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Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and
The Jackson Laboratory, Bar Harbor, ME 04609
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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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The sources of H Ags and the mechanisms by which H Ag polymorphisms affect the T cell response have been debated for decades but are now beginning to be revealed. The mitochondrial ND1 protein was the first H gene product to be identified, from which a N-formylated peptide was presented by the nonclassical H2-M3 MHC class Ib molecule to CD8+ T cells (6). Different alleles of the ND1 protein differed by a single amino acid substitution within this peptide sequence and were recognized bidirectionally by T cells in appropriate donor/host strain combinations. Since then, two other mitochondrial proteins, COI and ATPase 6, have been found to be sources of peptides presented by H2-M3 and rat RT1a molecules, respectively (7, 8). By contrast, the unidirectional female anti-male CD8+ T cell response (anti-HY) was recently shown to be specific for peptide/MHC complexes derived from the nuclear Y chromosome-encoded Smcy or the Uty genes (9, 10, 11). Again, multiple amino acid substitutions within the antigenic peptides in the corresponding female Smcx and Utx homologues presumably allowed the male peptides to be recognized as nonself. Furthermore, the HY-encoding genes are ubiquitously expressed, and their gene products are immunogenic in mouse, rat, and primates (12). Thus, identifying the precursor source proteins of H Ags in one species may also be relvant to H Ags in other species as well.
The relative simplicity of the mitochondrial and the Y chromosomes led to the identification of these H loci and their gene products. In contrast, the autosomal H loci have been rather difficult to identify by traditional positional cloning approaches due to the high gene density of the autosomal chromosomes. As a consequence, the role of polymorphic autosomal H loci in tissue rejection reactions has been difficult to elucidate. For example, several intriguing autosomal Ags have been detected in the MHC-matched C57BL/6 anti-BALB.B response, with hierarchical immunogenicity among themselves and relative to the sex-linked HY loci (13, 14, 15, 16, 17). Possible mechanisms that could account for their immunodominance include substitutions in antigenic peptide sequences and/or in the relative abundance of peptide/MHC expressed in the donor strain. Alternatively, homologous peptides in the host strain could cause differences in frequency of the responding T cell precursors by influencing the development of the T cell repertoire (18, 19, 20, 21). To test these hypotheses and to understand how genetic polymorphisms yield immunogenic peptide/MHC complexes, it is necessary to identify the H peptides, and their precursor proteins in the donor strain, and to determine their relationship to the corresponding host homologues.
We recently used a novel expression cloning strategy to define the autosomal H13 locus at the molecular level (22, 23, 24, 25). Here we apply this strategy to define a new autosomal H locus defined by C57BL/6 anti-BALB.B CTLs, designated H60.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All the indicated inbred mouse strains were obtained from or bred at The Jackson Laboratory (Bar Harbor, ME). The BALB.B and (BALB/c x BALB.B)F1 mice were bred in the animal care facility at the University of California, Berkeley, CA, from The Jackson Laboratory stocks.
Cell lines
Cell lines were maintained in RPMI 1640 medium (Life
Technologies, Gaithersburg, MD) supplemented with 2 mM glutamine, 1 mM
pyruvate, 50 µM ß-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 10% FBS (HyClone, Ogden, UT) or in DMEM supplemented
as described (26). LMtk- (C3H, H-2k)
and its MHC-expressing transfectant derivatives have been described
previously (27). EL4-B7 (H-2b), RMA (H-2b), and
its TAP- derivative RMA/S cell lines were obtained
from Drs. J Allison and D. Raulet (University of California, Berkeley,
CA). To generate anti-BALB.B or anti-CXB-K-specific CTL, B6
mice were primed i.p. with 2 x 107 splenocytes from
H2b-matched BALB.B or CXB-K/By recombinant inbred (RI)
mice. After 7 to 14 days, primed responder cells were harvested and
restimulated in mixed lymphocyte culture (MLC) with 5 x
106 200-Gy-irradiated CXB-K cells in modified DMEM medium
supplemented with 10 to 30 U/ml rIL-2, using established conditions
(28). For restimulation in vitro, 4 x 106 cells from
the mixed lymphocyte cultures were cocultured with 5 x
106 200-Gy-irradiated B6 spleen cells pulsed with 10 nM
LYL8 peptide for 30 min at 37°C and then washed once in DMEM
medium. All CTL lines were maintained by weekly stimulation with BALB.B
spleen cells and 10 to 30 U/ml rIL-2, using established conditions
(29). The lacZ-inducible T cell hybrid BCZ39.84 was
generated by fusing the anti-BALB.B CTL line with the BWZ.36/CD8
fusion partner, as described (30).
T cell activation assays
T cell responses specific for peptide/MHC were measured by the production of ß-galactosidase (lacZ) activity in the T cell hybrids (30, 31). T cell hybrids (3–10 x 104) were cocultured overnight with APC (2–5 x 104) either expressing the Ag endogenously, transfected with Ag cDNAs or with exogenous peptides in 96-well plates. The peptide/MHC-induced T cell response was assayed as lacZ activity using the substrate chlorophenol red ß-galactoside (CPRG), as described (30). The conversion of CPRG to chlorophenol red was measured at 595 nm and 655 nm as a reference wavelength with a 96-well microplate reader (Bio-Rad, Richmond, CA). Data show the mean absorbance of replicate cultures and are representative of at least three independent experiments. For detection of cytolytic activity, 4-h 51Cr release assays were performed. Peptides were tested using 51Cr-labeled TAP-deficient RMA/S cells that were incubated with 10 nM LYL8 (H60) or with the VSV (RGYVYQGL) (32), at 30 min at 37°C, and washed twice to remove unbound peptide. Effector cells were then added to target cells in V-bottom plates at varied E:T cell ratios. Percent specific lysis, calculated from the amount of 51Cr released into the culture supernatant, is shown as the mean of triplicate cultures.
cDNA library and expression screens
A unidirectional cDNA library was constructed (Superscript
Choice System, Life Technologies) using poly(A)+ mRNA from
Con A-stimulated (BALB.B x BALB/c)F1 spleen cells in
the BstXI/NotI sites of the mammalian expression
vector pcDNA1 (Invitrogen, San Diego, CA) (23, 33). The cDNAs were
screened by transforming competent bacteria with recombinant plasmids
and culturing in pools of 
30 to 100 cfu in 96-well U-bottom plates.
Aliquots of cDNA, prepared by the alkali lysis method directly in the
96-well plates (34), were transiently transfected into 3 x
104 LMtk- cells cotransfected with the
relevant MHC Class I cDNA (10 ng/ml) and B7-2 cDNA (5 ng/ml). Two days
later, 10 x 104 BCZ39.84 T cells were added per well
and cocultured overnight. Positive pools were identified by adding CPRG
and scoring pools with above background absorbance. The plasmid
encoding the antigenic activity was identified by repeating the screen
with individual colonies obtained from the positive cDNA pool. The
H60 cDNA sequence is available from the NCBI GenBank with
the accession No. AF084643.
Expression constructs and peptides
Deletion constructs were generated by amplifying DNA fragments
of the 28.64 plasmid with a vector-specific T7 forward primer
(5'-AATACGACTCACTATAG-3') and three reverse primers R1
(5'-AGCAGTAGTGTGAAATCTCTTTAC), R2 (5'-GTATCGTAGATGTTTTATCCACTT), or
R3 (5'-TTCATTGATGGATTCTGGGCCATC), using Pfu polymerase (Stratagene, La
Jolla, CA). PCR fragments were digested with BamHI in the 5'
flanking region of the vector and cloned into the
BamHI/EcoRV sites of pcDNA1. Minigene constructs
MLYL8 (MLTFNYRNL), MIFL8 (MILSLFILL), and MFFI8 (MFIDGFWAI) shown in
Figure 4
were prepared using complementary oligonucleotides
corresponding to the indicated sequences. The synthetic peptides
LTFNYRNL (LYL8), SIINFEKL (SL8) (35), RGYVYQGL (VSV) (32), ANYDFINV
(AFNV8) (36), and SLVELTSL (SEL8) (33) were prepared using solid phase
F-moc chemistry on the ABI Model 433 synthesizer (Perkin Elmer,
Foster City, CA), purified by HPLC, and confirmed by mass spectrometry.
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Southern and Northern blots were prepared according to
established procedures (34). For Southern blots, genomic DNA from the
indicated strains were either purchased from The Jackson Laboratories
DNA resource or prepared from mouse liver. Genomic DNA was digested
with BamHI or XbaI before electrophoresis and
transferred to nylon membranes (Zeta-bind, Bio-Rad). Following DNA
cross-linking to the membranes by UV irradiation (Stratalinker;
Stratagene), the blot was probed with 32P-labeled DNA
inserts of 28.64 or 22.26 plasmids containing the antigenic peptide,
washed at 0.1x SSC at 60°C, and exposed for 24 to 48 h before
autoradiography. For Northern blots, total RNA from resting or Con A +
LPS-activated spleen cells was fractionated, transferred to nylon
membranes, and probed with 32P-labeled 22.26 cDNA fragment,
washed, and autoradiographed, as above. For simple sequence length
polymorphism (SSLP) analysis, splenic genomic DNA was amplified by PCR
according to standard procedures (37), using the primer pairs for
D10Mit2 (5'-CTGCTCACAACCCATTCCTT-3') and
(5'-GTTCATTTGAGGCACAAGCA-3') purchased from Research Genetics
(Huntsville, AL). PCR products were run on 3% agarose gels and
visualized with a UV transilluminator, following ethidium
bromide (EtBr) staining. For mapping of the H60 gene,
a single strand conformational polymorphism (SSCP) that distinguished
parental B6 and SPRET/Ei strains was used to type The Jackson
Laboratory BSB panel of (C57BL/6 x SPRET/Ei)F1 x
C57BL/6 DNAs (38). PCR primer pairs H60-F
(5'-GTGTGATGACGATTTGTTGAG-3') and H60-R
(5'-ATTGATGGATTCTGGGCCATC-3') amplified a 195-bp genomic DNA
fragment from the 3' region of the 28–64 cDNA. PCR amplification was
performed in the presence of [-32P]dCTP (Amersham,
Arlington Heights, IL), and the DNA samples were heat denatured
and placed on ice before gel electrophoresis. MDE gels were run
according to the manufacturer’s (FMC Bioproducts, Natick, MA)
recommended protocols, and the SSCP was visualized by autoradiography.
MHC binding assays
The synthetic LYL8 peptide was compared with AFNV8, SEL8, and SL8 peptides for its Kb-stabilization ability using RMA/S cells. RMA/S cells were first incubated overnight at 31°C to stabilize the "empty" Kb molecules and were added to varying concentration of the peptides in a 96-well U-bottom plate. The plate was incubated at 31°C for 30 min, allowing peptides to bind to previously stabilized Kb molecules. After this incubation period, unbound peptides were washed off. The cells were then incubated at 37°C for 4 to 5 h to cause dissociation of Kb molecules that remained empty or were weakly bound to the peptide. The residual Kb was measured by staining the cells with the anti-Kb mAbs Y3 or 5F1 and using flow cytometry. In the second assay, the ability of different peptides to compete with the SL8 peptide for binding to Kb MHC was measured using the recently described Ab, 25D1.16 (a kind gift of Drs. A. Porgador and R.N. Germain, National Institutes of Health, Bethesda, MD), which recognizes the SL8/Kb complex (39). To RMA/S cells, previously incubated at 31°C overnight, the SL8 peptide (50 nM) was added in the absence or presence of varying concentrations of each competitor peptide. Peptide-cell mixtures were incubated at 31°C for 4 to 5h, washed, and then stained with 25D1.16 ascites (1:500) to quantitate the relative levels of the same SL8/Kb complex. The decrease in expression of SL8/Kb complexes is a measure of Kb MHC binding by the competitor peptides.
Extraction and HPLC analysis of naturally processed peptides
Total acid soluble peptide pool from C3 cells was extracted as described (40, 41). Briefly, 1.5 to 3 x 108 C3 cells were washed with PBS and extracted with 1 ml of 1% trifluoroacetic acid (TFA) with boiling for 5 min. An irrelevant 17-mer peptide was added at 1 µM concentration to the extract as a carrier to prevent nonspecific losses. Cellular debris was removed by centrifugation, and the extract was fractionated by HPLC after filtration through a 10-kDa Millipore filter to remove large m.w. species. A narrow bore reverse phase C18 column (Vydac, 2.1 x 250 mm, 5 µm) was run in 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B). Flow rate was maintained at 0.35 ml/min and seven drop fractions were collected, dried in a vacuum centrifuge, and resuspended in 50 µl PBS + 10% DMSO. Serial dilutions of each fraction were assayed for stimulating BCZ39.84 T cells with Kb-L cells as APC, in a total volume of 200 µl, as described above. Mock injections with sample buffer alone were performed before each extract sample, using the same column and identical run conditions to demonstrate absence of cross-contamination between samples. The fractions from mock injections were assayed in the same experiment, using the same APC and T cells in parallel with fractions from the cell extracts and synthetic peptide standards. The minimal LYL8 peptide concentration required for BCZ 39.84 activation was typically 1 to 10 pM.
The abundance of naturally processed peptide was estimated by
comparison with the synthetic LYL8 standard curve and taking procedural
losses into account. Peptide loss during extraction was estimated by
spiking the H60-negative EL-4 cells with known amounts of LYL8 and
determining its recovery after TFA extraction and ultrafiltration by
comparison with a synthetic LYL8 standard curve run in parallel (e.g.,
see Figure 4
D). Likewise, known amounts of LYL8 peptide were
injected into the HPLC, and peptide recovery in the active fractions
was determined. In three independent measurements, the LYL8 activity
recovered after extraction and ultrafiltration was 68.7 ± 8.8%
(mean ± SD) and after HPLC fractionation, 67.8 ± 11%. The
overall recovery was therefore judged to be 47% and was used to
extrapolate the peptide activity in HPLC fractionated cell extracts to
copies of LYL8 peptide/cell.
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Results |
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The CD8+ BCZ39.84 T cell hybrid was obtained from B6
(H-2b) mice immunized with spleen cells from the MHC
identical BALB.B mice. The BCZ39.84 T cells responded to BALB.B spleen
cells but not to the MHC congenic BALB/c (H-2d) nor to the
host B6 spleen cells (Fig. 1A). The Ag expression was
restricted by the Kb MHC class I molecule because the
BCZ39.84 response was inhibited by anti-Kb (Y3), but
not anti-Db (B22.249.R1) MHC class I or by
anti-Ab (28.16.8S) MHC class II Abs (Fig. 1B). The BCZ39.84 T cell thus defined a polymorphic BALB Ag
presented by the Kb MHC molecule.
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A). The
screen was repeated with individual plasmids isolated from this cDNA
pool, and one of the positive clones, termed 28.64, was selected for
further analysis (Fig. 2B). The plasmid DNA from clone 28.64
and from another similarly isolated clone, 22.26, allowed generation of
the BCZ39.84 ligand, in a dose-dependent manner, but only in recipient
cells that expressed the Kb but not the Db MHC
molecule (Fig. 2, C and D). As shown below, both
28.64 and 22.26 clones encoded the same polypeptide sequence, which was
thus a candidate precursor of the peptide/Kb complex
recognized by the BCZ39.84 T cells.
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To determine whether the BCZ39.84 T cells detected a polymorphic
H locus represented by the isolated cDNAs, we compared the
host B6 and donor BALB DNA in a Southern blot analysis. A distinct set
of bands was detected by the 22.26 cDNA probe in
BamHI-digested B6 and BALB.B DNAs (Fig. 3A). Neither the strong (4.7,
7.2 kb) nor weakly (9 kb) hybridizing bands in the BALB.B DNA
matched the single 5-kb band in the B6 DNA, demonstrating that the cDNA
clone did detect a restriction enzyme site polymorphism among these two
strains. The corresponding transcripts were then analyzed by Northern
blot analysis of total splenic RNA (Fig. 3B). With the 22.26
probe, three different transcripts (approximately 1.0, 1.5, and 4.6 kb)
were detected in the BALB.B splenic blasts, but only the single 5-kb
transcript was found in the resting BALB.B spleen. In contrast,
transcription of this locus was undetectable in either resting or
blasted B6 spleen cells. As a positive control, the same blot was
stripped and rehybridized with the recently discovered H13
cDNA probe (25). As expected for H13, both the B6 and the
BALB spleen cells expressed these transcripts, demonstrating that the
RNA samples were intact. We conclude that the 22.26 cDNA defined a
polymorphic H locus that was differentially transcribed in
the B6 and the BALB.B strains.
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C). Comparison of the 28.64 and 22.26 sequences showed that
both clones were identical in their coding sequence but differed in the
length of their 5' untranslated sequence. Because an in-frame
termination codon was not found at the 3' end, it is likely that these
cDNA clones represent partial transcripts encoding 244 amino
acids of a protein truncated at its C terminus. The relationship of the
cDNAs and the putative polypeptides encoded by the three transcripts
observed in the Northern blot is not yet known. Interestingly, a search
of the nucleotide or the predicted protein sequence in the NCBI
GenBank did not yield any significant matches in the nonredundant
or the EST databases, demonstrating that these cDNAs represent a novel
gene. However, a putative endoplasmic reticulum (ER)
translocation signal sequence (aa 1–29), a transmembrane region (aa
213–241), and seven potential N-linked glycosylation sites were
predicted by the Prosite program, indicating that this antigenic
precursor polypeptide could be a transmembrane protein. We conclude
that these cDNAs defined a polymorphic H locus, encoding a
previously unknown, possibly transmembrane protein, whose first
described function is to serve as a precursor for an antigenic
peptide/Kb MHC complex. Identification of the antigenic peptide within the cDNA
The minimal antigenic peptide within the cDNA clone was defined by
deletional analysis. The vector encoded 5' forward primer, and three
reverse primers were used to generate 3' deletions of the 22.26 cDNA by
DNA amplification (Fig. 4A).
All three 3' deletion constructs were highly active in generating the
BCZ39.84 ligand in transiently transfected cells (Fig. 4B).
The antigenic activity was therefore located within the first 530 nt
that encoded three peptides (see Figure 3C,
ILSLFILL,
LTFNYRNL, and
FIDGFWAI) with the
Kb binding motif, xxxx[F,Y]xx[I,V,L,M] (42). Minigene
constructs with the oligonucleotide sequences encoding each of these
octapeptides were transfected into Kb-expressing recipient
cells and tested for stimulation of BCZ39.84 T cell. Among these, only
one construct, encoding the peptide
M-LTFNYRNL
(referred to as LYL8) was active (Fig. 4C). That the LYL8
coding sequence itself, without the translation initiator methionine,
was sufficient to generate the BCZ39.84 stimulating activity was
confirmed with the synthetic LYL8 peptide. In an exogenous assay with
Kb-L cells as APC, the LYL8 peptide stimulated the BCZ39.84
T cells at <10 picomolar concentration (Fig. 4D). None of
the other Kb-restricted T cells tested, e.g., OVA257–264
(SL8)-specific, B3Z, or the H4b-specific T cells (not
shown) were stimulated by the LYL8 peptide at even 10,000-fold higher
concentration. We conclude that the antigenic ligand for the BCZ39.84 T
cells was defined by the octapeptide LYL8/Kb complex.
Natural abundance and Kb binding activity of the LYL8 peptide
It has been suggested that the immunodominance of H
peptides could be due to their high abundance in the donor tissues or
due to their ability to form stable complexes with the MHC molecules
relative to other peptides (13). To test these hypotheses, we analyzed
the naturally processed LYL8 peptide in cell extracts. Despite being
unrelated in their background genes, spleen cells from either BALB.B or
129/J strains stimulate BCZ39.84 T cells (Fig. 1, and data not shown).
Attempts to extract the antigenic peptide from the BCZ39.84-stimulating
spleen cells of BALB.B or 129/J strains were however unsuccessful,
suggesting that the LYL8 peptide was expressed in spleen cells at a low
level. But in the extract of the pre-B cell line, C3, derived from the
(B10 x 129/J)F1 mice, a single peak of
BCZ39.84-stimulating activity was found after HPLC fractionation (Fig. 5A). This single peak of
activity in the C3 extract precisely matched the retention time of the
synthetic LYL8 peptide, strongly suggesting that the two were identical
(Fig. 5B). Furthermore, by comparing the activity of the
recovered natural peptide with synthetic LYL8 peptide, and taking
procedural losses into account (see Materials and Methods),
the natural abundance of the LYL8 peptide in three independent extracts
of 263, 150, and 232 x 106 C3 cells was estimated to
be 5.2, 6.8, and 14.6 copies/cell or an average of 9 copies/cell. We
conclude that the synthetic LYL8 peptide coelutes with its naturally
processed analogue and is expressed at the very low end of the
abundance scale of peptide/MHC complexes.
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, A and B).
However, this standard assay is sensitive to the influence of the
peptide sequence on the conformational epitope recognized by the
anti-MHC mAb (43, 44). To obviate this potential artifact, we also
measured the ability of peptides to compete with SL8 for Kb
binding as assessed by expression of the SL8/Kb epitope on
the RMA/S cells using the recently described mAb 25D1.16 (39). As shown
in Figure 6C, the expression of the SL8/Kb
complex was specifically detected by the mAb 25D1.16 and was inversely
proportional to the concentration of the competing peptides.
Interestingly, superimposible inhibition curves were obtained with each
of the three Kb binding peptides. This result indicates
that, despite apparent differences in its ability to generate the Y3 or
5F1 epitopes, the LYL8 peptide was comparable to the SEL8 (33) and
AFNV8 (36) peptides in its ability to compete with SL8 for binding
Kb MHC. We therefore infer that, among the peptides tested,
the Kb binding capacity of the LYL8 H peptide was
indistinguishable from other foreign (AFNV8) or alloreactive (SEL8)
peptides.
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To define the chromosomal location of this H locus, we
used The Jackson Laboratory mapping panel, which consists of 94
(C57BL/6 x SPRET/Ei)F1 x C57BL/6 backcross DNAs
(38). Oligonucleotide PCR primers from the 3' end of 28.64 cDNA were
designed to amplify a 195-bp region, and sequence polymorphisms were
detected by SSCP analysis (see Materials and Methods). The
results summarized in Figure 7A showed that the 28.64 cDNA
corresponds to a gene on mouse chromosome 10 that cosegregates with the
D10Mit2 marker located 6.4 cM proximal to
D10Hun15 and 7.5 cM distal to D10Hun14. None of
the known minor H loci is consistent with this chromosomal position,
with the possible exception of one linked to the male-sterile mutation
(mshi) (45). The mshi locus can, however, be
excluded because it was detected in H-2d rather than the
H-2b mice analyzed here and because polymorphisms at this
locus are detected only by skin grafts onto mutant mice as opposed to
splenic expression of H60 shown here. Furthermore, none of B6.C
bilineal congenic strains used to characterize unmapped minor H Ags are
consistent with this map position (4, 46). The BCZ39.84 antigenic cDNA
therefore defines a new H locus we termed H60.
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B). This strain distribution pattern
was also observed by simple sequence length polymorphism analysis of
the RI strains using PCR primers that amplify the cosegregating
chromosome 10 locus D10Mit2 (data not shown). This analysis
further confirms the genetic polymorphism of the H60 locus
and clearly showed that the CXB-K was the only RI strain among those
tested that carried the BALB alleles at the D10Mit2 as well
as the H60 loci. The LYL8 epitope is dominant among B6 anti-BALB.B CTL
To determine the relative immunogenicity of the H60
locus, and whether any of the previously described B6 anti-BALB.B
CTL activities could be attributed to H60, bulk CTL lines
were generated by priming B6 mice with BALB.B spleen cells. The CTL
lines were tested for their lytic specificity using RMA/S cells loaded
with the LYL8 peptide or with the Kb binding VSV peptide
(32), as a negative control, as well as splenic blasts from B6, BALB.B,
and the same panel of CXB RI strains as above (Fig. 7C).
After the first wk of culture, high levels of anti-LYL8 (H60)
peptide-specific lytic activity was detected, with the bulk CTLs using
RMA/S cells as targets. Furthermore, these CTLs preferentially lysed
the BALB.B, CXB-G, and CXB-K targets but lysed the CXB-I, CXB-J, and
self-B6 weakly or not at all. Previous analysis of the B6
anti-BALB.B CTLs have shown that the CXB-K strain expresses the
CTT-1 and CTT-5 Ags (13). To determine whether either of these two Ags
corresponded to the H60 Ag, the LYL8-specific CTLs were further
enriched by restimulating them for another 2 wk with LYL8-pulsed B6
cells. Again, the LYL8-pulsed RMA/S cells, as well as the BALB.B and
CXB-K target cells, were specifically lysed, and no lysis of self B6 or
the other four CXB RI cells was observed. That only the BALB.B and
CXB-K cells expressed the H60 Ag was further confirmed by their ability
to stimulate the monoclonal LYL8/Kb-specific BCZ39.84 T
cell hybridoma (Fig. 7C). In an independent immunization of
B6 mice with BALB.B spleen cells, T cell hybrids were derived, and five
of seven Kb-restricted hybrids were found to recognize the
LYL8/Kb complex (data not shown). These results demonstrate
that the anti-LYL8 specificity is predominant among primary B6
anti-BALB.B CTLs and that, among the RI strains tested, only the
CXB-K strain expresses the LYL8/Kb complex. Taken together
with the previous analysis of the same RI strains by Wettstein and
colleagues, our data suggest that their CTT-5 Ag, which is also
restricted by the Kb MHC, is in fact the LYL8 peptide
derived from the H60 locus described here.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sources of minor histocompatibility Ags
After decades of uncertainty, the identity of T cell-defined minor H loci is now being revealed. Among the available methods, genetic strategies, and in particular the expression-cloning strategies, are proving to be rather successful in providing not only the identity of the antigenic peptide but also, as an essential tool, the cDNA clone of the protein precursor as well (6, 7, 8, 9, 11, 25). The availability of the precursor gene then allows a genetic analysis of the H locus, which provides the molecular explanation of their polymorphic behavior. By contrast, the sequence of the antigenic peptide, obtained after its biochemical purification, does not necessarily yield the identity of its donor protein if no match is found in the sequence data bases (47, 48).
As sources of H peptides, all four known sex-linked and autosomal H loci encode novel gene products. Both the Y chromosome-encoded Smcy and Uty genes, based upon their sequence motifs, are likely to be located in the nucleus. Sequence analysis of the two autosomal H Ags, H13 and H60, suggests that these are likely to be membrane-bound proteins. These cellular locations are, however, also shared by other precursors of MHC class I binding peptides, such as the influenza nuclear protein or the surface hemagglutinin glycoprotein (49). Given that no intracellular compartment is excluded as a source of antigenic peptides, the finding that a variety of different proteins serve as sources of antigenic H peptides extends and reinforces this notion to minor histocompatibility Ags as well. The high likelihood that the yet unknown normal cellular functions of the H proteins will also vary suggests that H precursors will not share any functional characteristics either. However, whether the H proteins and their processed peptide/MHC complexes are distinguished by their distribution among different tissues, particularly the professional APCs and/or different target tissues, remains to be explored. Furthermore, the existence and immunogenicity of H60 homologues among different mouse strains and other species remain to be determined.
The LYL8/Kb complex
The antigenic peptide derived from the H60 Ag precisely matched the Kb octapeptide consensus motif, xxxx[F,Y]xx[I,L,M,V] (42). Although exceptions to the MHC consensus motifs have been found among minor H as well as other antigenic peptides (25, 33, 50), knowledge of the Kb motif did narrow the search for the LYL8 octapeptide within the H60 coding sequence. The HPLC retention time of the synthetic LYL8 peptide also precisely matched the single peak of antigenic activity in the naturally processed peptide pool of the C3 (B10 x 129/J)F1 cell line. Because the H60 cDNA was isolated from the BALB background, it is likely that its homologue is also present in the distinct 129/J background. Remarkably, the amount of naturally processed LYL8 peptide was found to range between 5 and 15 copies/cell, which makes it a member of the rare rather than abundant set of peptides expressed on the cell surface (51). Furthermore, the MHC binding capacity of the LYL8 peptide was comparable to other peptides derived from foreign, viral, or allogeneic Ags. Thus, neither the MHC binding ability nor the natural abundance of the LYL8 peptide distinguish it from other known antigenic peptides. Among the other H peptides, the natural abundance of the mitochondrial ND1, COI, ATPase 6, or the HY-derived peptide/MHC complexes are yet unknown, but our previous analysis of the H13a-derived SVL9/Db complexes had shown that it too was expressed at less than 50 copies/cell (25). Whether the expression of these antigenic H peptides in different cell types will reveal substantial differences in their natural abundance remains to be determined, but our findings strongly argue against the hypothesis that the potent immunogenicity of H Ags is due to their unusual MHC binding ability or due to their overall high abundance.
Polymorphisms of H loci
The ability of H loci to induce T cell immunity suggested that they were polymorphic between the donor and host strains. Comparison among the known H loci, however, reveal different mechanisms for their antigenic polymorphism. At one extreme are the male HY-encoding loci of the Y-chromosome, where their X-chromosome homologues in the female host contain amino acid substitutions within the relevant peptides presented by the MHC (9, 11). For the murine Smcy/Kk and Uty/Db peptides, the substitutions in the Smcx and Utx homologues were drastic enough to prevent their ability to bind the relevant MHC, and they would therefore be absent from the repertoire of self peptides in the female host. The human Smcy/HLA-B7 and Smcy/HLA-A*0201 peptides may also be similar to their murine counterparts (10, 52). On the other extreme, polymorphic substitutions within the mitochondrial ND1 peptides presented by the nonclassical H2-M3 do not prevent their presentation, and T cell responses can be elicited in either direction (6)
The only two autosomal H loci known so far fall within these two extremes. We recently showed that a single conservative valine to isoleucine amino acid substitution within the murine autosomal H13 peptide SSV[I,V]GVWYL/Db explained its bidirectional immunogenicity in strains expressing either the H13a or the H13b alleles (25). The H13 locus is therefore similar to the mitochondrial H loci in this respect. The autosomal H60 locus described here conceptually resembles the HY-encoding loci because its expression was not detected in the host B6 strain, making expression of any LYL8 homologue extremely unlikely. The reasons for this differential expression of the H60 locus and its functional consequences, if any, are not yet known. Because transcription of H60 was up-regulated in mitogen-stimulated spleen cells, it also suggests that H60 may be an example of a transcriptionally regulated H gene in contrast to other identified H loci that appear to be constitutively expressed.
In conclusion, we have identified a new murine H60 histocompatibility locus that encodes an immunodominant H Ag. Knowledge of this differentially expressed locus and its processed peptide now provides the genetic and biochemical tools to determine why, among thousands of estimated polymorphic loci (4, 53), only a small set is apparently immunogenic (13, 14).
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Footnotes |
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2 The seqnence reported in this paper has been deposited in the GenBank database under accession No. AF084643.
3 Address correspondence and reprint requests to Dr. Nilabh Shastri, University of California, LSA 421, Division of Immunology, Department of Molecular and Cell Biology, Berkeley, CA 94720. E-mail address:
4 Abbreviations used in this paper: H, minor histocompatibility; RI, recombinant inbred; B6, C57BL/6; CPRG, chlorophenol red ß-galactopyranoside; SSCP, single strand conformational polymorphism; TFA, trifluoroacetic acid; NCBI, National Center for Biotechnology Information.
Received for publication March 3, 1998. Accepted for publication June 2, 1998.
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