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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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Until recently, the molecular definition of H Ags had remained elusive because of the technical challenges of identifying T cell-stimulating Ags (9). Only 10 unique antigenic precursors are known in mice, where H loci have been extensively studied by genetic, biochemical, and immunological methods and have served as models for the study of graft rejection and the immune system itself. Two of these (ND1, COI) are encoded by the mitochondrial (8, 10), another three (Smcy, Uty, and Dby) by the Y (11, 12, 13), and four others by autosomal chromosomes (14, 15, 16, 17). For each of these H loci, the donor and host differ in transcriptional regulation or in polymorphic amino acid substitutions within the antigenic peptides, explaining why host CD8 T cell responses were induced to donor peptide-MHC complexes (11, 12, 16). Interestingly, if both the donor and the host expressed polymorphic H Ag peptide-MHC complexes, reciprocal CTL responses were elicited in most cases (8, 10, 14, 15), suggesting that the mere presence of distinct H peptides was sufficient for immunogenicity.
Here we provide the molecular definition of the polymorphic murine H47 locus that encodes the CD8 T cell epitope of the classically defined H4 transplantation locus first detected four decades ago (18, 19). Unlike previously identified H loci where single amino acid substitutions elicit bidirectional CD8 T cell responses, the immunogenicity of the H47 allelic products was profoundly influenced by other neighboring H loci. Thus, the results provide new insights into the unique peptides that allow self-nonself discrimination and the mechanisms of histoincompatibility.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred mice were obtained from or bred at The Jackson Laboratory
(Bar Harbor, ME) and have been described previously (19).
Immunizations, generation of CTL lines, 
-galactosidase
(lacZ)-inducible T cell hybrids, and the maintenance of the cell lines
has been described (14, 19, 20). Briefly, the
H47a-specific CTL was generated by immunizing the
congenic B10.129-H46bH47b (21M) with
B10 splenocytes. All experiments were performed in compliance with the
institutional Animal Use and Care Committee guidelines.
T cell activation assays
LacZ-inducible T cell hybrids were cocultured with APC that either were transfected with Ag cDNAs or were pulsed with exogenous peptides. The lacZ activity was measured as described previously (20). CTL lysis assays were performed with target cells, either 51Cr-labeled Con A-stimulated lymphoblasts from indicated strains or T2-Db cells loaded with 100 nM SII9 or SFI9 peptide. All T cell activation data shown are the mean of triplicate cultures.
cDNA library and expression screens
An EL4 cDNA library was screened in pools of 
50–100 CFU by
transforming bacteria with recombinant plasmids as described previously
(14). Briefly, cDNA pools were cultured, isolated, and
screened in 96-well plates by transient transfection into recipient
APCs cotransfected with the relevant MHC class I cDNA and B7-2 cDNA.
The sequence of 1163 cDNA is available from the National Center for
Biotechnology Information GenBank with the accession number AF335543.
Expression constructs and peptides
All plasmid constructs were in the expression vector pcDNA1
unless otherwise noted. Nested deletions were made by PCR of the 1163
cDNA with a vector-specific forward primer and an 1163-specific reverse
primer H47aR4 (5'-TCACAGAACAGTCTCGGCTTGGT-3'); H47aR7
(5'-TCACTGCCTCAAAGCCCTCAGTC-3'); and H47aR8
(5'-TCAGAGTAGGATGCAGCTGAAGAG-3') with PFU polymerase (Stratagene,
La Jolla, CA). PCR fragments generated by RT-PCR were amplified with
the H47aF1 forward with an internal BamHI site for cloning
(5'-GCTATGGATCCGCGGTGGCCGCCATGGAT-3') and either the H47aR1
(5'-GCCTTCAACCTACCAACTAG-3') or H47aR4 primer. The genomic DNA PCR
fragments presented in Fig. 4
were amplified with the H47FPE
(5'-CGATTCCTGCACGTGACAG-3') forward and H47RPE
(5'-ACAACATCAGGTTCCAGAAC-3') reverse primers and sequenced
directly. The hemagglutinin (HA) epitope (YPYDVPDYASL)-tagged H47
construct was generated by fusing an oligonucleotide cassette (coding
strand,
5'-CTGGCGGCTACCCCTACGACGTGCCTGACTACGCCAGCCTCTAGCAT-3';
noncoding strand,
5'-CTAGAGGCTGGCGTAGTCAGGCACGTCGTACGGGTAGCCGCCAGATG-3') encoding the
epitope into an internal PflMI site (restriction enzyme site) at
the C terminus. The Minigene constructs MFI10 (Met-FSCILLYIVI),
MSII9 (Met-SCILLYIVI), and MSFI9 (Met-SCILLYFVI) were prepared
by using complimentary oligonucleotides corresponding to the indicated
sequences and followed by a translational stop codon. Minigene
construct MSXI9 (Met-SCILLYXVI) was prepared as described above except
X encoded any amino acid (X = NNg/c). The synthetic peptides SII9
(SCILLYIVI), SFI9 (SCILLYFVI), NP (ASNENMETM), AFV8 (ANYDFICV),
and WI9 (WMHHNMDLI) (12) were purified by HPLC and
confirmed by mass spectrometry.
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Southern blot RFLP analysis indicated in Fig. 4b
was with
genomic DNA purchased from The Jackson Laboratory DNA resource and
probed with 32P-labeled 1.2-kb 1163 DNA by
standard methods. The 1163 cDNA was mapped between the gene pink-eyed
dilution (p) and the distal locus albino
(c) on the (129/J x B10) x 129/J backcross mouse
panel (19), and Mpmv1 location was determined
as described (21). High-resolution mapping of 1163
was done with The Jackson Laboratory (B6 x
SPRET/Ei) x SPRET/Ei (BSS) backcross panel
with the primer pair H47aF2 (5'-CATCTGGCGGCTGAAACTAA-3') and H47aR6
(5'-GACGAAGTCAAGCTCACAAA-3') (14).
RMA/S stabilization and biochemical analysis
Peptide/MHC I binding was measured by RMA/S stabilization assay (14). For immunofluorescence microscopy, COS cells were transiently cotransfected with cDNAs expressing Kb and H47-tagged at the C terminus with an epitope from HA, fixed in 4% paraformaldehyde with 0.1 M glycine, permeabilized for 4 min in 0.2% saponin, and stained with HA.11 mAb (Babco, Berkeley, CA) followed by polyclonal anti-Kb Ab. Secondary staining was with donkey anti-mouse Ab conjugated to Texas Red (Jackson ImmunoResearch, West Grove, PA) and goat anti-rabbit Ab conjugated to FITC (Caltag, Burlingame, CA). Cells were visualized on the digital Nikon (Melville, NY) microphot FX microscope. In vitro translations were all done with the TNT rabbit reticulocyte lysate system (Promega, Madison, WI). Translation reactions were done in the presence of [14C]leucine with or without canine pancreatic microsomes as per the manufacturer’s instructions. After translation, the microsomes were pelleted by centrifugation at 65,000 rpm for 15 min at 4°C over a sucrose cushion (1 M KOAc, 500 mM sucrose, 50 mM HEPES). Pellets were digested with proteinase K (100 µg/ml) with or without 1% Triton X-100 as indicated. Reactions were stopped by adding 1 mM PMSF and directly analyzed by SDS-PAGE. Synthetic SII9 peptide was back-extracted by spiking 129/J spleen cells with the equivalent of 105 peptide copies/cell and recovery determined by T cell activation assay with 1AZ.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify the T-cell stimulating H47a gene
product, we generated the lacZ-inducible, T cell hybrid 1AZ by fusing
the H47a-specific CTL line (19) with
the lacZ-inducible fusion partner, BWZ.36/CD8
(20).
Similar to the parental 1A line, the 1AZ hybridoma recognized B6 spleen
cells as well as EL4, a thymoma cell line of B6 origin. Furthermore,
this recognition was specifically inhibited by
anti-Db mAbs (not shown). The lacZ-inducible
1AZ T cell hybrid was used as a probe to screen for the
Db-restricted Ag gene in an EL4 cDNA library
(14, 22). The cDNA pool, 224-11C, was identified by its
ability to stimulate the 1AZ T cell hybrid when transfected into
LMtk-recipient cells (Fig. 1a). The response of the 1AZ
hybrid to pool 224-11C was dose dependent and occurred only when the
Db but not Kb MHC was
cotransfected. The 224-11C cDNA pool was further subdivided into
individual colonies by transforming bacteria and several cDNAs that
stimulated the 1AZ hybrid were isolated (Fig. 1b). One of
these clones, designated 1163, stimulated 1AZ when cotransfected with
the Db MHC I but not with the irrelevant
Kb MHC I cDNA (Fig. 1c). We conclude
that 1163 cDNA encodes a Db-restricted antigenic
activity recognized by the 1AZ T cell hybrid.
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H47 encodes a CD8 T cell epitope and is part of the
H4 complex on chromosome 7 based on tissue-graft rejection
and in vitro CTL reactivity assays (19, 23). Genetic
mapping was performed to establish whether the 1163 cDNA maps to the
chromosome 7-encoded H47 locus, a gene that regulates the
expression of H47 or a gene encoding an unrelated but
antigenically cross-reactive H Ag. Southern blot RFLP analysis of
XbaI-digested genomic DNA from mouse strains previously
typed for H47 Ag polymorphisms were probed with the 1163 cDNA. Unique
4- and 0.4-kb RFLP bands were found in the
H47a strain B10 in contrast to 1.8- and
5-kb bands in the prototypic H47b strain
129/J (Fig. 1d). Most significantly, RFLP patterns of DNAs
from 21M
(B10.129-H46bH47b)
mice the congenic segment of which spans the H4 complex and
the recombinant strain RC6 (B10.129-H47b) that
retains H47b but not
H46b were identical to that of the 129/J
strain. This demonstrated that the 1163 cDNA probe identified a gene in
the H4 congenic interval. Higher resolution mapping
consisting of RFLP analysis of a (129/J x B10) x 129/J
backcross panel previously typed for the H47a Ag
(19) as well as typing of The Jackson Laboratory mapping
panel with the 1163 cDNA (Fig. 1e), indicated that the H47
Ags and the 1163 cDNA cosegregated and colocalized within the
H4 complex 0.9 ± 0.6 cM distal to the pink eyed
dilution locus, p. Consistent with this map position, simple
sequence length polymorphism analysis of The Jackson Laboratory
interspecific BSS mapping panel
(http://www.jax.org/resources/documents/cmdata/) with 94
(B6 x SPRET/Ei) x SPRET/Ei backcross mice mapped the 1163
cDNA near p and 1.06 ± 1.06 cM from Pcsk6
and Tjp1 (data not shown). Together the results proved that
the 1163 cDNA was derived from the H47 locus.
H47 is a membrane-bound cell surface protein
The H47a cDNA encoded a novel 188-residue
protein with no exact matches in the current nonredundant sequence
databases (Fig. 2a;
http://www.ncbi.nlm.nih.gov/blast/). The closest relative of
H47a, with a striking 64% amino acid identity,
was a hypothetical protein from the human adrenal gland (accession
number AF157317). Hydropathicity analysis of predicted H47 amino acid
sequence suggested that it contained a putative transmembrane region,
Tm (Fig. 2a). To test this prediction, we used the cDNAs in
an in vitro translation assay including
[14C]leucine and microsomes. The H47 cDNA
yielded a 24-kDa band, close in size to the predicted 21-kDa product
that was membrane-bound, as it associated with the microsomal pellet
(Fig. 2b). To determine whether this polypeptide was
translocated into the lumen of the microsomes, the pellet was treated
with proteinase K, which resulted in a reduction of the product size to
a single 5.5-kDa fragment. Disruption of the membranes with Triton
X-100 in the presence of proteinase K caused a further reduction in
band intensity and size, indicating that the 5.5-kDa fragment was
protected from hydrolysis in intact microsomes. The protease-sensitive
18-kDa and protease-resistant 5.5-kDa fragments correspond closely to
those expected if the N-terminal 48-residue peptide, including the
transmembrane residues 29–48, were sequestered within the microsomal
membranes, and the C-terminal residues 49–188 were in the cytoplasm
and accessible to proteinase K. These results directly confirmed the
predictions of the sequence analysis and showed that H47 is a type I
transmembrane protein.
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c).
We conclude that H47 is an integral membrane protein that is expressed
on the cell surface. The SCILLYIVI nonpeptide defines the antigenic activity of the H47a locus
To define the self-nonself relationship between the donor and host
strains, we first identified the minimal antigenic peptide encoded by
the H47a cDNA by deletion analysis (Fig. 3a). Positive response to the
deletion construct 
R4 mapped the 1AZ stimulating activity to the
first 229 nucleotides of clone 1163 (Fig. 3b). Surprisingly,
this region did not contain a sequence with the canonical
Db consensus motif xxxx[N]xxx[L,I,M]
(24). Differential responses to additional deletion
constructs R7 and R8 further narrowed the antigenic activity to
be contained within or to overlap residues 44 and 60 (Fig. 3c). Minigenes encoding either the decamer peptide
FSCILLYIVI (FI10) or the nonamer peptide SCILLYIVI (SII9) and an
additional methionine codon for translational initiation were then
tested for their ability to stimulate the 1AZ hybrid. Both
minigenes stimulated strong and comparable 1AZ T cell
responses (Fig. 3d). We conclude that the 1AZ T
cell-stimulating activity was defined by the noncanonical SII9
nonapeptide contained within the transmembrane region (Fig. 2a).
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Phe substitution defines antigenic
polymorphism of the H47 locus
Northern blot analysis of the donor B6
(H47a) and host 129/J
(H47b) mRNA with a 1163 cDNA probe confirmed
that the H47 genes were transcribed in both strains (data
not shown). To determine the molecular basis of the allelic
polymorphism, B6 (H47a), 129/J
(H47b), and BALB/c (H47a)
mRNA as well as Spretus (H47a) genomic DNA were
used as template for PCR with H47-specific primers. The PCR products
were cloned into an expression vector, transfected into recipient APCs
with Db MHC I cDNA, and tested for their ability
to stimulate the 1AZ T cell. All the constructs, except that from the
H47b 129/J strain, stimulated the 1AZ T cell
hybrid (Fig. 4a). Nucleotide
sequence of the 129/J construct revealed that a single nucleotide
change had resulted in the amino acid substitution from Ile to Phe at
the p7 residue of the SII9 peptide. Remarkably, nucleotide sequences of
this region in 15 other mouse strains showed that all, including the
evolutionarily distant Mus spretus- and Mus
castaneus-derived strains, could be typed as
H47a or H47b by the
presence of the Ile or the Phe residue, despite other nucleotide
sequence differences that were reflected in their distinct RFLP band
patterns (Fig. 4b). Thus, the S[I/F]I9 coding sequences
appear to be conserved in the suborder Mus.
To directly test the functional significance of the p7 Ile-to-Phe
substitution within the antigenic peptide, an expression construct
encoding the H47b allelic peptide SCILLYFVI
(SFI9) was directly compared with the construct encoding
H47a peptide SII9 in an endogenous
presentation assay. Only the M-[SII9] stimulated the 1AZ T cell
hybrid, and the response to the M-[SFI9] construct was virtually
indistinguishable from vector alone (Fig. 4c). To rule out
the possibility that the inability of the M-[SFI9] minigene to
stimulate 1AZ was attributable to inefficient processing of this
antigenic precursor, we tested synthetic peptides in an exogenous
presentation assay for their ability to stimulate the 1AZ hybrid.
Again, the SII9 peptide was recognized by the 1AZ T cells, and at a
1000-fold lower concentration than the allelic SFI9 peptide (Fig. 4d). These results directly confirmed the assignment of the
antigenic activity to the SII9 peptide and demonstrated that the single
amino acid substitution between the two alleles was sufficient to
determine T cell specificity.
We determined whether the inability of the SFI9 peptide to stimulate
the 1AZ T cell was attributable to its inability to bind to the
Db class I MHC molecule or an effect on TCR
recognition. Despite the lack of a Db binding
consensus motif, both peptides specifically stabilized the
Db MHC I molecule on the surface of RMA/S cells
(Fig. 4e). Interestingly, the SFI9 peptide was apparently
5-fold more effective than the SII9 peptide. However, both H47 peptides
were far less efficient than the influenza NP or the
HY-derived WI9 peptides, both of which conform to the
Db consensus motif (Fig. 4e).
Conversely, neither SII9 nor SFI9 peptides stabilized the
Kb MHC I molecule that was stabilized by a
Kb-restricted peptide AFV8 (Fig. 4f).
We conclude that the antigenic polymorphism at the H47 locus
was attributable to a single amino acid substitution within poor
Db binding S[I,F]I9 peptides that determined
the ability of T cells to recognize the
peptide/Db complex.
The p7 polymorphism influences TCR specificity
To define the stringency with which the T cell specificity
was influenced by the polymorphic amino acid substitution in the H47
alleles, we generated M-S[X]I9 minigene constructs encoding the
minimal H47a antigenic peptide SCILLY[X]VI
with a redundant p7 codon, NNG/C (N = A, C, G or T). This allowed
32 different codons at p7 and included all 20 amino acids designated as
X. Individual plasmid DNAs from the M-S[X]I9 panel were tested for
their ability to stimulate the 1AZ hybrid. The frequency of individual
clones that could stimulate the 1AZ T cell was higher than the 1:16
expected if the naturally occurring isoleucine was the only amino acid
that could be recognized by T cells at p7. To determine which amino
acids were compatible with TCR recognition, several positive clones
from the primary screen were sequenced and the p7 codon was found to
specify several amino acids including valine, leucine, alanine, and
methionine (Fig. 5a). However,
in a stringent DNA titration test of these constructs for their
antigenic activity we found that the natural isoleucine residue could
only be effectively substituted with the valine and to a 10-fold lower
extent with the leucine residue (Fig. 5b). The response to
Ala and Met substitutions remained below the detection limit. Because
most amino acid substitutions were detrimental to TCR recognition, we
conclude that the p7 residue was a key determinant of TCR
specificity.
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Reciprocal immunizations were conducted to determine the
relative immunogenicity of the H47 allelic homologues. Immunization of
21M (H47b) mice with spleen cells of its
congenic partner B10 (H47a) generated CTLs that
specifically lysed the donor B10 but not host 21M cells as effectively
as the H47a-specific 1A CTL line (Fig. 6, a and b). Both
the bulk and the 1A CTLs also lysed targets incubated with the
H47a peptide SII9 but did not recognize targets
incubated with the H47b peptide SFI9 (Fig. 6, c and d). In contrast, reciprocal immunization of
B10 (H47a) mice with 21M
(H47b) cells generated vigorous CTL responses
which lysed the donor 21M target cells (Fig. 6, e and
f) but failed to lyse the corresponding SFI9 peptide-coated
target cells (Fig. 6, g and h). The
SFI9/Db-specific lytic activity also was barely
detectable in CTLs generated in 21M mice grafted with B10 skin.
However, after in vitro restimulation of these CTLs in the presence of
SFI9 peptide CTLs specific for the SFI9/Db
complex were detected and were found to be as effective as
SII9/Db-specific CTLs in their lytic activity
(Fig. 6, i–l). Therefore, we conclude that both
H47 alleles can be immunogenic but the immunogenicity of
H47b is profoundly influenced by the existence
of at least one additional H locus encoded within the
congenic region of 21M mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several different approaches, including positional cloning, biochemical purification, expression cloning, and mimotope development, have been used to identify T cell-stimulating histocompatibility Ags (25, 26). Among these, the expression cloning approach has proved to be particularly powerful because it does not require any prior assumptions of genomic location and reveals not only the identity of the antigenic peptide, but that of the precursor gene as well (27). The H Ag cDNA as shown here is an essential tool for defining the underlying genetic mechanisms of polymorphism as well as structural and functional analysis of the precursor protein. Notably, despite repeated attempts, we failed to detect the extremely hydrophobic SII9 peptides in cell extracts (not shown). In our hands, this failure is unique to H47 and can be accounted for by the low (<0.5%) recovery of the SII9 peptide in HPLC fractionated cell extracts that were spiked with known amounts of the synthetic peptide (not shown). Because the biochemical purification approach depends on the ability to detect the antigenic activity in cell extracts, hydrophobic peptides may not be accessible to this method. Note also that like the H13 peptides we had identified earlier (14), the Db-presented H47 peptides do not conform to the Db peptide binding motif xxxx[N]xx[I,L,M] (24) and thus would be inaccessible to the mimotope approaches as well (26).
The high hydrophobicity of the S[I,F]I9 peptides is a consequence of
their location within the transmembrane region of its precursor protein
(Fig. 2). This location is most unusual among peptides presented by
classical MHC class I molecules (28). However,
presentation of hydrophobic materials to T cells is characteristic of
nonclassical MHC I molecules; N-formylated peptides are
presented by H2-M3 (8), signal peptides by Qa1
(29), and lipids by CD1 molecules (30, 31).
Thus, the presentation of the transmembrane H47 peptides by
Db may be analogous to the presentation of signal
peptides by Qa1 molecules. Interestingly, the hydrophobic signal
peptides presented by Qa1 serve as ligands for both the TCR of
CD8 T cells as well as the NKG2A,C,E receptors of NK cells (29, 32, 33). Recently, it was discovered that the H60
histocompatibility Ag that we had earlier identified by its ability to
elicit CD8 T cell responses (16) is also a ligand for the
NKG2D receptor (34, 35). Therefore, it is conceivable that
the highly conserved S[I,F]I9/Db complexes or
the H47 gene product could play a role in self-nonself
discrimination beyond eliciting CD8 T cell responses.
The difference in relative immunogenicity of the H47 alleles was
striking. Both the H47a (SII9) and
H47b (SFI9) peptides were presented by
Db MHC and were recognized by CD8 T cells.
However, although CTLs specific for SII9/Db were
readily elicited after immunization of 21M (H47b)
mice with congenic B10 (H47a) cells, CTLs
specific for SFI9/Db complex were rather
difficult to elicit in B10 mice responding to 21M cells (Fig. 6).
Instead, the B6 anti-21M CTLs were specific for another
Kb-restricted Ag(s) encoded in the 21M congenic
region (not shown), possibly the CTT2 epitope originally described by
Wettstein (36). Thus, unlike the other known murine
biallelic H loci (ND1, H13, and H3a), relative
immunogenicity of the foreign H47 alleles varied
dramatically and was affected by one or more linked H loci.
How this putative locus might affect the immunogenicity of H47 Ags is
intriguing. With analogy to the earlier analysis of immunodominance
among H loci (23, 37), and potential viral
epitopes (38), it is possible that this H locus
in the 21M strain encodes an inherently immunodominant epitope that
overwhelms the anti-H47b response. The same
locus in B10 mice would then be predicted to be a "null" allele or
to encode a subdominant epitope. An alternative but less likely
possibility is that the H46b locus, which also
maps to the chromosome 7 congenic interval and regulates the MHC class
II-restricted CD4 T cells (19), may be selective in its
helper function in being able to help the
anti-H47a but not the
H47b CTL responses analogous to the selective
interactions among Th cells and Ab-secreting B cells (39).
Currently, these hypotheses cannot be resolved because the identities
of the H Ags recognized by the anti-21M CTL or the
anti-H46 CD4 Th cells are unknown. However, they are now accessible
by the strategies described here and those recently described by
Simpson and her colleagues (13).
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Acknowledgments |
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Footnotes |
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2 Current address: Marywood University, Department of Science, 2300 Adams Avenue, Scranton, PA 18509-1598.
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 94720-3200.
4 Abbreviations used in this paper: H, histocompatability; lacZ, -galactosidase.
Received for publication October 24, 2000. Accepted for publication January 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactosidase. I. The fine specificity of T cells dictates the fine specificity of antibodies directed to conformation-dependent determinants. Eur. J. Immunol. 15:345.[Medline]This article has been cited by other articles:
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T. Kanaseki and N. Shastri Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing Regulates Quality of Processed Peptides Presented by MHC Class I Molecules J. Immunol., November 1, 2008; 181(9): 6275 - 6282. [Abstract] [Full Text] [PDF]
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D. Duffy, S. M. Sparshott, C.-p. Yang, and E. B. Bell Transgenic CD4 T Cells (DO11.10) Are Destroyed in MHC-Compatible Hosts by NK Cells and CD8 T Cells J. Immunol., January 15, 2008; 180(2): 747 - 753. [Abstract] [Full Text] [PDF]
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 M. A. de Witte, M. Toebes, J.-Y. Song, M. C. Wolkers, and T. N. M. Schumacher Effective graft depletion of MiHAg T-cell specificities and consequences for graft-versus-host disease Blood, May 1, 2007; 109(9): 3830 - 3838. [Abstract] [Full Text] [PDF]
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R. Yadav, Y. Yoshimura, A. Boesteanu, G. J. Christianson, W. U. Ajayi, R. Shashidharamurthy, A. K. Stanic, D. C. Roopenian, and S. Joyce The H4b Minor Histocompatibility Antigen Is Caused by a Combination of Genetically Determined and Posttranslational Modifications J. Immunol., May 15, 2003; 170(10): 5133 - 5142. [Abstract] [Full Text] [PDF]
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A. Cerwenka, C. A. O'Callaghan, J. A. Hamerman, R. Yadav, W. Ajayi, D. C. Roopenian, S. Joyce, and L. L. Lanier Cutting Edge: The Minor Histocompatibility Antigen H60 Peptide Interacts with Both H-2Kb and NKG2D J. Immunol., April 1, 2002; 168(7): 3131 - 3134. [Abstract] [Full Text] [PDF]
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D. A. Ostrov, M. M. Roden, W. Shi, E. Palmieri, G. J. Christianson, L. Mendoza, G. Villaflor, D. Tilley, N. Shastri, H. Grey, et al. How H13 Histocompatibility Peptides Differing by a Single Methyl Group and Lacking Conventional MHC Binding Anchor Motifs Determine Self-Nonself Discrimination J. Immunol., January 1, 2002; 168(1): 283 - 289. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). 1A CTL is an
established long-term anti-H47a CTL line
(•). e–h, Bulk B10 CTLs were generated by
immunization of B10 mice with congenic 21M spleen cells (
) and the
B10 CTL plus graft cells were generated from B10 mice previously
tail-grafted with 21M skin and stimulated with 21M cells in a MLC
(
). i–l, B10 CTL plus graft/restim were generated
with an additional restimulation in vitro with SFI9-loaded syngeneic
cells. All CTLs were analyzed for their lytic specificity on
51Cr-labeled B10 lymphoblasts, 21M lymphoblasts,
SII9-loaded T2-Db targets, and SFI9-loaded
T2-Db targets as indicated.