Indirect allorecognition of HLA class I peptides by CD4+ cytolytic T lymphocytes

ELSEVIER

Indirect Allorecognition of HLA Class I Peptides by CD4+ Cytolytic T Lymphocytes B. Susskind, Michael R. Iannotti, Michael D. Shornick, Nancy S. Steward, John Gorka, and T. Mohanakumar ABSTRACT: T-cell responses to alloantigens can occur either by “direct” recognition of donor MHC molecules, or “indirect” recognition of MHC peptides in association with self-MHC. To evaluate human T cells mediating indirect allorecognition, a CD4’ XL and clones specific for HLA-Al or HLA-B8 (residues 60-84) were generated from normal PBLs (A2,29 B62,- DR1,4 DQ3). Most clones were Al specific (16 out of 17 tested), HLA-DR4 restricted (8 out of S), and lysed targets pulsed with Al peptide (16 out of 16). An amino acid substitution at position 86 of the DR4 8 chain (G -+ V) abrogated the capacity of CD4‘ CTLs to lyse target cells. Chloroquine

ABBRJWIATIONS APC antigen-presenting cell c totoxic T lymphocyte CTL ‘H-TdR [.YHlthymidine LCL lymphoblastoid cell line MHC major histocompatibility complex

treatment of Al-pulsed targets reduced their susceptibility to lysis, indicating a requirement for peptide processing. The TCL and clones were stimulated to proliferate by cells bearing intact HLA-Al when autologous APCs were present, indicating that the epitope contained within the Al 60-84 peptide being recognized is produced when APCs process native HLA-Al. Furthermore, the clones and TCL did not recognize HLA-Al on target cells carrying this allele plus self-HLA-DR4. These studies suggest a much wider role for CD4’ T cells in allograft immunity.

MR PBL SR TCL TCR

maximum release peripheral blood lymphocyte spontaneous release T-cell line T-cell receptor

INTRODUCTION Recognition of polymorphic, non-self major histocompatibility complex (MHC) molecules by T cells is the basis of organ allograft rejection. However, the specific form of alloantigen recognized during an in vivo allograft response, is not well defined. There are two distinct pathways by which alloantigens expressed on the allograft may be recognized by the recipient host’s immune system [l--4]: (a) the direct pathway, where donor MHC class I and II molecules are recognized as intact antigens

FromtbeDepartmentofSurgerv(B.S., M.R.I., M.D.S., N.S.S., T.M.) and Department of Molecular Biology and Pharmacology f’J.G.), Washington Univwsity Medical School, St. Louis, Missouri, USA. B. Susskind’s current address is Division of immunology and Organ Transplantation, University of Texas He&h Science Center, 643 1 Fannin, MSB 6.254, Houston, TX 77030, USA. Address reprint requests to Dr. T. Mohanakumar, Department ofSurgety, Washington University Medical School, Box 8109, CSRB 3326, 4939 Children’s Place, St. Louis, MO 63110, U.S.A. Received October6, 1995; accepted December 1 I, 1995. Human Immunology 46, l-9 (1996) 0 American Society for Histocompatibiliry

and Immunogenerics,

1996

on cells of the allograft (part of the target structure may be endogenous or even host peptides occupying the antigen-binding site); or (b) the indirect pathway, where MHC antigens shed from the allograft are treated like convention exogenous antigens, i.e., processed by autologous antigen-presenting cells (APCs) and presented in association with host HLA molecules. Recognition of allo-class I by CD8’ cytotoxic T lymphocytes (CTLs) is primarily by the direct pathway. The role of the indirect pathway in the CD4-mediated allograft response is a matter of intensifying debate. Initial allograft recognition by CD4’ T cells probably involves interactions with donor class II MHC on passenger leukocytes via the direct pathway 12, 31. It has been postulated that after passenger leukocytes have emigrated or been eradicated from the graft, allograft cells which express donor MHC class II may not be immunogenic due to lack of costimulator activity, and might even induce anergy of the recipient’s CD4’ T cells 13, 5-71. Therefore, it is conceiv0198~8859/96/$15.00 SSDI 0198~8859(95)002 15-4

2

B. Susskind

able

that

CD4’ that

protracted

alloreactivity

T cells which are processed

fessional” that lograft

A few recent

responses

reaction

associated

host

of such

To evaluate alloantigens

via the

cell clones

capable

the context

during

{3, 4, 8).

however,

of T cells that

indirect

pathway,

of recognizing

of self-class

In this

that the CD4’

T cells that recognized

in association

with

capable

of both

Moreover,

proliferative

the

HLA-Al

peptide

Al

was processed in vivo

imply

that

pathway tion

and

immunogenic

the occur

self-class

by the

during

CD4’

could

epitope

both

of the allograft

as could results

negative

HEPES.

regula-

IL-2

cloned

(Cetus,

HLA

typing. The

stimulator

Lymphoblastoid tained for

from

sented

in Table

crocytotoxicity

phenotypes

in the various

1. HLA testing

typing

of the

clones

experiments

and

are pre-

was performed

by mi-

CC,].

TCL

and

methods

clones we

peripheral

were

have

blood

heparinized

produced

lymphocytes

whole

blood

by

(PBLs)

by density

and clones.

a modification

described

[lo].

of

In

brief,

were isolated

from

gradient

tained

centrifuga-

land,

blood

domain 1640

stimulated

of HLA-Al culture

with

derived

from

a combination

residues

60-84

and -B8 (10 pg/ml

medium

containing

15%

(Y,

in RPM1

normal

USA)

1

HLA

phenotypes

of relevant

human

Ranch0

10 mM

were

A

B

DR

DQ

MDS MS cw

2, 20 I, i 1, 2

62,8,27 8, w62

1.4 l,? i.-

I,4 I, -3, 6 2

*0101, *0101, ND“

62, 35, GO, 51,-

4,l.4, i,-

8 5 8 7

*040 1 *0101 *0404 *0407

2, II, 24, 31,

” ND, nor detrrm~ned

25-mer

cells

CO,,

Costa

and

Iscalf

2 mM

streptomy-

representing

cells

the

of HLA-A

peptide

1,

synthesis

reversed

phase

x 10”) in 250

with

250

washed 10%

four times newborn

assays were conducted

ofeffector

pCi of

CA, USA) for one hour at

containing

with

calf seby adding

cells and 5 x 10’ “Cr-labeled

1O:l

or 5:l

to wells

in a total

volume

5 hours

effector:target

supernatants counter.

release

(MR)

ratios

of 96-well

V-bottom

of 0.2

of culture

of incubation

a gamma

maximum

(2-4

incubated

cells were then

(typically

using

USA),

60-84)

by analytic

Mesa,

100 pl of culture

counted

CA,

peptides

were

in triplicate After

ob-

Grand

100 pgiml

by solid phase

release

plates

virus

All LCLs were

15%~ newborn

(residues

for purity

medium

used)

from

by trans-

acid analysis.

(ICN,

numbers

medium.

with

Dominguez,

medium

Chromium

MDS)

Technologies,

CTL m-say. Target

The target

(Boleth,

Epstein-Barr

penicillin,

region

checked

RPMI-1640 rum.

with

ob-

Society

HEPES.

and amino

NaL5 ‘CrO,

either

ml

at 37°C were

in 5%’

harvested

Spontaneous were

and release

determined

by

adding target cells to wells containing 0.2 ml of culture medium or 1% Triton X-100, respectively. Percent spe-

cells

Name

BOLETH WTI OOBIS PE117 JHAF

was

prepared

(MS, CW,

(Life

100 U/ml

hypervariable

37°C.

or were

supplemented

(Biocell,

HPLC

(SR) TABLE

as medium

were

cell line B95-8.

in RPMI-1640

NY,

graded

of alloof the

each),

USA)

Immunogenetics

donors

of B lymphocytes

[ 111 and

culture

were

intervals

recombinant

(MDSxAl/B8)

LCLs

and JHAF)

-B7, and -B8 were made

DR

peptides

autologous

of the American

and

PE117,

L-glutamine,

target

1,4),

(LCLs).

from the marmoset

cultured

tion over Ficoll-Hypaque (Pharmacia, Piscataway, NJ, USA). PBLs from a normal donor, MDS (A2,29 B62,-, MHC

restimulated

at weekly CA,

2 mM

streptomy-

dilution.

Histocompatibility

F’Cr-r&z~e

previously

were

+ 20 U/ml

Emeryville,

ceb lina

ul of culture

Production of- antigen-specific T-cell line (TCL)

cells

the cell repository

WTlOOBIS,

third

METHODS

HLA

cells used

USA),

and irradiated

The TCL thus obtained

by limiting

cin, and

response.

AND

The

50% MLR supernatant

human

MO,

100 pgiml

restimulations

Peptides. Synthetic MATERIALS

City,

the peptides

PBLs. Two subsequent

serum

via the indirect

and

10 mM

1 week with

formation

HLA-

The

and

PBLs of normal

within

native

pathway,

respond

positive

were

activities.

rejection.

T cells which

exert

HLA-

contained when

exogenous

transplant

we

II MHC

cytolytic

was produced

in

report

Al

Kansas penicillin,

T-

class I allopeptides

II molecules.

cin,

supplements.

HLA

we generated

Biologic, 100 U/ml

included

The

recognize

(RJO

after

is limited.

demonstrate peptide

“pro-

an in vivo al-

II molecules

serum

glutamine,

have demonstrated

in humans,

the functions

those

class I and class II peptides

class

reports

by

molecules

by the recipient’s studies

can develop

to allogeneic

with

number

is caused

shed allo-MHC

and presented

APCs.

T-cell

recognize

et al.

DRpl *0401 *0401

cific lysis was defined

as Sample

9& specific

lysis = (

CPM

MR CPM

- SR CPM - SR CPM

x 100 1

Prolifiration

assays. Twenty-five thousand MDSxAliB8 TCL cells or derived clones were cultured in flatbottomed microtiter plates (Falcon 3042) in 200 pl of culture medium. Irradiated PBLs (3000 R) were used as APCs at lo5 per well; LCL stimulator cells were irradi-

Indirect Allorecognition

3

by Human CD4’ CTLs

ated (10000 R) and used at 25 x lo3 per well. After 48 hours of culture, the plates were pulsed with 1 pCi of 13H]thymidine (3H-TdR) (2 mCi/mm, New England Nuclear). Cultures were harvested after 18 hours (PHD harvester, Cambridge Technology, Cambridge, MA, USA) and their radioactivity measured by liquid scintillation spectroscopy. Results given are the mean of triplicate cultures * SE.

RESULTS Proliferative response of “indirect” alloreactive CD4’

clones.

Four weeks after stimulation of PBLs from a normal donor (A2,29 B62,-, DR 1,4) with both HLA-Al and -B8 peptides, a CD4’ TCL (MDSxAl/B8) was established that proliferated in the presence of the combination of allopeptides. The response of the TCL to Al + B8 peptides required the presence of HLA-DR compatible APCs (Fig. 1). In the next step, the CD4’ MDSxAl/B8 TCL was cloned under limiting dilution conditions at one cell per well in the presence of autologous APCs and both A 1 and B8 peptides. Positive wells were expanded and screened for recognition of the individual peptides on the basis of proliferation. Seventeen of 24 clones tested displayed a measurable proliferative response to the peptides. All but one (I 6 out of 17) of the peptide-responsive MDSxAl /B8 clones were Al specific. Representative data on eight clones are shown in Fig. 2. The B8-reactive clone shown in Fig. 2, clone lOB8, failed to grow enough for further testing.

FIGURE 1 MDSxAl/B8 TCL (25 x 103) was stimulated with Al and B8 peptides in combination, at the indicated concentrations, in the presence of lo5 irradiated autologous (MDS) or allogeneic (CW) APCs. Cultures were pulsed with 1 pCi of C3H]thymidine after 48 hours and harvested 18 hours later. Results given are the mean of triplicate cultures.

2AlO 6C8

io

Al/B8 Peptides @g/ml)

jo

2B1 4D114All

3DlO lOB8

Clone FIGURE 2 Clones (25 x 103) were stimulated with Al or B8 peptide (30 pg/ml) in the presence of lo5 irradiated autologous APCs. Cultures were pulsed with 1 pCi of E3H]thymidine after 48 hours and harvested 18 hours later. Stimulation index is defined as the ratio of CPM in the presence of peptide divided by CPM in the absence of peptide. Cytotoxic activity of “indirect” alloreactive CD4’

TCL

and

clones. The MDSxAl/B8

TCL and clones derived from it were tested for CTL activity using DR-matched targets cells (MS LCL) in the presence of either the Al or B8 peptides. Lysis of the MS LCL was detected only when the Al peptide was present, and even the uncloned TCL exhibited little or no reactivity toward the B8 peptide (Fig. 3). Besides being peptide specific, target cell sensitization was also peptide dose dependent (Fig. 4). The MS LCL target cells used for the CTL assays in Figs. 3 and 4 co-expressed HLA-Al (and HLA-BS) as well as the DRl and DR4 alleles of the clones (DRBl*OlOl and DRB*0401). However, the MDSxAl/ FIGURE 3 Cytolytic activity and peptide specificity of human CD4’ clones. Cloned human CD4’ T cells were assayed for cytolytic activity at a 5:l effector:target ratio in a 4-hour “Cr-release assay using MS LCL for target cells in the presence of 10 pg/ml Al or B8 pepride. The results represent the mean of three replicates.

-20 v

6

6F2

/ TCL 2A10

2B1

6F2

6C8

CLONE

6B9

ID4

8C7

4

B. Susskind

et al.

Peptide IA1 17 BS IB7

TCL 6C8 7E9 9D7 6F2 7D4 6B9 8C7 2E8 0

3

1

CLONE

10

Peptide Concentration

@g/ml)

FIGURE 4 Titration ofpeptides required for target cell sensitization and peptide specificity. Human CD4’ T-cell clone 6F2 was assayed for cytolytic activity at a 5:l effector:target ratio in a 4-hour “Cr-release assay using MS LCL for target cells in the presence of Al, B8, or B7 peptides. The results represent the mean of three replicares.

FIGURE 5 HLA restriction of indirect clones. Cloned human CD4’ T cells were assayed for c tolytic activity at a 5:l effector:target ratio in a 4-hour “Cr-release assay using WTl 00 (DRB 1*0 101) or Boleth (DRB 1*040 1) homozygous LCLs for target cells in the presence of 10 pgiml Al peptide. The results represent the mean of three replicates.

a single chain

B8

TCL

and

compatible Al

clones

MS LCL only

peptide.

Therefore,

cell did not result which

derived

was incorporated

it

synthesis

the

DR-

of exogenous

by the

MS target

of a constituent

into the DRl

pathway

of the target

lysed

in the presence

Al

in derivation

by the endogenous on the surface

from

or DR4

peptide

amino

acid substitution

of DR4

specific, get cells,

DR4

B 1*040 l-restricted

presumably

of the peptide tide

by affecting

by the clones,

by the DR4

presented

cell.

Al

determine

phic

the MDSxAliBS

of recognizing

pressing either the DRl of the cells was assessed ing either

syngeneic

Cells expressing allele

were

ever,

of eight

HLA-DR

capable

5). and

in the presence

the DRBl*OlOl

of presenting

Al-specific

restricting

stricted (Fig. Specificity

peptide

clones

element, affinity

Al

of Al peptide. or DRBl*O401

to the TCL;

characterized all were

of peptide

groove.

zygous LCLs expressing or Val-86 were capable

We determined DR4 alleles of functional

how-

for their

HLA-DR4

binding

DR4 is known to be affected by polymorphisms due 86 of the B chain, an influential component antigen-binding

the recognition of the pep-

CTL clones. To determine

CD4’

antagonist

in order

CTL clones,

chloroquine,

of antigen

whether

Al pep-

to be recognized

processing,

by the

a lysosomotrowas used.

Target

on cells ex-

or DR4 allele, cytolytic activity against homozygous LCLs bear-

allele

either

cell lines and clones

the Al

either

or the binding

peptide haJ to be processed in order to recognized hy the

indirect CD4’ indirect

whether

of Al-

CD4 ’ CTL to lyse tar-

molecules

and subsequently

HLA restriction and fine specificity oj-the indirect ~-lones. To capable

86 of the p

the capacity

molecule.

tide had to be processed

were

at position

(G -+ V) abrogated

whether containing presentation

FIGURE 6 HLA fine specificity of indirect clones. Cloned human CD4’ T cells were assayed for cytolytic activity at a 5: 1 effector:target ratio in a 4-hour “Cr-release assay using Boleth (DRBl *O&)1), PE117 (DRB1*0404), or JHAF (DRB1*0407) homozygous LCLs for target cells in the presence of 10 pgiml Al peptide. The results represent the mean of three replicates.

re-

to HLAat resiof the homoGly-86 of the

Al 60-84 antigenic epitope(s). As shown in Fig. 6, the MDSxAlIB8 clones manifest Al-specific cytolytic activity against Boleth (DR4*0401) and JHAF (DR4*0407) homozygous LCLs, whose DR4 alleles contain Gly at residue 86 of the B chain, but failed to lyse PE117, which expresses DR4*0404 and contains Val-86. Thus,

Boleth

(*0401)

PEll7(*0404)

TARGET

JH4F

CELL (DRBl)

(‘0407)

Indirect Allorecognition

by Human CD4’ CTLs

5

cells were pretreated overnight with medium, chloroquine alone, Al peptide alone, or Al peptide plus chloroquine. After washing, the cells were labeled with 51Cr and used as target cells in the CTL assay in the presence or absence of exogenous Al peptide. Normal and chloroquine-treated targets were not killed in the absence of exogenous peptide, and both were killed equally as effective in the presence of peptide. Peptide pretreated targets were killed without addition of peptide to the assay, but chloroquine treatment significantly blocked presensitization of the MS target cells (Fig. 7). Chloroquine-treated targets were killed as effectively as nontreated targets when exogenous peptide was included in the assay. Thus chloroquine treatment does not simply render target cells resistant to cytolysis. Furthermore, we also found that glutaraldehyde fixation of LCL significantly inhibited their ability to present Al peptide to the clones (not shown). These results indicate that the Al peptide has to be processed by the target cells for recognition by the clones. Response of MDSxAl

lB8 cells to HLA-Al

in the presence of autologous

APCs.

on stimulator

cells

determine if the epitope contained within the Al 60-84 peptide was produced when native HLA-Al was processed, MDSxAliB8 TCL and cloned cells were tested for proliferation when stimulated with a natural form of Al. Al’ LCLs were used as the source of native HLA-Al. As shown in Table 2, the TCL and clones proliferated in response to this source of Al only in the presence of autologous APCs. DR-mismatched APCs could not support the proliferative response to Al-bearing allogeneic LCLs. These results indicate that an immunogenic epitope is contained within the Al 60-84 peptide which is produced when natural Al is processed by autologous APCs.

None

Clq

PRETREATMENT

Al

Al + Clq

OF TARGET

CELLS

FIGURE 7 Al peptide has to be processed in order to recognized by the indirect CD4’ CTL clones. MS LCL cells were treated overnight with chloroquine (0.1 mM), the Al peptide (10 pgiml), chloroquine plus Al peptide, or left untreated. After washing, the cells were labeled with 5’Cr, then used as target cells in a CTL assay in the absence or presence of 10 pgiml Al peptide. Effector cells were the MDSxAl/BS TCL used at a 5:l effector:target ratio. Results represent the mean of three replicates.

To

DISCUSSION In this study T cells form a DRB1*0101’,DR4*0401’ individual were sensitized to allo-MHC peptides derived from residues 60-84 of the (Ye domain of HLA-Al and HlA-B8. A CD4’ cell lines was produced and clones were derived that responded to peptides in association with self-class II. Nearly all of the clones characterized were specific for the Al peptide. The CD4’ T cells that recognized Al peptide in association with self-class II MHC were capable of both proliferative and cytolytic activities. Although MHC class II-restricted CD4’ T cells are thought to be characterized primarily by “helper” functions while cytolytic activity is considered to be predominately a property of MHC class-I-restricted CD8’ T cells, recent studies in both mice and humans have demonstrated that CD4’ T cells can develop into “cytolytic T lymphocytes” (CTLs) [12-151. Thus, we and others El] have shown that CD4’ T cell clones that

recognize alloantigenic HLA peptides in combination with self-HLA are cytotoxic for peptide-pulsed cells. Chloroquine treatment of target cells significantly inhibited their ability to present the synthetic 25-mer Al peptide, indicating that the allopeptide had to be processed in order to be presented to the Al-specific TCL and clones. This was surprising, as peptides of this length are capable of direct association with MHC class II molecules { 16}. Suciu-Foca and co-workers { 17, 181 similarly found that processing was required for the emergency of antigenicity with a 20 amino acid class II allopeptide ultimately presented in association with self HLA-DR. Apparently, further processing of these peptides is required for optimal class II binding to occur, which is consistent with the observation that the peptides displayed in association with MHC class II molecules usually consist of less than 20 amino acids 1161. Our finding that almost all of the responding clones were Al specific and that the bulk TCL did not manifest significant B8 peptide reactivity indicates that epitopes contained within the B8 60-84 peptide are not immunogenic for this particular individual. This could reflect the fact that there is a greater disparity between the Al peptide sequence and the responder’s HLA-A phenotype (4-6 amino acids), compared to the B8 peptide sequence and the responder’s HLA-B62 (2 or 3 amino acids). Alternatively, the B8 peptide or products of processed B8 may have failed to associate with the individual’s DRl or DR4. Because peptides derived from the third hypervariable region of some HLA class I molecules (which in-

B. Susskind et al.

6

TABLE

2

Proliferation

in response

to native

HLA-Al

in the presence

of autologous

APCs

Antigen (CPM t SE ‘H-TdR Experiment

T cells

I

TCL TCL ID7 9D7 4-6~ 4-6M TCL TCL

II III IV

?APC

Al peptide 194 2998 59.3 3508 936 1224 10185 1188

+

+ + (DR4’) + (DR4-)

* + + t i + k *

I4 24 12 21 111 124 144 404

Al + LCL‘ 381 1175 782 1686 653 1646 2993 566

t i + i f i 2 t

208 200 180 174 20 41 122 311

incorporated) Al-

LCL”

256 ? 59 i 655 + 548 t 584 i 799 i ND ND

86 16 86 40 14 27

” MS LCL. ii Boleth LCL in experiments

I and IV, MDS LCL in rxperments

II and 111.

eludes residues 60-84) have been shown to produce suppression of T-cell responses, a third possibility is that the B8 peptide itself may have prevented the development of T-cell clones that could react with immunogenic epitopes derived from the peptide [19, ZO]. A single amino acid substitution at position 86 of the p chain of DR4 (G + V) abrogated the capacity of Al-specific, DR4 B 1*0401-restricted CD4’ CTLs to lyse target cells. This limited polymorphism at residue 86 of the p chain is known to affect specificity and affinity of peptide binding to HLA-DR4 [21]. Alternatively, peptide binding could occur, but with an altered conformation that affects the epitope of the peptide recognized by the clones. The MDSxAl/B8 TCL and clone cells responded to HLA-Al on stimulator cells in the presence of autologous APCs (DRB*OlOl,DRB*0401), indicating that contained within the Al 60-84 peptide is an immunogenic epitope which is also produced when natural Al is processed by autologous APCs. However, the MDSxAli B8 cells did not recognize HLA-Al on target cells expressing this allele plus HLA-DRl (DRB*OlOl) and/or HLA-DR4 (DRB*0401). This was somewhat surprising, in light of the fact that among the naturally occurring peptides bound to HLA-DR molecules, there is a predominance of MHC-derived peptides, and that both MHC class-1 and -II-related peptides are permiscuous in binding to DR 1163. These results are also in contrast to the work of others who found that CD4’ T cells sensitized to allo-HLA class I or class II peptides, presented in association with autologous HLA class II molecules, could recognize cells carrying the foreign HLA allele plus self-class II [I, IS]. The reasons for the discrepancies between these reports is unclear, particularly as the experimental protocols employed were similar. Only limited data of this kind have been reported for human CD4’ T cells that mediate self-restricted allorecognition by the indirect pathway. In our studies, the MS LCL

apparently failed to express a functional complex of endogenous Al peptide bound to the DR molecules at the cell surface. This suggests that the immunogenic epitope contained within the Al 60-84 peptide was not produced when constitutive HLA-Al was processed by the endogenous pathway. Alternatively, the peptide products of endogenously processed Al either failed to associate with the DRl or DR4, they were produced in too small an amount, or the amount of the complex formed was limiting due to competition for antigen-binding sites on the DR molecules from various other endogenous as well as exogenous peptides. Not all antigenic epitopes contained within self-MHC molecules are expressed at the cell surface in association with self-MHC class II molecules by the pathway for presenting endogenous peptides {l]. Our results indicate that exogenous and endogenous MHC antigens are processed differently. Graft rejection is usually associated with T cells, and both CD4’ and CDS’ T cells are capable of direct recognition of alloantigens. Whereas allogeneic MHC antigens presented on cells of the graft are potent immunogens, the immunogenicity of isolated MHC antigens is no greater than that of conventional antigens {17, 22]. It is perhaps for this reason that the role of polymorphic donor-derived peptides recognized in association with self-HLA antigens was not widely regarded as an important immunologic processes in graft rejection until recently. Passenger leukocytes, which more effectively stimulate T cells that respond by direct allorecognition than other cells from the graft (especially dendritic cells [2]), emigrate or are eliminated from the graft within the first several weeks after transplant. The organ is repopulated with recipient leukocytes [S, 231, and host APCs process and present donor MHC antigens emanating from the graft, activating T cells that respond to allopeptides bound to self-HLA antigens 13, S]. Based on this premise it has been proposed that T cells recognizing alloantigen by the direct pathway are important in trig-

Indirect Allorecognition

by Human CD4’ CTLs

gering early rejection episodes, but subsequent to the departure of donor passenger leukocytes, recipient CD4’ T cells responding to donor alloantigens by the indirect pathway become the dominant mediators of allograft rejection 12, 3, S}. Evidence that self-restricted allorecognition by the indirect pathway occurs in vivo is the finding that T cells primed by the allograft subsequently respond specifically to peptides derived from MHC molecules of the allograft 13, 4, S]. A principal role in graft rejection of CD4’ T cells that respond by the indirect pathway is as helper cells for the synthesis of anti-HLA antibodies, activation of CD8’ CTLs, and mediation of a DTH-like response 13, 4, 241. If T cells responding to peptides derived from MHC molecules of the graft ultimately are more important than T cells that perceive alloantigen as intact MHC molecules on grafts, then this could explain previous results demonstrating that the MLR is not an accurate indicator of host allograft tolerance 125, 261. T cells that recognize alloantigens by the “indirect” pathway may also function as regulatory cells that influence allograft tolerance. Potential roles for MHC classII-restricted CTLs in immunoregulation can be proposed, based on the demonstrations that CD4’ T cells can lyse APCs bearing the appropriate antigen in the context of autologous MHC class IIc 127, 28) our results). In this manner, CD4’ CTLs could effect a role in downregulation by eliminating the residual antigen stimulus, leading to cessation of the immune response. Moreover, human T cells are capable of MHC class II expression and are themselves also susceptible to killing by autologous CTLs [28-30). Exposure to donor HLA peptides “preprocessed” in the milieu of a site of active rejection could render T cells susceptible to CD4’ CTL-mediated lysis by virtue of binding to class II on the surface of the T cells. Moreover, donor class-I-specific CDS+ T cells may be specifically susceptible to elimination by self-restricted CD4’ CTLs due to uptake of soluble donor alloantigens through the T-cell receptor (TCR). CDS’ CTLs are able to bind and respond to soluble class I MHC molecules, and human T cells are capable of processing antigens 130-331. Thus self-restricted CD4’ CTLs may also play a role in the downregulation of alloimmune T-cell responses by lysing autologous cells that display donor-specific peptides. Such an occurrence may account for the clonal deletion of CD8’ CTLs in liver and long-term kidney allograft recipients that we have previously described [25, 341. Understanding the forms of allogeneic MHC antigens arising from an allograft that are recognized by the recipient’s T cells has important implications toward controlling the initiation, perpetuation, and regulation of organ transplant rejection. The capacity of T cells to mount a strong primary response to allo-MHC antigens

implies

that direct allorecognition is a polyclonal re135, 361. If MHC molecules on the graft endure as the most important target structures for T cells that mediate organ allograft rejection, the reaction will be difficult to control because of the magnitude of the T-cell response. If, on the other hand, the role of the direct pathway diminishes after passenger leukocytes subside, then the indirect allorecognition ultimately becomes the important pathway for organ transplant rejection. Constituent peptides of MHC molecules that are derived by antigen processing, bind to a given class II molecule, and activate T cells, apparently are limited to a few immunodominant antigenic determinants f5, 22). Therefore, T cells that respond through indirect allorecognition would be expected to employ a limited selection of TCR V, genes. Results from animal and human studies support this premise [S, 17, 37, 38). The practical implications of this would be that anticlonotypic reagents could be used for immunotherapy, allowing selective elimination of the indirect alloreactive T cells. Alternatively, selfrestricted allorecognition by the indirect pathway may also be amenable to tolerance induction, e.g., by the use of altered TCR peptide ligands f39,40]. Further study is required to elucidate the relevance of this “indirect pathway of allorecognition” in CD4’ T cells insofar as its role in acute vs chronic graft rejection, or conversely, allograft tolerance. sponse

ACKNOWLEDGMENTS

The authors are grateful for Dr. Nancy Poindexter

for critical reading of the manuscript and to Billie Glasscock for her secretaria assistance in preparing the manuscript. This work was supported by NIH Grant AI-26934.

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