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,
*
Department of Microbiology and the Beirne Carter Center for Immunology Research, and
Department of Pathology, University of Virginia, Charlottesville, VA 22908; and
Department of Chemistry, University of Virginia, Charlottesville, VA 22901
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most of these studies examined limited numbers of CTL clones that may not be representative of the bulk alloreactive response. Analysis of large numbers of short term alloreactive CTL specific for either HLA-A*0201 or H-2Dd demonstrated that 80 to 90% recognized epitopes dependent upon the presence of endogenous peptides that are absent or significantly reduced on the Ag-processing mutant T2 (17, 18). Since the HLA-A*0201 molecules on T2 cells are occupied by peptides produced directly in the endoplasmic reticulum (19, 20), this suggests that this fraction of alloreactive CTL directed against this Ag recognizes one or more specific peptides whose expression depends upon the normal Ag-processing pathway. Although such endoplasmic reticulum-derived peptides may not be presented by other MHC molecules (20), the ability of CTL to discriminate murine and human target cells expressing H-2Dd suggests that the majority of alloreactive CTL directed against this Ag are also peptide specific.
In addition to the controversy concerning the recognition of specific peptides, there is also uncertainty regarding how many different peptides are recognized, and whether a small number might be immunodominant. Some studies have suggested that T cells stimulated by a single alloantigen are directed at different peptides presented by that Ag, and among the clones evaluated there is no evidence for two with identical peptide specificity. Other studies have shown that single peptide species were recognized by large fractions of the alloreactive CTL clones that responded to the foreign MHC molecule used as a stimulator (10, 21). In an effort to address some of these discrepancies, we have now evaluated the role of peptides in Ag recognition by short term bulk HLA-A*0201- and HLA-B*0702-reactive CTL lines, which are more representative of the alloreactive T cell repertoire than selected clones. We demonstrate that these alloreactive lines are not only peptide dependent, but are also peptide specific, and that the number of different peptide Ags recognized is very high. These data indicate that individual alloreactive T cells are exquisitely specific for individual peptides presented by MHC molecules, but the collective response is directed at a wide array of different peptides.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bulk alloreactive T cell lines were established by culturing 2 x 106 HLA-A*0201-negative, Ficoll-Hypaque-separated PBLs with 1 x 105 irradiated JY cells (homozygous for HLA-A*0201, HLA-B*0702) in 2 ml of RPMI 1640 supplemented with 10% FBS and 2 mM L-glutamine in 24-well Linbro plates (Flow Laboratories, McLean, VA). After 1 wk, viable cells were harvested and restimulated with fresh irradiated JY cells at a ratio of 20:1 in medium containing 20 U/ml of human rIL-2; 20 U/ml of rIL-2 was also added on day 4 following each restimulation. This protocol was repeated weekly for 3 to 4 wk until HLA-A*0201 specificity was established. Alloreactive CTL lines were also initiated by stimulating 2 x 106 HLA-A*0201- PBLs with 1 x 106 irradiated HLA-A*0201+ PBLs, followed by three or four weekly restimulations using irradiated PBLs from different HLA-A*0201+ donors. All bulk lines were used in assay between 4 and 10 wk.
Generation of alloreactive CTL clones
HLA-A*0201-specific alloreactive bulk CTL lines, 3 to 4 wk of age, were plated in graded numbers (1–1,000 cells/well) in a 96-well U-bottom plates (Flow Laboratories) containing 2,000 irradiated JY cells and 50,000 irradiated PBLs/well, 20 U/ml rIL-2, and 1 µg/ml PHA. On day 7, another 20 U/ml rIL-2 was added. On day 14, cultures were screened for positive growth and tested on HLA-A*0201-positive and -negative targets. HLA-A*0201-specific alloreactive clones were expanded by stimulating 2 x 106 CTLs with 5 x 106 EBV-lymphoblastoid cell lines and 1 x 107 PBLs in 80 ml of RPMI 1640 supplemented with 15% FCS and 20 U/ml rIL-2. On day 5, another 40 ml of medium with the same concentrations of FCS and rIL-2 was added. CTLs were harvested on day 7 and either used immediately or frozen in aliquots for later use.
Extraction of endogenous peptides
HLA-A*0201 molecules were immunoaffinity purified from 2 x 1010 JY cells using mAb BB7.2 as previously described (22), except that 1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) was substituted for Nonidet P-40 in all preparative steps. Peptides were eluted from HLA-A*0201 molecules with 10% acetic acid, pH 2.1, and separated using a 5000-dalton cut-off filter. HLA-B7-associated peptides were extracted using the same protocol, except an HLA-B7-specific Ab ME1-1.2 was used in the immune purification. A portion of the extract corresponding to 5 x 107 cell equivalents was concentrated in a Speed-Vac (Savant Instruments, Farmingdale, NY) to near dryness and resuspended in 100 µl of RPMI 1640 (Life Technologies, Grand Island, NY) containing 5% FCS and 2 mM glutamine (RPMI medium) for in vitro reconstitution assays. In some cases, peptide extracts were fractionated by HPLC on a reverse phase C18 column (7-µm particles, 300-Å pore size, 2.1-mm inner diameter, 3-cm length; Brownlee, Rainin Instruments, Braintree, MA), using an Applied Biosystems (Foster City, CA) model 130a Separations System with a gradient of acetonitrile and 0.1% trifluoroacetic acid. The concentration of acetonitrile was increased from 0 to 9% (0–5 min), 9 to 36% (5–55 min), and 36 to 60% (55–62 min; v/v), and 200-µl fractions were collected every minute. One-half microliter of each fraction, corresponding to 1 x 108 cell equivalents, was diluted into RPMI medium, as described above, for use in in vitro reconstitution assays.
Synthetic peptides
Peptides were synthesized by solid phase F-moc methodology using
Applied Biosystems or Gilson Medical Electronics (Middleton, WI) AMS422
peptide synthesizers. Peptides were purified to >90% homogeneity by
reverse phase HPLC, and their identities were confirmed by mass
spectrometric analysis. For in vitro reconstitution assays, equal
amounts of 18 synthetic peptides corresponding to naturally processed
or other peptides known to bind to HLA-A*0201 (22, 23) or nine
synthetic peptides corresponding to naturally processed or other
peptides known to bind to HLA-B*0702 (24) (Table I
) were mixed in DMSO, and 1 µl was
diluted into 125 µl of RPMI 1640 medium. Assuming the average m.w. of
these peptides to be 1000, the final concentrations of these synthetic
peptide mixtures were 1 and 0.5 µM, respectively, and were equivalent
to the molar quantities of endogenous HLA-A*0201- or
HLA-B*0702-associated peptides extracted from 5 x
107 and 2.5 x 107 JY cells,
respectively.
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HLA-A*0201-positive T2 cells or T2-B7 cells that also express transfected HLA-B*0702 were labeled with 51Cr for 2 h, washed, and then incubated with 250 µl of 300 mM glycine/1% BSA, pH 2.5, for 3 min at room temperature (25). The acid was immediately neutralized with 15 ml of RPMI medium, and the cells were centrifuged at 700 x g for 3 min and resuspended in 1 ml of RPMI medium containing 5 µg/ml brefeldin A to prevent expression of newly synthesized class I molecules. One thousand cells in a final volume of 100 µl of RPMI medium supplemented with 3 µg/ml of human ß2m were pulsed with peptides for 2 h at room temperature. CTLs were added at an E:T ratio of 10, and incubation was continued for 4 h at 37°C before measuring 51Cr release into the supernatant.
Cell surface expression of HLA-A*0201
Untreated or acid-treated T2 cells were incubated with saturating amounts of an HLA-A*0201-specific mAb BB7.2 and fluorescein-conjugated F(ab')2 of sheep anti-mouse IgG. All buffers contained 0.02% azide to prevent new synthesis and expression of HLA-A*0201. The mean log fluorescence was determined by FACS analysis. The value for cells without acid treatment was considered the maximum, while the value for cells stained with secondary Ab only was used as the minimum: % maximal = [(mean fluorescence - minimum)/(maximum - minimum)] x 100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the role of specific class I MHC-associated peptides in
the formation of epitopes recognized by class I MHC-alloreactive CTL,
we developed a system in which the presence and the composition of such
peptides at the cell surface could be regulated.
HLA-A*0201+ T2 cells were briefly incubated at pH 2.5
to denature the MHC complexes. As shown in Figure 1A, acid-treated T2 cells were
no longer recognized by the HLA-A*0201-specific Ab BB7.2. However,
HLA-A*0201 was re-expressed to about 80% of pretreatment levels within
4 h at 37°C. This re-expression did not occur if the cells were
incubated at 25°C or if they were incubated at 37°C in the presence
of brefeldin A. This indicates that re-expression was dependent upon
newly synthesized HLA-A*0201 molecules, while those expressed on the
surface at the time of acid treatment remained denatured, even if
incubated at a temperature that confers stability on peptide-free class
I molecules (6).
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). In
the presence of brefeldin A, peptides, and ß2m, up to
25% of the denatured HLA-A*0201 molecules refolded in 4 h at room
temperature into a conformation recognizable by BB7.2 (Fig. 1B). The incubation at room temperature was critical
for the refolding; if incubated at 37°C immediately after acid
treatment, no increase in HLA-A*0201 expression was observed. However,
molecules that were expressed after refolding at room temperature
remained stable upon shifting the temperature to 37°C, consistent
with their occupancy by added peptide (data not shown). The refolding
of HLA-A*0201 molecules was also dependent on the peptide concentration
and was maximal when the cells were incubated with 0.2 µg/ml of total
peptide (Fig. 1C).
Acid treatment of T2 cells also abolished expression of
HLA-A*0201-dependent epitopes recognized by T cells. We previously
identified a peptide called 1049 that is recognized by a
HLA-A*0201-specific xenogeneic murine CTL clone AHIII12-2 and is
expressed on T2 cells despite their Ag processing defect (26). The
AHIII12-2 clone no longer recognized T2 cells after acid treatment and
incubation in the presence of brefeldin A (Fig. 2). However, when peptide 1049 was
incubated with acid-treated T2 cells at room temperature for 2 h
in the presence of brefeldin A and ß2m, recognition by
AHIII12-2 was restored to normal levels. We obtained similar results
using other defined HLA-A*0201-restricted peptides that were recognized
by a melanoma-specific CTL line (data not shown) or by CTL clones
directed at three other
epitopes.4 It is important to
note that in the absence of brefeldin A, re-expression of these
epitopes to >50% of pre-acid treatment values occurred due
to the processing and the presentation of relevant endogenous peptides
(Fig. 3). Therefore, treatment of cells
with acid and brefeldin A followed by addition of peptides provided us
with the ability to manipulate the composition of peptides associated
with cell surface class I molecules. It also allowed us to
systematically evaluate the dependence of alloreactive CTL on the
expression of endogenous peptides.
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The peptide-dependent refolding of acid-denatured HLA-A*0201
molecules on the surface of T2 cells allowed us to examine the
importance of specific peptides in the formation of epitopes recognized
by alloreactive CTLs. We evaluated two HLA-A*0201-specific alloreactive
CTL lines at 4 wk of age: line MHN anti-JY and line MHN
anti-PBL. At an E:T ratio of 10, both lines gave >60% specific
lysis of C1R-A2.1 cells and >30% specific lysis of T2 (Fig. 4). However, upon acid treatment,
recognition of T2 cells by these CTL was reduced to a level that was
not significantly higher than the background lysis of the
HLA-A*0201-negative cell line C1R. This result strongly suggests that
no significant fraction of these CTL lines is able to recognize a
peptide-independent conformation of this class I MHC molecule
(4).
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) that induced refolding of HLA-A*0201 as
detected by Ab. However, incubation of target cells with this mixture
did not restore recognition by either of the two HLA-A*0201-specific
alloreactive lines (Fig. 5). On the other
hand, if the mixture of all endogenous peptides extracted from
immunoaffinity-purified HLA-A*0201 molecules expressed on JY cells was
used in place of the synthetic peptides, they effectively reconstituted
recognition in a dose-dependent manner. Similarly, the epitopes
recognized by the HLA-B*0702-specific alloreactive CTL line ST
anti-PBL were lost by brief acid extraction of T2-B7 cells and were
restored using a mixture of all endogenous HLA-B*0702 peptides, but not
a mixture of 9 HLA-B*0702 binding peptides (Table I and Fig. 5C). These data indicate that the proper Ag
conformation necessary for recognition by these alloreactive CTL lines
is not conferred by mixtures of small numbers of structurally distinct
MHC-associated peptides, but instead requires the presence of other
peptides that are present in a more complex mixture. This result
suggests that alloreactive T cell recognition involves specific
contacts with these peptides.
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To characterize the endogenous peptides recognized by the
alloreactive T cells, HLA-A*0201-associated peptides extracted from JY
cells were fractionated by reverse phase HPLC, and a portion of each
fraction was evaluated for the ability to restore CTL recognition of
acid-treated target cells. Contrary to the reconstitution of
recognition by unfractionated endogenous peptides shown in Figure 5, HPLC-fractionated peptides were no longer able to reconstitute
recognition by any of the bulk CTL lines, even though the quantities
added to the target cells were the same (Fig. 6, A–C). This was true
regardless of whether the line was developed using JY cells or PBLs as
stimulators (data not shown). Apparent minor "peaks" that could be
discerned in some assays (Fig. 6C) were not
reproducible from assay to assay. The inability of HPLC-separated
peptides to reconstitute recognition by alloreactive CTL lines was not
due to the loss of specific peptide species, because repooling the HPLC
fractions led to the recovery of T cell recognition (Fig. 6D). On the other hand, recognition by three
HLA-A*0201-specific alloreactive clones derived from the CTL line MHN
anti-JY was reconstituted by individual HPLC fractions that eluted
at different positions in the gradient (Fig. 7). When the fractions that contained
this reconstituting activity for CTL clone MHN24 were omitted, and the
remaining fractions were pooled, this pool was unable to reconstitute
recognition for the CTL clone (Fig. 6D). This
demonstrates that each clone is exquisitely peptide specific, and that
each recognizes a distinct peptide in the mixture extracted from
HLA-A*0201. These data suggest that bulk alloreactive CTL lines consist
of a broad spectrum of T cells with distinct peptide epitope
specificities, such that no specificity is sufficiently well
represented to give a discernible peak of reconstituting activity when
only one or a few appropriate peptides are present in an HPLC
fraction (27).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies extended observations by several other groups who
demonstrated reconstitution of epitopes for small numbers of class
I-specific CTL clones (8, 9, 10, 11, 12, 13). Although it has been reported that in
some instances alloreactivity may not depend on the recognition of a
precise self peptide but on a class I epitope influenced by the peptide
(15), we believe that this is an exception rather than the rule, based
on our data from three alloreactive bulk CTL lines in which the
majority of alloreactive T cells are specific for endogenous peptides.
Our results stand in strong contrast to a recent report by Smith et al.
(4), who reported that up to 20% of primary and essentially all
secondary alloreactive CTL primed by skin grafting were able to
recognize H-2Kb molecules expressed in T2 cells even
after acid treatment and concluded that these CTL recognized a
conformation of the class I molecule that was independent of peptides.
One possible explanation for the discrepancy between their results and
those of many other groups, including our own, may lie in their
observation that the generation of peptide-independent CTL was enhanced
by maneuvers that are known to select for CD8-independent, and thus
high avidity, CTL (28, 29). Such CTL may be able to recognize a
residual level of native class I MHC molecules after acid treatment.
However, it should also be noted that this group apparently did not
prevent re-expression of new class I molecules on the acid-treated
cells by the use of brefeldin A. As shown convincingly here (Fig. 3),
re-expression of class I molecules during the time course of a CTL
assay in the absence of brefeldin A is substantial and more than
sufficient to allow CTL recognition.
A second issue raised by this study is the complexity of the alloreactive epitopes. It is possible to easily discern up to 11 peaks of reconstituting activity in short term bulk CTL lines reactive with human melanoma cells (30, 31). Therefore, the failure to see any significant peaks of reconstituting activity in HPLC-fractionated peptide extracts for the alloreactive CTL lines examined in the present study suggests that the number of distinct Ags that they recognize is substantially higher than this (30, 31). Our results contrast with other recent studies that have shown that the 2C peptide presented by H-2Ld and the Qdm peptide presented by Qa-1b are immunodominant in alloreactive responses to these class I MHC molecules (10, 21). In addition, we observed that the 1049 peptide recognized by the clone AHIII12-2 is also recognized by a substantial fraction of HLA-A*0201-specific murine anti-human xenoreactive CTL (26). These observations have raised the alternative possibility that a single peptide with high abundance can create a high determinant density on APC that activates a large number of T cell clones. However, the expression of H-2Ld on cells is low, and the other two systems cited are not typical alloantigenic responses. It remains to be determined whether the immunodominance of a small number of peptides reflects some unique features of H-2Ld or Qa-1b molecules or is a consequence of the relatively weak xenoreactive response (32, 33). In any case, our estimate of the complexity of the epitopes recognized in more conventional alloantigenic responses to HLA-A*0201 and HLA-B*0702 is consistent with the idea that the strength of these responses is a result of recognition of multiple unique peptide-MHC complexes.
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Footnotes |
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2 Present address: Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff, Wales CF4 4XX.
3 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Department of Microbiology and the Beirne Carter Center for Immunology Research, Charlottesville, VA 22908.
4 C. J. Luckey, G. M. King, B. F. Maier, V. L. Crotzer, J. Shabanowitz, D. F. Hunt, and V. H. Engelhard. Proteasomes can either generate or destroy MHC class I epitopes: evidence for non-proteasomal epitope generation in the cytosol. Submitted for publication.
Received for publication May 15, 1997. Accepted for publication October 10, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and 2 domains. B. Dupont, ed. In Immunology of HLA Vol. 2:140. Springer-Verlag, New York.
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) or without (
) a mixture of
HLA-A*0201 binding synthetic peptides, as listed in Table I
), or with the xenogeneic
peptide epitope 1049 (ALWGFFPVL; •). The peptide 1049-specific CTL
clone AHIII12-2 was added, and the plate was further incubated at
37°C for an additional 4 h before assessing 51Cr
release. Untreated T2 cells (
) or endogenous peptides (
, Amounts corresponding to 2 x 108
cell equivalents from each of HPLC fractions 1 to 60 were pooled
together, vacuum centrifuged to a volume of 50 µl, and then used in a
reconstitution experiment analogous to that described for A
through C using CTL line MHN anti-JY. A relative peptide
concentration of 1 corresponds to 1 x 108 cell
equivalents. •, An identical reconstitution assay was conducted
except that CTL clone MHN24 was used instead of CTL line MHN
anti-JY, and fractions 16 and 17, which contain the sensitizing
activity for this clone (see A), were omitted when
the HPLC fractions were pooled and concentrated.