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CUTTING EDGE |
,§
*
Department of Microbiology and Beirne B. Carter Center for Immunology Research, University of Virginia, Charlottesville, VA 22908;
Department of Chemistry, University of Virginia, Charlottesville, VA 22901;
Department of Immunohematology and Bloodbank, Leiden University Medical Center, Leiden, The Netherlands; and
§
Department of Pathology, University of Virginia, Charlottesville, VA 22908
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptide Ags displayed by class I MHC molecules have been successfully identified either by the construction and screening of cDNA libraries (reviewed in Ref. 9) or the analysis of class I-associated peptides extracted directly from the cell (10, 11). Both approaches employ cytotoxic T cells to screen either transfected cells or peptide fractions, and both have been successfully used to identify mHag (reviewed in Refs. 4 and 8). By using the direct peptide extraction approach in conjunction with HPLC fractionation and mass spectrometry (MS), our groups have successfully identified four human classical mHag (12, 13, 14, 15). Two of these peptides are human male-specific mHag restricted by HLA-B*0702 and HLA-A*0201 and are derived from the product of SMCY, a gene on the Y chromosome. Simpson and coworkers used the cDNA cloning strategy to identify two murine HY Ags, one originating from Smcy (16) and the second derived from another Y chromosomal gene Uty (17). Collectively, these results raise the question of whether additional Y chromosomal genes give rise to HY T cell epitopes.
To address this issue, we have implemented the combination of nanoflow liquid chromatography with electrospray ionization (ESI) on a Fourier transform mass spectrometer (FTMS). This allows detection of peptides at levels as low as 2–10 amol with mass measurement accuracy in the millimass range (18). With the inherent resolution of this instrument, assignment of the charge state to ions is easily accomplished, and coeluting peptides of similar mass are readily resolved (18). In this manuscript, we describe the use of this novel analytical system for identification of an HY Ag restricted by HLA-A*0101 (A1-HY).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CD8+ CTL clone A42 has been shown to specifically recognize HLA-A*0101-positive male cells (19). It was maintained as described previously (19), or by using a rapid expansion protocol (20). The HLA-A*0101+ male (Rp) and female (C1R-A1) B lymphoblastoid cell lines were grown in RPMI 1640 containing 10% FCS and 3 mM L-glutamine.
Extraction and HPLC fractionation of HLA-A*0101-associated peptides
HLA-A*0101 molecules were immunoaffinity purified from male Rp
cells (HLA-A*0101, A*0201, B8, B27), and their associated peptides were
extracted as previously described (11, 12, 13, 14, 15). Iodoacetamide
was omitted from the protease inhibitor mixture to avoid potential
amidocarboxymethylation of free cysteine residues. HLA-A*0101 was
purified by first removing the HLA-B and -C molecules with the mAb
B1.23.2 (21), then depleting the HLA-A2 molecules with mAb
BB7.2 (22), and finally isolating the HLA-A*0101 molecules
with mAb W6/32 (23). Peptides were separated from class I
H chains and ß2-microglobulin by elution in
10% acetic acid and passage through a 5-kDa cutoff filter. One Rp
extract was separated as previously described (15). A
second Rp extract was fractionated on a HAISIL C18 column (2.1 x
40 mm, 5 µm particles, 300 
pore size) (Higgins, Winter Park,
FL) using a linear gradient of 15–60% solvent B for 50 min. Solvent A
was 0.1% TFA (HPLC grade; Applied Biosystems, Foster City, CA) in
NANOpure water (Barnstead, Dubuque, IA), and solvent B was 0.085% TFA
in 60% acetonitrile (HPLC grade; Mallinckrodt, Paris, KY). Fractions
were collected every 40 s at a flow rate of 200 µl/min. Active
fractions were pooled and run through a second round of chromatography
with the identical column and gradient, but using heptafluorobutyric
acid (HFBA) as the ion-pairing agent.
Epitope reconstitution assays
Aliquots of each HPLC fraction were incubated with 2000 51Cr-labeled C1R-A1 target cells for 30 min at 37°C and then tested for their recognition by A42 CTL in a standard chromium release assay (13). Synthetic peptides were assayed using the same protocol, except that HBSS containing 1% BSA and 50 mM HEPES was employed as the medium to avoid cysteinylation of free cysteine residues (15).
Peptide analysis using an on-line effluent splitter and an FTMS
Biologically active second dimension HPLC fractions were analyzed by ESI on an FTMS equipped with nanoflow liquid chromatography and a modified on-line effluent splitter (11, 18, 24). Samples were fractionated using an on-line microcapillary HPLC column at a total flow rate of 825 nl/min. Thirteen-fourteenths of the effluent were deposited into wells of a microtiter plate containing 50 µl of 0.1% acetic acid and reserved for epitope reconstitution assays. The remaining one-fourteenth was directed to the FTMS and analyzed by ESI MS.
Sequence analysis of candidate Ags
Collision-activated dissociation (CAD) mass spectra were recorded on selected peptide candidates using a Finnigan ion trap mass spectrometer (LCQ) equipped with sheathless nanoflow HPLC ESI as previously described (18). Data were acquired by manually switching from MS-only mode to MS/MS mode after the chromatographic elution of a marker peptide. In MS/MS mode, the ion of interest was isolated using a 3.0 atomic mass unit isolation window and fragmented using 35% collision energy.
Synthetic peptides
Peptides were synthesized and cysteinylated as described (15), except the cysteinylation time was 45 min. Oxidation of Met residues to the corresponding sulfoxide was accomplished by incubation with performic acid for 1 min at room temperature. The reaction mixture was diluted with 0.1% acetic acid and purified by reverse phase HPLC. Sequences of all synthetic peptides were confirmed by MS.
Class I MHC peptide binding assays
Quantitative, cell-free peptide binding assays were performed essentially as described (15), except that we used HLA-A*0101 molecules purified from the B lymphoblastoid cell line HAR and the iodinated indicator peptide used had the sequence YTAVVPLVY (25).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HLA-A*0101-associated peptides were purified from male Rp cells
and fractionated by reverse phase HPLC using HFBA as the ion-pairing
reagent. Epitope reconstitution was performed by adding aliquots of
these fractions to the HLA-A*0101+ female target
C1R-A1 and assaying with the A1-HY-specific CTL clone A42. A single
peak of reconstituting activity was observed (data not shown). Active
fractions were pooled and rechromatographed using TFA in place of HFBA,
and, again, a single peak of reconstituting activity was seen (data not
shown). Candidate masses for the HY Ag were identified by an on-line
effluent splitter analysis of these active fractions as previously
described (11, 12, 13, 14), except that an FTMS was employed in
place of a triple quadrupole mass spectrometer instrument.
Identification of candidate peptides was made after plotting the
abundances of ions observed in spectra recorded on peptides from wells
that showed epitope reconstitution activity. The abundances of only
five ions were considered to be similar to the CTL lysis profile (Fig. 1
). Two of these (m/z
669.786+2 and 611.232+2)
were analyzed by CAD on the LCQ instrument, and their sequences were
determined to be MoxTEXYDYPKY (data not shown)
and XVDC*XTEMoxY (Fig. 2A), respectively. X
represents either Ile or Leu, which cannot be differentiated by the
instrument; C* represents a Cys residue covalently modified by the
attachment of a Cys via a disulfide linkage; Mox
represents Met in which the sulfur has been oxidized to a sulfoxide.
When incubated with C1R-A1 targets and CTL clone A42,
XVDC*XTEMoxY reconstituted activity, while
MoxTEXYDYPKY did not (Fig. 3A). These results suggested
that XVDC*XTEMoxY represents the A1-HY
epitope.
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To establish the impact of cysteinylation and methionine oxidation
on CTL recognition of the peptide, several peptides with different
combinations of oxidation and cysteinylation were synthesized and
evaluated for their ability to reconstitute the epitope recognized by
the A42 CTL clone. The peptide with the highest immunological activity
(half-maximal lysis at a concentration of 3.5 pM) contained a
cysteinylated Cys, but a nonoxidized Met (Fig. 3
B).
Oxidation of Met resulted in a 130-fold reduction in immunological
activity in three independent experiments (Fig. 3B and data
not shown). Interestingly, removal of the cysteinylation from the
peptide with a nonoxidized Met had a much more modest impact, ranging
from a 2- to 15-fold reduction in three independent experiments.
However, in peptides with an oxidized Met, removal of the
cysteinylation led to an 
400-fold reduction in biological activity
(Fig. 3B). These results demonstrate that the structure of
the amino acids at P4 and P8 are both important for immunological
activity.
The immunological activity results suggested that the oxidation of Met
in the peptide we had identified was quite deleterious for recognition
by the A42 CTL clone. Consequently, we prepared a second HLA-A*0101
peptide extract from Rp under conditions that minimize the possibility
of oxidation during purification. Analysis of immunologically active
second dimension fractions by FTMS failed to detect an ion of
m/z 611.232+2 corresponding to the
previously identified peptide with an oxidized Met. We then searched
for an ion corresponding to the cysteinylated peptide with a
nonoxidized Met (calculated m/z =
603.244+2) and detected a candidate signal of
m/z 603.254+2. A CAD spectrum recorded
on this ion confirmed the expected sequence (Fig. 2B). We
also searched for ions corresponding to XVDCXTEMY and
XVDCXTEMoxY, but neither was detected. We
conclude that Met oxidation represents an artifact of peptide
extraction in one preparation, and that the true A1-HY epitope is
represented by the sequence XVDC*XTEMY.
The A1-HY epitope is derived from the DFFRY gene encoded on the Y chromosome
A search of the known protein sequence databases generated two
matches for the XVDC*XTEMY sequence. These two sequences, IVDCLTEMY and
IVDSLTEMY, represent amino acid residues 1521–1529 and 1512–1520 of
the genes DFFRY and DFFRX (26, 27),
which are located on the Y and X chromosomes, respectively. Allowing
for the inability to distinguish Ile and Leu on the mass spectrometer,
and the cysteinylation of the cysteine residue at P4, the peptide
identified by MS was an exact match for the sequence from the
DFFRY gene. Support for this was obtained by synthesizing
the four possible variants of the XVDC*XTEMY peptide based on
substitution of either Leu or Ile for X. When spiked into an aliquot of
the naturally processed peptide extract, only the form containing Ile
at P1 and Leu at P5 coeluted with the naturally occurring biological
activity (data not shown). The DFFRX homologue of the
peptide that we had identified differed from the DFFRY
sequence by the substitution of a Ser residue for the cysteinylated
Cys. To provide additional evidence that the sequence we had identified
was the A1-HY Ag, both the Y and X chromosomal homologues were
evaluated for their ability to reconstitute the epitope for the A42 CTL
clone. Recognition of the nonoxidized X homologue required 1500-fold
more peptide than did recognition of the nonoxidized Y peptide,
regardless of whether the latter was cysteinylated (Fig. 4). These observations suppport the
conclusion that the sequence of the A1-HY epitope is IVDC*LTEMY and is
derived from the DFFRY gene.
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and 4, the levels of this
peptide on the cell surface are 10 times lower than those of the
noncysteinylated male form. Thus, the apparently modest difference in
CTL recognition of the noncysteinylated and cysteinylated male peptides
is due to the fact that the latter binds 10-fold less well. Instead,
the A1-HY-specific CTL recognize the male cysteinylated form
substantially better than the noncysteinylated form. This result
provides additional support for designating the cysteinylated male
peptide as the A1-HY epitope. Both the male and female forms of the A1-HY epitope are present at the cell surface
Male cells generally express the protein products of both X and Y
chromosomes (28). Accordingly, the homologous peptide
derived from the DFFRX gene was expected to be present on
the surface of an HLA-A*0101+ male cell. To
evaluate this possibility, synthetic IVDSLTEMY was chromatographed
under the same conditions as the first dimension separation of the
A1-HY+ peptide extract, and its elution point
corresponded to fraction 31 of the first dimension fractionation.
Fractions 29–33 of the Rp peptide extract were screened on the LCQ
instrument for charge states corresponding to the mass of the
homologous X chromosome peptide. An ion of m/z
535.698+2 was present in fraction 33, and its
amino acid sequence (IVDSLTEMY) was confirmed by CAD analysis (data not
shown). Based on coelution of the naturally occurring peptide with a
known amount of synthetic IVDSLTEMY, we calculated the female peptide
to be present at about 10 copies per cell. Similarly, we calculated
that 30 copies of the male peptide are present per B lymphoblastoid
cell line.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This is the first demonstration that DFFRY encodes an HY Ag. Previous studies identified two other Y chromosomal genes, SMCY and Uty, as sources of HY epitopes. These three are among nine genes in the nonrecombining region of the human Y chromosome that are ubiquitously expressed and have homologous X chromosomal counterparts (28). Furthermore, each of these three genes encodes a product of 1100–2500 amino acids and thus may give rise to numerous additional HY epitopes. It remains to be determined whether any of the six remaining genes in this region also give rise to HY Ags.
The identification of A1-HY was facilitated by the development of new MS technology (11, 13, 14). The previously described effluent splitter (11, 12, 13, 14) was modified to deliver a lower flow rate (50 vs 800 nl/min) to the ESI source, and the triple quadrupole mass spectrometer was replaced by the FTMS, which is 1000 times more sensitive (detection limit = 2–10 amol vs 3–5 fmol). Sample consumption is reduced by more than a factor of 10, and masses of Ags present at the single copy per cell level can be determined from <108 cells. The mass measurement accuracy of the FTMS is in the millimass range; thus, coeluting peptides that differ by several hundredths of a mass unit are easily resolved. This greatly reduces the probability that a mass will be eliminated as a candidate because it coelutes with a second peptide of similar mass. The higher resolution of the FTMS makes it possible to assign charge states to ions based on the observed mass separation between 12C and 13C isotope peaks and to eliminate candidates whose molecular mass is outside the range expected for class I-associated peptides. Combined with the on-line effluent splitter device, these improvements allowed us to identify the 611.23+2 ion mass as one of five primary candidates for A1-HY.
The principal advantage of using MS for Ag identification is the direct determination of the structure of the presented peptide. A1-HY was initially found to have a cysteinylated Cys at P4 and an oxidized Met at P8. Cysteinylation is a common feature of class I-associated peptides, including another HY epitope restricted by HLA-A*0201 (15). In that instance, two different T cell clones showed strong (>100-fold) preferential recognition of either the cysteinylated or noncysteinylated peptides. When relative binding affinities are taken into account, the A1-HY CTL clone used here recognizes the cysteinylated male peptide 10–100 fold better than the noncysteinylated form. Because we have not confirmed the presence of the uncysteinylated IVDCLTEMY peptide, it remains to be determined whether this is present on the cell surface or recognized by other A1-HY-specific CTL.
Oxidation of Met in naturally processed class I-associated peptides has not been previously described. The Met residue at P8 in A1-HY is predicted to be accessible to both solvent and the TCR (25, 30). Consistent with this orientation, oxidation led to a substantial reduction in T cell recognition. Although the exact cause of this oxidation is unclear, our results suggest that it is most likely a result of the peptide extraction procedure. Nevertheless, if such oxidation reactions occur under other conditions in tissue, they may give rise to additional epitopes with the potential to be recognized by the immune system.
In sum, this study of the identification of A1-HY provides further insight into modifications that affect T cell recognition, while its origin establishes a new gene that encodes HY epitopes. Such studies continue to be important in establishing the basis for the existence and recognition of human mHag and to their potential use in improving the outcome of transplants between MHC-identical, mHag-mismatched individuals.
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Footnotes |
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2 R.A.P., E.D.F., and J.M.M.d.H. made equal contributions to this work and the order of their listing should be considered arbitrary.
3 Address correspondence and reprint requests to Dr. Victor H. Engelhard, University of Virginia Health Sciences Center, MR4 Box 4012, Charlottesville, VA 22908. E-mail address:
4 Abbreviations used in this paper: mHag, minor histocompatibility Ag; A1-HY, HLA-A*0101-restricted HY Ag; GVHD, graft-vs-host disease; HFBA, heptafluorobutyric acid; MS, mass spectrometry; FTMS, Fourier transformation mass spectrometer; LCQ, ion trap mass spectrometer; CAD, collision activated dissociation; ESI, electrospray ionization; C*, cysteinylated cysteine residue; Mox, oxidized methionine residue.
Received for publication August 27, 1999. Accepted for publication October 25, 1999.
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