that are taken up by self-APC, which then present the peptides to CD4 and CD8 cells by hIHC-restricted mechanisms.

as intercellular chaperones by transfering immunogenic peptides to self-APC that then present these peptides to T cells. Two pathways can be considered for hsp-dependent T cells. The hsp70 (eg, grp78)dependent pathway involves self-APC that, through class II-restricted mechanisms, present peptide to CD4+ cells. In the grp94-dependent pathway, the self-APC would use a class I-restricted presentation of peptides to CD8+ cells. This model reflects the concept that the release of hsp-peptide complexes by stressed tissue may represent what Matzinger’“’ calls a “danger” signal that elicits a T-cell response. What is the functional significance of hsp-dependent lymphocytes in transplant rejection? By definition, such cells react with self-APC that must first interact with stress proteins presumably released by injured cells in the graft. However, we have found that the hsp70-dependent responsiveness is very low for lymphocytes directly isolated from the allograft unless IL2 is added to the culture.g This lack of responsiveness might reflect some in situ anergic state which can be reversed by IL2. A likely source of IL-2 would be the graft-infiltrating alloreactive T

Shss IWeins in Transplant Immunily

cells that have been activated by donor-derived professionalARC through direct allorecognition. The increasedintragraft levels or cytokines such as IL2 would promote the expansionof hsp-dependentlymphocytesand their differentiation into effector cells that would amplify cellular rejection. Without the appropriate cytokines, an anergic state of hspdependentcellscould be maintainedthrough continuous release of low amounts of stress proteins by allograft tissue. Thus, hsp-dependent lymphocytes might play a dual role in graft surveillance. These cells may play a protective role, but, depending on the cytokine environment within the allograft, they might be stimulated to become effector T cells mediating an inflammatory response.In addition, cytokinessuchasinterferon-y can increasehspexpressionin APC,‘O”which might promote hsp-dependent T-cell activation. What kinds of peptides would be recognized by hsp-dependentT cells?Although there are no data, we might consider two possibilities.These cells may react with allopeptides from donor tissues,which would represent an hsp-dependent mechanism of indirect allorecognition. Second, these lymphocytes may recognize autologous-peptidesrrom self-proteins or conserved proteins shared by donor and recipient, which would reflect an autoimmune response. Grp78 has rather high binding affinity towards peptides containing hydrophobic amino acid residues.95,sG These sequencesare generally seenin the inner coresof proteins and would constitute a group of silent or cryptic antigenic determinants. Sercarz et alto2have shown that, during the development of a cellular immune responseto a protein, the T-cell repertoire spreadsto include recognition of cryptic determinants. This spreading is believed to be related to alternate mechanismsorantigen processing and presentation, especiallyduring an inflammatory state. Accordingly, grp78-dependent T cells might recognizecryptic determinants aspart of the spreading of a cellular immune responseassociatedwith the progressionof transplant rejection.

Relevance Transplant

of Intracellular Immunity

Hsp70 in

Many reports have indicated that ARC stressedby heat, chemical treatment, or ultraviolet irradiation lose their ability to stimulate in mixed leukocyte culturesto3-to5 and that animalsinjected with stressed allogeneiccells often exhibit prolonged graft survivals and even transplant tolerance.‘OG’Os Because

183

stressedcells have increased hsp expression, it is possiblethat these effects might be related to hsp mediated immunomodulation. Recent data from K.rensky et al’@’illustrates the involvement of hsp in the immunosuppressioninduced by certain HL4 class I peptides.togThese peptides inhibit T-cell function and promote rat allograft survival, which correlateswith their binding to the constitutively expressedhsp70and the heatinducible hsp70.Noninhibitory peptidesdo not bind these hsp. Nadler et alito have reported that the immunosuppressiveeffects of deoxysperguahn also involve the binding to hsp70 and that this drug interferes with antigen presentation.“’ Therefore, certain members of the hsp70 family have been proposedto representa third classof immunophilins in addition to the cyclophilins and FK-binding proteins.ias

Conclusions In this review, we have presentedevidencethat stress proteinsare important in transplant immunity. Their major role seemsrelated to antigen presentation. The stressprotein concept in transplant immunity will offer new perspectivesof the various immune mechanismsleading to rejection, chronic dysfunction, and, conversely, transplant tolerance and how these processesare affected by infection and ischemia/reperfusion injury.

References I. Dalchau R, Fangmann J, Fabrc JW: Allorccognition of isolated, denatured chains of class I and class II major histocompatibilitycomplcx molecules. Evidence for an important role Tar indirect allorecognition in transplantation. EurJ Immunol 1992,22:669 2. Fangmann J, Dalchau R, Sayer GJ, et al: T cell recognition of donor major histocompatibility complex class I peptides during allogrart rejection. EurJ Immunol 1992,22: 1525 3. Let RS, Grusby MJ, Glimchcr L.H, et al: Indirect recognition by helper cells can induce donor-specilic cytotoxic T lymphocytes in viva. Exp Med 1994,179:865 4. Shinvan H, Learner M, Wang H, Ed al: Pcptidcs derived from alpha hclices of allogeneis class I major histocompatibility complex antigens arc potent inducers ofCD4+ and CDE+ T cells and B cell responses after cardiac allograft rejection. Transplantation I995,59:40 I 5. Shoskics D, Wood K: Indirect presentation olMHC antigens in transplantation. Immunol Today 1994, IS:32 6. Warrens AN, Lombardi G, Lechler RI: Presentation and recognition or major and minor histocompatibility antigens. Transplant Immunol I994,2: 103

184

Duqupsnoy

7. Watschinger B, Gallon L, Carpenter CB, et al: Mechanisms of ago-recognition. Recognition by in viva-primed T cells of specific major histocompatibility complex polymorphisms presented as peptides by responder antigen-presenting cells. Transplantation 1994,57:572 8. Moliterno R, Woan M, Bentlejewski C, et al: Heat shock protein-induced T lymphocyte propagation from endomyccardial biopsies in heart transplantation. J Heart Lung Transplant 1995,14:329 9. Moliterno R, Valdivia L, Pan F, et al: Heat shock protein reactivity of lymphocytes isolated from heterotopic rat cardiac allografts. Transplantation 1995,59:598 10. Duquesnoy RJ: Stress protein research in transplantation, a report of presentations at the Fourth Basic Sciences Symposium ofThe Transplantation Society. Cell Stress and Chaperones, 1996, I:3 Il. Ritossa FM: A new puffing pattern induced by a temperature shock and DNP in Drosophila. Expetientia 1962,18:571 12. Tissieres A, Mitchell HK, Tracy UM: Protein synthesis in salivary glands of Drosophila melanogaster. Relation IO chromosome puffs. J Mol Biol 1974,84:389 13. Lindquist S, Craig EA The heat-shock proteins. Annu Rev Genet 1988,22:63 I 14. Polla BS: A role for heat shock proteins in inflammation? Immunol Today 1988,9: 134 15. Ellis RJ, de Vies SMV: Molecular chaperones. Annu Rev Biochem 1991,60:32 I 16. Georgopoulos C, Welch WJ: Role of major heat shock proteins as molecular chaperones. Annu Rev Cell Biol 1993, 9601 17. GethingJR, SambrookJF: Protein folding in the cell. Nature 1992,355:33 18. Morimoto RI, Tiisieres A, Georgopoulos C: The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Monograph Series. Plain View, NY, Cold Spring Harbor Laboratory, 1994 19. Lee AS. Mammalianstress response: Induction of the glucoseregulated protein family. Curr Opin Cell Biol 1992;4:267 20. Haas I: BiP (GRP78), an essential hsp70 resident protein in the endoplasmic reticulum. Expcrientia 1994,50:1012 21. Little E, Ramakrishnan M, Roy B, et al: The glucoseregulated proteins (GRP78 and GRP94): Functions, gene regulation, and applications. Crit Rev Eukatyot Gene Expr 1994,4:1 22. Flajnik MF, Cane1 C, Kramer J, et al: Which came first. MHC class I or class II. Immunogenetics 1991,133:295 23. Rippmann F, Taylor WR, RothbardJB, et al: A hypothetical model for the peptide binding domain of hsp70 based on the peptide binding domain of HLA. EMBO J 1991, IO: 1053 24. Polla B, Mariethos E: More evidence for a case ofchaperones in antigen processing. Immunol Today 1992, 13:421 25. DeNagel DC, Pierce SK Heat shock proteins in immune responses.Crit Rev1mmunol1993,13:71 26. Manara GC, Sansoni P, BadialideGiorgi L, et al: New insights suggesting a possible role or a heat shock protein 70-kD family-related protein in antigen processing/presentation phenomenon in humans. Blood 1993,82:2865 27. Williams DB, Watts TH: Molecular chaperones in antigen presentation. Curr Biol Immunol 1995,7:77 28. Eden WV, Young DB: Stress Proteins in Medicine. New York, NY, Marcel Dekker, 1996 29. Kaufmann SHE, Schoel B: Heat shock proteins as antigens

et al

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45. 46.

in immunity against infection and self, in Morimoto RI, Tissieres A, Georgopoulos C (eds): The Biology or Heat Shock Proteins and Molecular Chaperones. Plain View, NY, Cold Spring Harbor Laboratoty, 1994, p 495 Hermann E, Lohse AW, Mayet w, et al: Stimulation of synovial fluid mononuclear cells with the human 65-kD heat shock protein or with live enterobacteria leads to prererential expansion or TCR++ lymphocytes. Clin Exp Immunol 1992,89:427 Salvetti M, Buttinelli G, Ristori M, et al: T-lymphocyte reactivity to the recombinant mycobacteriaI65 and 70 kDa heat shock proteins in multiple sclerosis. J Autoimmun 1992, 5:69l Birnbaum G, Kotilinek L, Albrecht L: Spinal fluid lymph+ cytes From a subgroup or multiple sclerosis patients respond to mycobacterial antigens. Ann Neural 1993,34:294 Hogervorst JM, Bwg CJP, Wagenaar JPA, et al: T cell reactivity to an epitope of the mycobacterial 65-heat shock protein (hsp65) corresponds with arthritis susceptibility in rats and is regulated by hsp65-specific cellular responses. Eur JImmunol 1991,21:1289 Anderton SM, Vandenee R, Prakken B, et al: Activation OTT cells recognixing sell 60-kD heat shock protein can protect against experimental arthritis. J Exp Mcd 1995,181:943 Elias D, Markovits D, ReshelT, et al: Induction and therapy of autoimmune diabetes in non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein. Proc Nat1 Acad Sci USA 1990,87:1576 Srivastava PK: Peptide-binding heat shock proteins in the endoplasmic reticulum: Role in immune response to cancer and in antigen presentation. AdvCancer Res 1993,62: I53 Udono H, Srivastava PK Comparison of tumor-specific immunogenicities ofstress-induced proteins gp96, hsp90 and hsp70. J Immunol 1994, I52:5398 Srivastava PK, Udono H, Blachere NE, et al: Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics 1994,39:93 Suto R, Srivastava PK A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science 1995,269:1585 Torigoe T, Tsuboi N, Wada Y, et al: Cellular stress- and transformation-association cell surface antigens expressed on human and rodent tumor cells. Jpn J Cancer Res 1993, 84~673 Tamura Y, Tsuboi N, Sato N, et al: 70 kDa heat shock cognate protein is a transformation-associated antigen and a possible target for the host’s anti-tumor immunity. J Immunol 1993, 151:5516 Yoshino I, Goedegebuure PS, Peoples GE, et al: Human tumor-infiltrating CD4+ Tcells react to B cell lines expressing heat shock protein 70. J Immunol 1994,153:4149 Harada M, Matsuzaki G, Yoshikai Y, et al: Autoreactive and hsp60-recognizing CD4+ T cells show anti-tumor activity against syngeneis sarcoma. Cancer Res I993,53: 106 Lukacs KV, Lowrie DB, Stokes RW, et al: Tumor cells transfected with a bacterial heat shock protein gene lose tumorigenicity and induce protection against tumors. J Exp Med 1993,178:343 Srivastava PK Heat shock proteins in immune response to cancer: The Fourth Paradigm. Experientia l994,SO: 1054 Kaul’mann S: Heat shock proteins and autoimmunity: A critical appraisal. Int Arch Allergy Immunol 1994, IO3:3 I7

StressProteinr in Traytdant Immunib

47. Udono H, Srivastava PK Heat shock protein 70-associated peptides elicit specific cancer immunity. T Exp Mcd 1993, 178:1391 48. Udono H, Srivastava PK Comparison of tumor-specific immunogenicities of stress-induced proteins gp96, hsp90, and hsp70. J Immunol 1994, 152:5398 49. Srivastava PK, Udono H: Heat shock protein-peptide complexes in cancer immunotherapy. Curr Opin Immunol 1994, 6728 50. Arnold D, Faath S, Rammensee H, et al: Cross-priming OP minor histocompatibility antigen specific cytotoxic T cells upon immunization with heat shock protein gp96. J Exp Med 1995, 182:885 51. Multhoff G, Botzler C, Wicsnet M, ct al: CD3large granular lymphocytes recognize a heat-inducible immunogenic determinant associated with the 72-kD heat shock protein on human sarcomacells. Blood 1995,86:1374 52. Currie RW, Sharma VK, Stepkowsi SM, et al: Protein synthesis in heterotopically transplanted rat hearts. ExpCell Biol 1987,55:46 53. Perdrizet G, Heffron TG, Buckingham FC, et al: Stress conditioning: A novel approach to organ preservation. Curr Surg I989,46:23 54. Perdrizet GA, Kaneko H, Buckley TM, et al: Heat shock and recovery protects renal allografts from warm ischemia injury and enhances hsp72 production. Transplant Proc 1993, 25: 1670 55. Perdrixet GA, Buckingham FC, Rewinsi MJ, et al: The heat shock response protects organs from preservation injury. Fourth Basic Science Meeting of Transplantation Society, 1995, p. 375 56. Qian J, Moliterno R, Donovan-Peluso M, et al: Expression or heat shock proteins and lymphocyte reactivity in rat cardiac allografts undergoing cellular rejection. Transplant Immunol 1995,3: I I4 57. Bonnerot C, Marks M, Cosson P, et al: Association with BiP and aggregation of class II MHC molecules synthesized in the absence orinvariant chain. EMBOJ 1994, 13:934 58. Suzuki CK, Bonifacino JS, Lin AY, et al: Regulating the retention oTTcell receptor alpha chain variants within the endoplasmic reticulum: Ca(P+)dependent association with BiP.JCellBiol 1991, II4189 59. Davis EA, Wang BH, Stagg CA, et al: Induction of heat shock protein in a model of acute cardiac agograft rejection. Transplantation 1996,61:279 60. Barrios C, Lussow AK, van Embden J, et al: Mycobacterial heat shock proteins as carrier molecules. II: The use orthe 70 kDa mycobacterial heat shock protein as a carrier ror conjugated vaccines can circumvent the need for adjuvants and Bacillus-Calmette-Guerin priming. Eur J Immunol, 1992,22:1365-1372 61. Alevy YG, Brennan D, Durriya S, et al: Increased expression of the I-IDJ-2 heat shock protein in biopsies of human rejected kidney. Transplantation 1996,61:963 62. Cruse I, Maines MD: Evidence suggesting that the two forms or heme oxygenase are products of different genes. J Biol Chem 1988,263:3348 63. Maines MD, Mayer RD, EwingJF, et al: Induction of kidney heme oxygenase- I (HSP32) mRNA and protein by ischemia/ reperfusion: Possible role ofheme as both promoter ortissue damage and regulator of HSP32. J Pharmacol Exp Ther 1993,264:457

185

64. Ewing JF, Raju VS, Maines MD: Induction of heart heme oxygenase-I (HSP32) by hyperthermia: Possible role in stress-mediated elevation ofcyclic 3’:5’-guanosine monophosphate.J Phar ExpTher 1994,271:408 65. Raju VS, Maines MD: Coordinated expression and mechanism of induction of HSP32 (heme oxygenase-I) mRNA by hyperthermia in rat organs. Biochim Biophys Acta 1994, I2 17~273 66. Duquesnoy RJ, SuzowJ, Qian J, et al: Increased expression of heat shock proteins during acute cardiac allograft rejection. Fourth Basic Science Meeting of Transplant Society. Extended Abstract Book, 1995, p 199 67. Routsias JG, Txiouras AG, Sakarellos-Daitsiotis M, et al: Calreticulin synthetic peptide analogues: anti-peptide antibodies in autoimmune rheumaticdiseases. Clin Exper Immunol 1993,91:437 68. Yokoi T, Nagayama S, Kajiwara R, et al: Effects orcyclosporin-A and D-penicillamine on the development of hepatitis and the production ofantibody to protein disulfide isomerase in LEC rats. Res Commun Mol Pathol Pharmacol 1994, 85:73 69. Nagayama S, Yokoi T, Tanaka H, et al: Occurrence of autoantibody to protein disulfide isomerase in patients with hepatic disorder. J Toxicol Sci I994,19: I63 70. Karska K, Tuckova L, Steiner L, et al: Calreticuli~The potential autoantigen in celiac disease. Bicchem Biophys Res Commun 1995,209:597 7 I. Jacquier-Sarlin MR, Fuller K, Dinh-Xuan AT, et al: Protective effects of hsp70 in inflammation. Expcrientia 1994, 50:1031 72. Latir N, Rose ML, Yacoub MI-I, et al: Association or pretransplant antibodies with clinical course Tollowing cardiac transplantation. J Heart Lung Transplant 1995,14:1 I9 73. Goral J, Mathews I-IL, ClancyJJr: Antibodies specific for the inducible form of hsp70 parallel the development of acute graR versus host disease (GVHD). Clin Immunol Immunopathol 1995,75:147 74. Duquesnoy RJ, Trager JDK, Zeevi A: Propagation and characterization of lymphocytes from transplant biopsies. CRC RevImmunol 1991, IO:455 75. Trieb K, Grubeck-Loebenstein B, Margreiter R: T cells Tram rejected kidney allografts respond to heat shock protein 72. Fourth Basic Science Meeting orTransplant Society, 1995, p 22 76. Panchamoorty G, McLean J, Modlin RL, et al: A predominance or the T cell receptor Vy2/V82 subset in human Mycobacteria-responsive T cells suggest germline gene encoded recognyion. J. Immunol 1991,150:3360 77. Vaessen LMB, Ouwehand AJ, Baan CL, et al: Phenotypic and functional analysis of T cell receptor $-bearing cells isolated from human hearts. Transplantation 1991, 147846 78. Born W, Harbeck R, O’Brien RL: Possible links between immune system and stress response: The role of y6 T lymphocytes. Semin Immunoll991,3:43 79. HoloshitsJ, Konig F, ColiganJE, et al: Isolation oFCD4CDB mycobacteria-reactiw T lymphocyte clones liom rheumatoid arthritis synovial fluid. Nature 1989,339:226 80. Wick G, Schett G, Amberger A, et al: Is atherosclerosis an immunologically mediated disease? Immunol Today 1995, 27~27 81. Duquesnoy RJ, Kaufman C, Zerbe TR, et al: Presence of CD4,CD8 double negative and T cell receptor-gamma, delta

186

82.

83.

84.

85.

86.

87.

88. 89.

90. 91.

92.

93.

94.

95.

Duqutmoy et al

positive T cells in lymphocyte cultures propagated from coronary arteries from heart transplant patients with graft coronary disease. J Heart LungTransplant 1992, 11:583 Liu K, Moliterno R, Fu X-F, et al: Identification of two types of autoreactive T lymphocyte clones cultured from rat cardiac allograft cells incubated with recombinant mycobactcrial heat shock protein 71. (submitted) Hunig T, Wallny H-J, HartleyJ, et al: A monoclonal antibody to a constant determinant of the rat Tccll antigen receptor that induces T cell activation. J Exp Med 1989,169:73 Tsuchida M, Hirahara H, Matsumoto Y, et al: Induction of specilic unresponsiveness to cardiac allogralis by short-term administration of anti-T cell receptor alpha beta antibody. Transplantation 1994,57:256 Heidcckc CD, Hancock WW, Jakobs F, et al: Alpha/beta-T cell receptor-directed therapy in rat cardiac allograft recipients. Treatment prior to alloantigen exposure prevents sensitixation and abrogates accelerated rejection. Transplantation 1995,59:78 Gething M-J, Blond-Elguindi S, Mot-i K, et al: Structure, function, and regulation of the cndoplasmic reticulum chap erone BiP, in Morimoto RI, Tissieres A, Gcorgopoulos C (eds): The Biology or Heat Shock Proteins and Molecular Chaperones. Plain View, NY, Cold Spring Harbor Laboratory, 1994, p I I I Hightower SE, Guidon PlJ: Selcctivc release from cultured mammalian cells 01 heat-shock (stress) proteins that resemble glia-axon transfer proteins. J Cell Physiol 1989, 138:257 Roman E, Morcno C, Young D: Mapping olHsp70-binding sites on protein antigens. EurJ Biochem 1994,222:65 Flaherty KM, DeLuca-Flahcrty C, McKay DB: Threedimensional structure orthe ATPasc Fragment of a 70-hcatshock cognant protein. Nature 1990,346:623 Wang T-F, Chang J, Wang C: Identification of the peptidebinding domain of hsp70. J Biol Chcm 1993,268:26049 Flaherty KM, Wilbanks SM, DcLuca-Flaherty C, ct al: Structural basis of the IO-kilodalton heat shock cognate protein ATP hydrolytic activity. J Biol Chem 1994,269: I2889 Murasc N, Starzl TE, Tanabc M, et al: Variable chimerism, graft-versus-host disease, and tolerance alter different kinds of’cell and whole organ transplantation Irom Lewis to brown Norway rats. Transplantation 1995,60: I58 Liu R, Murase N, Suzow J, et al: Evidence Ior heat shock protein immunity in a rat cardiac allogrart model of chronic rejection. Fourth Basic Science Meeting oITransplant Society, 1995, p 25 Feige U, Polla B: Hsp70-A multigcnc, multi-structure, multi-hmction family with potential clinical applications. Experientia 1994,50:979 Flynn GC, Pohl J, Flocco MT, et al: Peptide-binding specilicityorthc molecular chaperone BiP. Nature 1991,353:726

96. Blond-Elguindi S, Cwirla S, Dower W, et al: Alhnity panning ol’ a library of peptides displayed on bactcriophagcs reveals the binding specificity oIBiP. Cell 1993,75:717 97. Nijenhuis M, NeeljesJ: Early events in the assembly ormajor histocompatibility complex II hctcrodimers from their lrec units. EurJ Immunol 1994,24:247 98. Li Z, Srivastava PK: Tumor rcjcction antigen gp96/grp94 is an ATPasc: Implications Ior protein folding and antigen presentation. EMBO J 1993, 12:3 143 99. Udono H, Srivastava PK: Cellular requirements Ior tumorspecilic immunity elicited by heat shock proteins: Tumor rejection antigen gp96 primes CD8+ T cells in viva. Proc Natl Acad Sci USA I994,9 I :3077 100. Anderson SL, Shen T, Lou J, et al: The cndoplasmic reticular heat shockprotein gp96 is transcriptionally upregulated in interferon-treated cells. J Exp Med 1994,180: 1565 101. Matzinger P: Tolerance, danger, and the extended family. AnnuRcvImmunol 1994,12:991 102. Sercarr. E, Lehmann P, Amctani A, et al: Dominance and crypticity ofTcell antigcnic determinants. Annu Rev Immunol 1993, I I:729 103. Brewer JA, Hank JA, Wcndel T, et al: Hcatcd lymphocytes express HLADR antigens despite their inability to stimulate MLC. Tissue Antigens I983,22:246 104. Boycr CM, Kostyu DD, Brissette CS, et al: Functional dclect of heat-inactiated human lymphocytes in mixed lymphocyte culture. Cell Immunol 1986, lOI: 105. Loertscher R, Abbud-Filo M, Lcichtman AB: DiRerential effects or gamma-irradiated and heat-treated lymphocytes on T cell activation and intcrleukin-2 and interleukin-3 relcasc in the mixed lymphocyte reaction. Transplantation 1987, &673 106. Baird MA, Bradley MP, Heslop BF Prolonged survival oC cardiac allogralts in rats following administration or hcattreated donor lymphocytes. Transplantation 1986,42: I 107. Freeman AJ, Baird: Immunoqupprcssion mediated by heattreated cells. Transplantation 1993,55: 1439 108. Martinelli GP, Horowitz C, Chiang R, et al: Prctransplant conditioning with donor-specilic translitsions using heated blood and cyclosporine. Transplantation 1987,43: 140 109. N6Rner E, Goldberg J, Naltzcr C, et al: HLAderived pep tides which inhibit T cell function bind to members of the heat-shock protein 70 1amily.J Exp Med 1996, 183:339 I IO. Nadlcr SG, Teppcr MA, Schacter 13, et al: Interaction of the immunosupprcssant dcoxyspergualin with a member of the hsp70 Family of heat shock proteins. Science I992,258:484 Ill. Hijgcr P, Tcppcr M, Faith A, et al: Immunosuppressant deoxyspergualin inhibits antigen processing in monccytes. J Immunoll994,l53