Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1020-1024
Direct Alloreactivity by Human Cytotoxic T Lymphocytes Can Be
Inhibited by Altered Peptide Ligand Antagonism
By
Scott R. Burrows,
Rajiv Khanna, and
Denis J. Moss
From the Queensland Institute of Medical Research and University of
Queensland Joint Oncology Program, Brisbane, Australia.
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ABSTRACT |
Alloreactive T lymphocytes that respond directly to foreign major
histocompatibility complex (MHC) molecules and bound peptide are known
to be central mediators of graft-versus-host disease (GVHD) and
allograft rejection. We have recently identified a peptide from the
human protein, cytochrome P450 (isotypes IIC9, 10, or 18), that is
recognized in association with human leukocyte antigen (HLA) B*3501 by
alloreactive cytotoxic T lymphocytes (CTLs). These CTLs with this
specificity were isolated from several unrelated individuals and were
found to express a common T-cell receptor (TCR). Synthetic analogs of
the cytochrome P450 peptide were generated by introducing single amino
acid substitutions at putative TCR contact positions. Four altered
peptide ligands were powerful competitive antagonists of these CTL
clones, reducing lysis levels of target cells expressing the
alloantigen HLA B*3501 by over 80%. This first demonstration that it
is possible to suppress CTL alloreactivity with structural variants of
allodeterminants raises the prospect that such TCR antagonists could be
exploited within the clinical arena to specifically modulate GVHD and
allograft rejection.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
T CELLS CAN RECOGNIZE foreign major
histocompatibility complex (MHC) antigens by two distinct routes. The
direct pathway involves the recognition of foreign MHC antigens as
intact molecules on the surface of allogeneic stimulator cells. In most
cases, endogenous peptides, which constitutively bind to MHC antigens, are thought to be an integral part of the epitopes recognized via this
route.1 The indirect pathway of allorecognition requires the presentation of peptides, derived from foreign MHC antigens, on an
MHC molecule shared with the alloreactive T cell.2 Although this self-restricted, indirect presentation of allopeptides may play an
important role in chronic allograft rejection, the direct pathway of
allorecognition is the principal contributor to cytotoxic T lymphocyte
(CTL) responses mediating graft-versus-host disease (GVHD) and early
allograft rejection episodes.3
Although the repertoire of T cells available for use in an alloresponse
against a single allo-MHC molecule is diverse,1 the actual
repertoire used may be highly selected. Studies of the clonal
heterogeneity of alloreactive T-cell populations infiltrating human
allografts undergoing rejection have shown highly selected T-cell
receptor (TCR) usage, with a single clone predominating in some
cases.4-6 In vivo clonal expansions of T cells with
selected TCR usage have also been identified during acute GVHD and
persistence of these T cells for up to 1 year has been
reported.7,8 Although the basis for this limited diversity
is unclear, it is possible that preexisting expansions of alloreactive
T cells, which have recently been demonstrated in healthy individuals,
could be involved. For example, a CTL clonotype with specificity for an
alloantigen was shown to be expanded in the periphery of a healthy
individual who had never been exposed to that alloantigen.9
This was thought to represent a cross-reactive memory T-cell population
that had been raised against a self-MHC-restricted foreign antigen,
which could not be identified.
Studies from our laboratory have also characterized alloreactive T-cell
expansions in healthy individuals. These were shown to be driven by
cross-reactive stimulation with the persistent herpes virus
Epstein-Barr virus (EBV).10,11 These
EBV/allo-cross-reactive CTL expansions were so large that such T cells
dominated conventional mixed lymphocyte cultures from some individuals.
This graphically illustrates how a prior history of infection with an
immunogenic virus such as EBV can influence an individual's level of
responsiveness to an alloantigen; such mechanisms may underlie the
observed clinical association between herpes virus exposure and
GVHD.12
The limited use of the TCR repertoire in GVHD and allograft rejection
may provide the opportunity to therapeutically disrupt the alloresponse
by targeting a selected T-cell population for inactivation, as has been
achieved in experimental animal models.13 In the present
report, we describe the inactivation of a CTL clonotype that displays
direct alloreactivity for HLA B*3501. We have found previously that
this clonotype recurs in unrelated healthy individuals and is
preexpanded due to cross-reactive antigenic stimulation with a
self-MHC-presented epitope from Epstein-Barr nuclear antigen 3A.11 A peptide corresponding to regions of the human
isoenzymes cytochrome P450 IIC 9, 10, and 18 is recognized by this
clonotype in association with the alloantigen HLA B*3501. We now show
that it is possible to use analogs of this cellular peptide to
specifically suppress the direct alloreactivity of these CTL clones,
via altered peptide ligand antagonism.
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MATERIALS AND METHODS |
Establishment and maintenance of cell lines.
Lymphoblastoid cell lines (LCLs) were established by exogenous
transformation of peripheral B cells with EBV derived from the B95.8
cell line.14 Phytohemagglutinin (PHA) blasts were generated
by stimulating peripheral blood mononuclear cells (PBMCs) with PHA
(CSL, Melbourne, Australia), and after 3 days, growth medium containing
supernatant from the MLA-144 cell line and recombinant interleukin-2
(rIL-2) was added. PHA blasts were propagated with biweekly replacement of rIL-2 and MLA-144 supernatant (PHA free) for up
to 8 weeks. The CTL clones used in this study have been described
previously.11 All cell lines were regularly screened for
mycoplasma contamination.
Cytotoxicity assay.
CTL clones were tested in duplicate for cytotoxicity in the standard
5-hour chromium release assay, using an effector:target ratio (E:T) of
2:1. Where synthetic peptide was involved, it was added directly to
51Cr-labeled targets and incubated for 1 hour before CTL
addition and remained present throughout the assay. Peptides were
synthesized by Chiron Mimotopes (Chiron Corp, Emeryville, CA) on a 1-mg
scale using Pin-Technology.15 Toxicity testing of all
peptides was performed before use by adding peptide to
51Cr-labeled PHA blasts in the absence of CTL effectors. A
beta scintillation counter (Topcount Microplate; Packard Instrument Co,
Meriden, CT) was used to measure 51Cr levels in assay
supernatant samples. The mean spontaneous lysis for target cells in
culture medium was always less than 20%, and the variation about the
mean specific lysis was less than 10%.
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RESULTS AND DISCUSSION |
Specific inhibition of T cells participating in alloimmune responses is
the ultimate goal of research in transplantation because currently
available immunosuppressive therapy is nonspecific, impairing the
entire immune system. Previous studies of self-MHC-restricted T cells
have shown that analogs of immunogenic peptides, so-called altered
peptide ligands (APLs), may profoundly reduce the magnitude of the
response to the wild-type epitope.16-22 To evaluate the potential of APLs as specific modulators of alloreactivity, a CTL
clonotype, previously characterized by our group, was examined further.
These CTL clones that use identical TCRs display direct alloreactivity
for HLA B*3501 and were isolated from two unrelated individuals.11 Although a variety of cell types that
express this alloantigen are lysed by these clones (including LCLs,
Burkitt's lymphoma cell lines, and a bladder cancer cell line), HLA
B*3501+ PHA-stimulated T-cell blasts are not recognized.
The peptide KPIVVLHGY, derived from human cytochrome P450 IIC 9, 10, or
18, was found to be recognized by these cross-reactive clones when bound to the alloantigen HLA B*3501 on PHA blasts.11
Fifty-seven monosubstituted peptide analogs of KPIVVLHGY were
synthesized in which the potential TCR contact residues at positions 6, 7, and 8 were sequentially replaced with all other genetically coded
amino acids. Because only analogs with significantly less activity than
the parent allodeterminant are potential antagonists of this CTL
clonotype, each analog was first screened for activity as an agonist by
testing for the capacity to sensitize HLA B*3501+ PHA
blasts to lysis. CTL clone JL12, a representative clone expressing this
alloreactive TCR, was used as an effector in a standard
51Cr-release assay against PHA blast target cells from
donor LP (HLA A2, A32, B*3501, B62) after pretreatment with
three different concentrations of each peptide. As shown in
Fig 1, 51 of 57 peptides were either
inactive or required peptide concentrations over 100-fold higher than
the parent peptide for comparable lysis levels by CTL clone JL12. The
only amino acid replacements tolerated well by the clone were
phenylalanine, histidine, methionine, or tyrosine instead of leucine at
position 6, and phenylalanine or tyrosine instead of histidine at
position 7. All amino acid substitutions at position 8 of KPIVVLHGY
resulted in either loss of, or a large reduction in, allospecific lysis
of the PHA blast target cells.
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| Fig 1.
Recognition by CTL clone JL12 of HLA
B*3501+ PHA blasts with the addition of monosubstituted
peptide analogs of KPIVVLHGY. Every one of the 20 genetically coded
amino acids was tested in each of positions 6, 7, and 8 within the
parent sequence KPIVVLHGY. The letter within each graph represents the
parent residue being replaced; the horizontal axis lists the residue
replacing the parent residue. Three different peptide concentrations
were used (200 µmol/L [ ], 2 µmol/L [ ], and 0.02 µmol/L
[ ]) and the E:T ratio was 2:1.
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The 51 monosubstituted peptide analogs of KPIVVLHGY that had
significantly less agonist activity than the parent peptide were then
tested for their ability to inhibit alloreactivity by this CTL
clonotype. LCLs from donor AF (HLA A1, A11, B*3501, B51) were treated with 200 µmol/L, 2 µmol/L, or 0.02 µmol/L of each peptide before being tested for lysis by CTL clone JL12. These LCLs were also
used as targets for the clone without peptide pretreatment, and the
level of allospecific lysis was 33.1%. Data are presented in
Fig 2 as percent inhibition of lysis,
relative to this lysis level observed without exogenous peptide
addition. As shown in Fig 2, many of the peptide analogs showed the
capacity to significantly reduce allospecific lysis below 33.1%. Eight
APLs were powerful competitive antagonists of these CTL clones,
reducing lysis levels of the HLA B*3501+ LCL target cells
by over 50%. In some cases, these antagonist peptides inhibited CTL
lysis most efficiently at 200 µmol/L, the highest concentration
(KPIVVAHGY, KPIVVGHGY, and KPIVVLHNY).
Other APLs (KPIVVQHGY, KPIVVTHGY,
KPIVVVHGY, KPIVVLMGY, KPIVVLHAY), showed
differential effects on the CTL clone, inhibiting lysis most
effectively at 2 µmol/L, but increasing lysis levels at 200 µmol/L
(negative inhibition values are shown as 0% in Fig 2).
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| Fig 2.
Inhibition of anti-HLA B*3501 allospecific lysis by CTL
clone JL12 with peptide analogs of KPIVVLHGY. An HLA
B*3501+ LCL was tested for lysis by CTL clone JL12 after
pretreatment with selected monosubstituted peptide analogs of
KPIVVLHGY. The level of lysis of these LCLs without peptide
pretreatment was 33.1%. The letter within each graph represents the
parent residue being replaced; the horizontal axis lists the residue
replacing the parent residue; the vertical axis displays the percent
inhibition of lysis relative to the level of lysis of the LCL without
exogenous peptide addition. Where peptide increased lysis of the
B*3501+ LCL, a value of 0% inhibition is shown. Three
different peptide concentrations were used (200 µmol/L [], 2 µmol/L [], and 0.02 µmol/L []) and the E:T ratio was
2:1.
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These eight APLs were then tested at six different peptide
concentrations for antagonistic activity using a different CTL clone
(JL20), isolated from the same individual, that shares an identical TCR
with JL12.11 The target cells used were LCLs from donor CS
(HLA A3, A23, B*3501, B44) and these were lysed at 32.7% without peptide addition. As shown in Fig
3, all peptides again showed strong antagonistic effects on the TCR. As
in the earlier experiment, some of the APLs displayed optimal
inhibitory effects at the highest concentration (Fig 3A), while others
blocked lysis most efficiently at 0.2 µmol/L or 2 µmol/L (Fig 3B;
data not shown for the 200-µmol/L peptide level where these APLs
showed agonistic activity).
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| Fig 3.
Antagonism of CTL clone JL20 with eight different APLs.
An LCL from donor CS (HLA A3, A23, B*3501, B44) was tested
for lysis by CTL clone JL12 after pretreatment with a range of
concentrations of synthetic peptide APLs. The data are presented as
percent lysis and percent inhibition of lysis relative to the level of
lysis of the LCL without exogenous peptide addition (32.7%). The E:T
ratio was 2:1.
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The discovery that TCR antagonist peptides can inhibit the function of
T cells specific for conventional self-MHC-restricted antigens has
raised hopes for their clinical application in modulating harmful
immune responses.16-22 Indeed, such novel reagents based on
the structure of autoantigenic epitopes have enjoyed some measure of
success in the treatment of experimental models of
autoimmunity.19-22 The present report reinforces such hopes
in relation to the potential use of APLs as specific therapeutics for
GVHD and allograft rejection in humans by showing, for the first time,
that CTL alloreactivity can be inhibited with analogs of an
allo-MHC-bound peptide ligand. Also supporting this notion is a recent
demonstration that indirect alloreactivity by T helper cells can be
suppressed by TCR antagonism.23 A report from Paul Allen's
laboratory also demonstrated the effectiveness of APLs in inhibiting
the proliferation and activation of alloreactive T helper
cells.24 The relevance of this latter model to human transplantation is less clear, however, because the TCR antagonist peptides were presented by a self-MHC molecule that was coexpressed on
the stimulator cells with the allo-MHC molecule. Nonetheless, this
murine study corroborates our basic observation that direct alloreactivity can be just as susceptible to APL antagonism as self-MHC-restricted T-cell reactivity. This result is surprising given
that recent studies have shown that TCR affinities for
allo-MHC-restricted ligands tend to be higher than for
self-MHC-restricted ligands.25,26
The present report describes the antagonism of a particularly
interesting class of alloreactive T cells, ie, clonotypes that are
preexpanded in healthy individuals due to cross-reactivity with common
environmental stimuli. Although the importance of such T cells in
transplantation is not yet clear, it seems possible that the APLs
defined in this study could ultimately be used clinically to block
these preactivated T-cell populations in appropriate transplant
recipients to modulate either the graft-versus-host or anti-allograft response.
Conclusion.
To achieve long-lasting, antigen-specific unresponsiveness is the
ultimate goal in transplant biology. As the underlying mechanisms that
contribute to allorecognition become more clearly defined, the prospect
of finding effective methods to specifically prevent the clinical
complications associated with alloreactivity is enhanced. The biggest
obstacle to such approaches is likely to be the potential diversity of
the TCR repertoire in the response to alloantigens. However, because
the alloreactive T cells infiltrating human allografts undergoing
rejection often use a highly selected TCR repertoire, it is certainly
possible that specific immunomodulating techniques such as APL
antagonism will find application in human transplantation.
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FOOTNOTES |
Submitted August 10, 1998; accepted October 2, 1998.
Supported by grants from the National Health and Medical Research Council.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Scott R. Burrows, PhD,
Queensland Institute of Medical Research, The Bancroft Centre, 300 Herston Rd, Brisbane 4029, Australia; e-mail: scottB{at}qimr.edu.au.
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Abstract
Introduction
