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2,*,
,
ugich4,*,,
* Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR 97006;
Laboratory of T Cell Development, Immunology Program, Memorial Sloan-Kettering Cancer Center, and
Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021
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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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carbon backbones of these molecules, ruling out major structural
variation, measurable movement of side chains and, occasionally, of the
main chain segments, was detected as an indirect result of peptide
interactions by x-ray crystallographic (5, 6, 7, 8), serological
(9, 10, 11, 12, 13, 14), and biophysical (15) studies.
Previously, we engineered and characterized an
H-2Kb variant, KbW9, devoid of the
central anchor ("C") pocket owing to a point mutation located on
the floor of the peptide-binding groove (V9
W). This mutation
drastically altered selection of bound peptides so that the
KbW9 molecule predominantly, if not exclusively, bound
nonamers, and new peptide anchor residues substituted for the loss of
the P5F/Y:C pocket interaction. Molecular modeling strongly suggested
that KbW9 bound nonamers bulge out of the groove and are
more exposed to TCR than Kb-bound peptides
(16). Results also indicated that the peptide repertoires
of Kb and KbW9 were largely nonoverlapping in
vivo (16). Therefore, these two class I molecules were
suitable for investigation of the effect of different peptide motifs on
MHC structure and TCR binding.
In this study, we compared conformation-sensitive Ab and alloreactive TCR-mediated recognition of Kb and KbW9. We found that the unique features of KbW9 floor which result in unique length and positioning of the peptide resulted in selection of peptide-independent, cross-reactive CTLs. Based on these results, structural correlates of cross-reactive, peptide-independent MHC allorecognition are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.A(4R) mice (H-24r, Kk, Ak, Db) were obtained from The Jackson Laboratory (Bar Harbor, ME) and were used at 8–16 wk of age. B10.A (4R).Kb and B10.A (4R).KbW9 (abbreviated as 4R.Kb and 4R.KbW9) transgenic mice were generated as described below.
mAbs and FCM analysis
Anti-H-2Kb mAbs Y3 (19), 5F1 (20), EH-144 (19), 28.13.3 (22), 100.30 (9), and AF6-88.5.3 (AF6, ATCC HB158; American Type Culture Collection, Manassas, VA) were used in the form of ascitic fluid. Secondary PE-conjugated goat anti-mouse IgG2a and IgG2b were obtained from Fisher Scientific (Malvern, PA). Cell lines and flow cytofluorometry (FCM) were described previously (17).
Generation of transgenic mice
To generate transgenic mice, 11-kb SalI fragments of the wild-type Kb and the mutant pKbW9 TCR neo construct (16) containing the wild-type or mutated gene under the transcriptional control of endogenous elements were isolated on a 0.6% low melt agarose gel and purified using Gelase agarase according to the manufacturer’s (Epicentre Technologies, Madison, WI) instructions. After phenol-chloroform extractions, the fragments were resuspended in Tris-EDTA buffer containing CsC at 1.68 g/ml and centrifuged at 90,000 rpm for 4 h. Peaks containing the DNA were collected and dialyzed 3–4 times in 4 L of injection buffer (5 mM Tris, 5 mM NaCl, 0.1 mM EDTA, pH 7.4). Purified constructs were injected by the Memorial Sloan-Kettering Cancer Center Transgenic Animal Core Facility into (CBA/Ca x B6)F2 fertilized eggs. Transgenic mice were screened by a combination of FCM and PCR methods, and the transgene was backcrossed for at least 10 generations onto the B10.A(4R) background. Expression of the B10.A(4R) endogenous and transgenic MHC molecules was confirmed by a combination of Southern blot and FCM techniques.
Peptides
Peptides were obtained from the Memorial Sloan-Kettering Cancer Center Microchemistry Core Facility or from Research Genetics (Huntsville, AL). Origins, sequences, and original references for the peptides OVA-8, HSV-8, FLK-8 (18), and VEP-9 (VEPVRLILL) VPY-9 (sequenceVAPYVRLLIL; Ref. 16). are given in respective references and in the abbreviations footnote.
Generation of alloreactive CTL and CTL assays
B10.A(4R) splenocytes (20 x 106) were mixed with 20 x 106 irradiated (3000 rad) splenocytes from either 4R.Kb or 4R.Kbw9 mice in upright 25-cm3 tissue culture flasks (Falcon; BD Biosciences, San Jose, CA) containing 10 ml of RPMI 1640-10% FCS. Seven days later, effector CTLs were counted, and 3 x 106 cells were restimulated with irradiated (3000 rad) 20 x 106 (B10.A(4R)-transgenic) splenocytes and 20 x 106 B10.A(4R) feeder cells in flasks containing 10 ml of RPMI 1640-10% FCS medium. Subsequently, CTL were maintained by weekly restimulation as described above, in the presence of cytokine-rich Con A supernatant. Effectors were harvested on day 5 after restimulation for use in 51Cr release assays. 51Cr release assays were performed as previously described (18). For detection of peptide-independent epitopes, TAP-deficient, class I-transfected cells were grown overnight at 28°C before 51Cr labeling or, alternatively, grown overnight at 29°C, collected, incubated for 30 min at 29°C with relevant peptides (5 µg/ml), and then labeled with 51Cr and processed at 37°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the purpose of analyzing the immune response restricted by
KbW9, we generated KbW9 (and control
Kb) transgenic mice and bred the transgene onto an
Kb-negative background using B10.A(4R) mice (Kk
Ak E0 Db), abbreviated as 4R.
Strains thereby obtained are referred to as 4R.Kb and
4R.KbW9. To assess conformational integrity of the
1 and 2 domains of Kb
and KbW9, we stained Kb and KbW9
with a panel of Kb-specific Abs. TgKb and
TgKbW9 were expressed at comparable levels (both were
expressed off the endogenous regulatory elements), differing by <15%
as assessed by the anti-Kb mAb Y3 staining (Fig. 1
). The Y3 (19) epitope is
on the right side of the 2 helix, adjacent to the C
terminus of the peptide. This Ab recognizes only properly folded and
peptide-occupied molecules and is not sensitive to the character of
bound peptide or to peptide-induced Kb movements and is
thus generally believed to provide an excellent measure of the overall
expression of Kb-like molecules (9).
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1
and 2 helices. All are more or less sensitive to the
changes in the bound peptide and the consequent indirect rearrangements
of the class I side chains and were extensively used to assess the
conformation and subtle rearrangements of the pMHC complex. A lack of
binding of one or more of these Abs is considered evidence for
significant alterations of the parts of the pMHC complex, whereas
decreases or increases in binding are indicative of subtle
rearrangements in the solvent-accessible portion of the pMHC complexes
(19, 20, 21, 22, 23). The 5F1 epitope maps to the left end of the
2 helix (19, 20, 21); the EH-144 (19, 20) epitope lies on the right end of the 1 helix,
in the vicinity of residues 77, 80, 89 and 90; 28.13.3 binds to the
"left" part of the TCR contact area, possibly contacting both
helices, whereas 100.30 has not been precisely mapped but has been
shown to be sensitive to alterations in the N half of the peptide
(9).
The binding efficiency of peptide-sensitive Abs can be evaluated by
calculating the staining intensities corrected for surface expression
of Kb and KbW9 detected by Y3, the Y3 index
(Fig. 2). Such analysis indicated that
the presence of nonamers bulging out of the groove affects the binding
of Abs, likely by rearranging the MHC side chains. This makes the
epitopes of AF6, EH144, 5F1, and 28.13.3 less optimal, and the epitope
of 100.3 more optimal for mAb binding. Therefore, KbW9
exhibits subtle differences in serological profile compared with
wild-type molecule, while retaining all the common Ab epitopes
tested. The fact that none of the mAbs exhibited a loss of recognition
suggested that KbW9 folds just like Kb and that
no massive alterations were introduced into the -helical parts of
KbW9 by virtue of the mutation at the floor of the
molecule. Variation of the Y3 index for other Abs was
consistent with the subtle rearrangement of different
KbW9 regions. Therefore, comparable TgKb and
TgKbW9 levels and absence of gross conformational
disturbances caused by the introduced mutation allowed us to study and
compare the peptide-presenting function of KbW9.
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To assess the ability of KbW9 to serve as a
restricting element for an Ag-specific CTL response, we immunized
4R.KbW9 mice with a synthetic peptide VPY-9 (VAPYRLLIL).
This peptide contains the preferred KbW9-binding sequence,
deduced from the bulk elution and sequencing of the
KbW9-binding self peptides (16), but it also
contains a noncanonical (and, presumably, non-self) Y at the position
P4, previously defined as a non-MHC contact for
KbW9-binding nonamers. The rationale for this experiment
was that P4 would be a potential TCR contact and that it should
therefore elicit an Ag-specific response. Indeed, a robust CTL response
was obtained in response to VPY-9 priming (Fig. 3). This response was specific and
KbW9 restricted, because neither peptide-negative control
KbW9 cells nor VPY-9-pulsed Kb cells were
lysed. These results indicate that KbW9 can serve as a
restricting element for an Ag-specific CTL response. They also imply
that this molecule is expressed on professional APCs and on the thymic
epithelium and that it can mediate both positive intrathymic selection
and Ag uptake and presentation in vivo.
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To study the TCR-mediated allorecognition of Kb and KbW9, we generated CTL lines against both the mutated and the wild-type molecules. We used splenocytes from B10.A(4R) mice as responders and splenocytes from B10.A(4R)-KbW9 or B10.A(4R)-Kb transgenics as stimulators in a standard MLR (see Materials and Methods).
We next compared the TCR specificities of the alloresponding T cell
populations by testing whether 4R CTL lines generated against
KbW9 and Kb cross-reacted with Kb
or KbW9, respectively (Fig. 4). Interestingly, although 4R
anti-Kb cells did not recognize KbW9,
alloreactive 4R anti-KbW9 CTL lines invariably (100%
cultures) strongly cross-reacted with Kb. It is noteworthy
that three in vitro restimulations were required to expand these
anti-KbW9-specific CTLs as the cytolytic reactivity of
MLR cultures at earlier stages was low and unremarkable. This indicates
that the frequency of KbW9-specific CTL precursors is low.
A similar alloreactivity pattern and similarly low CTL precursor
frequency were observed when BALB/c CTLs were generated against
Kb and KbW9 transfectants in vitro (not shown).
These experiments revealed distinct T cell populations responding to
the two class I molecules: the alloreactive anti-Kb
subset with monospecific TCRs and the less frequent
anti-KbW9 alloreactive cells that are cross-reactive
and respond to both wild-type and engineered MHC.
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Two possible reasons could account for the observed
cross-reactivity. The shared determinants recognized by
anti-KbW9 CTLs could be found exclusively on the MHC
molecules themselves. Alternatively, a subset of peptides binding to
both molecules in a similar conformation could be recognized by the
anti-KbW9 CTLs. This would be consistent with our
previous finding that a subset of synthetic peptides that bind well to
KbW9 can also bind (albeit 100-fold worse) to
Kb (16). To test the first hypothesis, we
explored the peptide dependency of MHC recognition by the
cross-reactive anti-KbW9 TCRs (Fig. 5). To that effect, we used TAP-deficient
cell lines expressing wild-type Kb (RMA-S) or
KbW9 (RS-W9) as targets in a standard Cr release
cytotoxicity assay. These cells express empty class I molecules that
are unstable at 37°C and can be stabilized by overnight shifting to
29°C. Moreover, when incubated with defined class I-binding peptides,
empty class I molecules capture those peptides and present them to T
cells (23). The cross-reactive anti-KbW9
CTLs lysed both RMA-S and RS-W9 cells expressing empty Kb
and KbW9 (stabilized by overnight culture at 29°C).
Furthermore, the cytotoxicity also occurred on both types of targets
for which the class I molecules were loaded with different exogenous
peptides, Kb or KbW9 binding peptides. As
expected, the killing of targets cultured at 37°C was lower,
corresponding to low levels of MHC expressed on their surface.
Cross-reactive CTLs also lysed S.bm8 (not shown), a cell line
expressing Kbm8, a natural Kb mutant containing
a groove mutation that alters the conformation of a subset of bound
peptides (17, 24). Overall, the cytotoxic response of
KbW9-induced cross-reactive T cells depended only on class
I ligand density and not the presence or identity of bound peptides.
Thus, the data show that anti-KbW9 CTLs cross-react
with Kb due to the peptide-independent recognition of MHC
determinants.
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If different MHC topology, formed because of the groove mutation
present in KbW9, is responsible for activating alloreactive
peptide-independent CTLs, we reasoned that under identical conditions
Kb should not activate the same CTL population. We repeated
the above peptide dependency experiment using anti-Kb
cells as effectors. Alloreactive anti-Kb CTLs thus
generated were peptide dependent (Fig. 5
); they failed to lyse RMA-S
cells carrying empty MHC molecules or Kb molecules loaded
with defined exogenous peptides. Thus, the ability to activate
alloreactive peptide-independent CTLs was a specific property of the
mutant KbW9 molecule.
Evidence that peptide-independent cross-reactive recognition of Kb and KbW9 molecules is mediated by the same TCRs
As mentioned, the cross-reactive, peptide-independent CTLs are
infrequent given that they require three in vitro restimulations for
expansion and reliable detection of its lytic activity. One, therefore,
might wonder about the relationship between the cross-reactivity
observed among KbW9 raised CTLs and peptide independency.
The design of our experiment is unequivocal in that regard;
cross-reactive CTL, raised against KbW9, are the ones for
which we show lack of peptide dependency in Fig. 5. Because such cells
grow by stimulation on KbW9 and can functionally recognize
both Kb and KbW9, one could at most envision
that there are two potential populations present within such lines, one
cross-reactive to both Kb and KbW9 and another
monospecific for KbW9. The results shown in Fig. 5
undoubtedly argue that the cross-reactive cells are peptide
independent; they are the only ones capable of recognizing both types
of targets in the first place (monospecific cells cannot recognize
Kb by definition). The potential presence of a
KbW9-monospecific population is irrelevant to that
conclusion. Moreover, although we cannot at this point categorically
exclude the presence of occasional monospecific cells among the
cross-reactive lines, our results strongly suggest that they would be a
minor population based on two lines of evidence. First, of the more
than 30 experiments in which independent lines against KbW9
were elicited, we had only 1 case in which cross-reactivity was weaker
than recognition of the original ligand; and in no experiment did we
see any evidence of cross-reactivity when the original stimulus was
Kb. Second, we used the line analyzed in Fig. 5 and
performed split stimulation; we passed this line on both Kb
and KbW9 stimulators and assayed its target specificity.
This change in stimulating ligands did not impact the recognition
pattern regardless of the stimulators; lysis of both Kb and
KbW9-expressing targets was within ±5% of the original
reactivity of the line before split stimulation (not shown). By
contrast, monospecific CTLs were indeed monospecific; CTL monospecific
for Kb failed to grow when stimulated by KbW9.
Therefore, we conclude that in cross-reactive lines analyzed above,
both allogeneic ligands are recognized by the same TCR in a
peptide-independent manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It could be argued that instead of direct recognition of allogeneic
molecules, our results reflect indirect presentation of a
Kb and/or KbW9 peptide(s) by other molecules
(e.g., Kd in a cytotoxic assay). This is unlikely to be the
case for three reasons. First, in the experiments, we used
P815-Kb and P815-KbW9 as targets in a CTL
assay, where presentation of a Kb and/or KbW9
peptide by Kd is theoretically possible, the responding
CTLs never saw that combination in the course of CTL priming. They were
raised against the Kb or KbW9 molecules on
Kk/Db background. It is highly unlikely that a
peptide from Kb and/or KbW9 bound to
Kk/Db would look similar to the CTLs as the
same (or even different) peptide bound to
Kd/Dd/Ld. Second, even if this was
so, we show that alloreactivity is peptide independent in the case of
KbW9, and it is difficult to see how would that be
compatible with recognition of a MHC-derived peptide on another MHC.
Finally, results in Fig. 5 show that the same CTL recognize
Kb and/or KbW9 molecules in the absence of any
other molecules on target cells (RMA-S cell transfectants), formally
ruling out indirect recognition.
The qualitatively conserved but quantitatively distinct patterns of Ab
binding to Kb and KbW9 suggest subtle
rearrangements in the pMHC structure in the mutant molecule. In
previous serological studies describing peptide-dependent class I
epitopes (9, 10, 11, 12, 13, 14), the change in Ab binding was explained
by the difference in amino acid composition of bound peptides that
induced subtle rearrangement of MHC side chains. Crystallographic data
demonstrated that bound peptides can alter MHC conformation. In the
HLA-A2 structure, the solvent-accessible side chain 167 adopts
different conformations when different peptides are bound
(7). Similarly, in different H-2Kb-peptide
complexes, solvent-accessible side chains 66, 146, 152, 155, and 167
adopt different conformations (5, 8), and MHC-bound
peptides may limit the rotamers of other solvent-accessible side chains
on the rim of the peptide-binding groove (13).
Furthermore, it has been argued that the MHC main chain conformation is
affected by bound peptide at HLA-A2 residues 144–151,
H-2Kb residues 142–151, and HLA-B53 residues 141–155, all
localized in the 2 domain short helix
(5, 6, 7). In our case, KbW9 binds nonamers,
rather than octamers (which bind to Kb). Increased peptide
length (9 instead of 8 aa customary for Kb) and the
elevated position of the middle of the KbW9 groove floor
are both likely to cause the bound peptide to be more exposed than in
complexes of octamers with Kb. This, in turn, has the
potential to cause subtle rearrangements in the adjacent -helical
residues or even in main chain positions of KbW9.
Using the biophysical technique of fluorescence resonance energy
transfer (FRET), Catipovic et al. (15) found no FRET
between different anti-Kb mAb when empty
H-2Kb molecules were analyzed, but a detectable FRET if
peptides were added, and the magnitude of FRET depended on the sequence
of the peptide used. Their results imply that empty H-2Kb
molecules are in a relatively extended conformation and that this
conformation becomes more compact when peptide is bound. One
characteristic of Kb structure with bound octameric
peptides is that peptides are heavily buried by the Kb
helices that come close together over parts of peptide. Given that the
helices of KbW9 are likely to be pushed apart by the
bulging nonamers, these complexes might mimic the loose structure of
empty class I molecules. Reportedly, mAb 100.30, sensitive to certain
H-2Kb conformers (9, 10), shows higher
reactivity not only toward Kb occupied with certain
peptides, but also toward empty Kb (10),
whereas 5F1 does not effectively bind to empty Kb molecules
(22). Interestingly, we detected a 2-fold increase in mAb
100.30 reactivity against the KbW9 molecule, an observation
similar to that made by Soldheim et al. (11) in the case
of Ld9v and the 34-1–2 mAb. Furthermore, reactivity of 5F1
mAb against the KbW9 was decreased, which is consistent
with empty class I conformation. The generation of MHC conformers in
KbW9 which mimic empty Kb molecules could be
due to indirect effects of an altered peptide spectrum, reduced
association with 
2-microglobulin (similar to the studies
by Ribaudo et al. (25) in the case of an Ld
mutant, or the combined effects of both. Studies are in progress to
discriminate between these alternatives.
Our data on TCR recognition of Kb and KbW9 have
revealed a population of alloreactive CTLs directed against
KbW9 that cross-react with wild-type Kb. These
CTLs were cross-reactive because they recognized MHC determinants,
while largely disregarding bound peptides. This is in sharp contrast
with the usual specificity of alloreactive CTLs (26, 27, 28)
including the Kb-induced alloreactive CTLs in this study.
Previous reports agree that most alloreactive CTLs are peptide
dependent (26, 27, 28), although a few behave as though they
can recognize class I molecules devoid of peptide (29).
Potter and colleagues (29) found low precursor frequency
of peptide-independent alloreactive cells. This is in full accord with
our observation that three rounds of in vitro restimulation were
necessary to expand a detectable number of anti-KbW9
CTLs. Although cross-reactive, these CTLs are detectable in vitro only
after in vivo priming by KbW9 and not by Kb
molecule. We explain this asymmetrical CTL reactivity as follows. Most
CD8 cells in common mouse strains are selected on class I molecules
that carry deeply buried peptides. Therefore, most of them will have a
propensity to react to allogeneic molecules that carry similarly buried
peptides. Thus, when stimulated by Kb, many CD8 cells
selected by, e.g., Kk, will react, giving rise to a brisk
and peptide-specific CTL response. By contrast, many fewer CTLs
educated on pMHC molecules with buried peptides will be able to
interact with pMHC molecules the peptide of which prominently arches
out of the groove. In fact, only those that are able to avoid the
peptide and still make meaningful contacts with the MHC helices will be
selected by this form of alloantigen. It is the low frequency of these
CTLs that dictates the slow nature of this response and that likely
accounts for our inability to detect such CTLs when conventional pMHC
molecules are used for stimulation. These observations imply that at
least in this subset of alloreactive CTLs, TCR-mediated recognition
depends on specific steric flexibility of TCR loops that need to
descend on critical MHC residues. One could also envision that similar
rules would guide allogeneic recognition between Kb and
KbW9-expressing animals, i.e., that only a small subset of
TCRs selected by Kb would be able to recognize
KbW9. Similarly, TCRs educated on KbW9 would
have difficulty recognizing Kb because they would contact
only the -helical parts of Kb in the absence of the
raised central portion of the peptide. Moreover, self-tolerance would
further reduce recognition of these distinct but still highly related
molecules. Indeed, this was found to be the case in that we were not
successful in eliciting CTL responses in either direction by in vitro
stimulation. Analysis of such CTL awaits experiments with repeated in
vivo stimulation by skin grafting.
The recently solved crystal structure of an allo-TCR coupled to its MHC ligand revealed that alloreactive TCRs interact with allo-MHCs in a mode similar to one used in self-MHC recognition (30), with a contact surface spanning diagonally across the peptide-binding groove. In light of this emerging universal principle for TCR interaction with both allo- and self-MHC molecules, it would be interesting to investigate the MHC contacts of TCRs that recognize self-MHC complexes in a peptide-independent manner. The question of physiological importance is whether self-MHC-restricted peptide-independent CTLs can escape negative selection and be found on the periphery, and if so, could they have any relevance for immunopathogenesis?
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Acknowledgments |
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ugich for expert flow
cytofluorometric analysis. |
Footnotes |
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.). 
2 V.J., K.R., and A.M. contributed equally to this work
and are therefore designated joint first authors.
3 Current address: Department of Microbiology and
Immunology, Albert Einstein College of Medicine, Yeshiva University,
Bronx, NY 10461.
4 Address correspondence and reprint requests to Dr. Janko Nikolich-ugich, Vaccine and Gene Therapy Institute, 505 185th Avenue, Beaverton, OR 97006. E-mail address: nikolich{at}ohsu.edu
5 Abbreviations used in this paper: pMHC, peptide-MHC; FCM, flow cytofluorometry; FLK-8, dominant Kk-restricted influenza nucleoprotein peptide 52–60; HSV-8, HSV glycoprotein B peptide 495–502 (SSIEFARL); OVA-8, OVA peptide 257–264; VPY-9, synthetic peptide VAPYRLILL-9, based on the pool sequence of natural peptides eluted from KbW9; FRET, fluorescence resonance energy transfer.
Received for publication December 26, 2001. Accepted for publication June 4, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex. Science 274:176.
atipovi, B., G. Talluri, J. Oh, T. Wei, X. Su, T. E. Johansen, M. Edidin, J. P. Schneck. 1994. Analysis of the structure of empty and peptide-loaded major histocompatibility complex molecules at the cell surface. J. Exp. Med. 180:1753.-
ugi. 1998. Peptide selection by an MHC H-2Kb class I molecule devoid of the central anchor ("C") pocket. J Immunol. 160:2815.-ugi. 1999. The mode of ligand recognition by two peptide:MHC class I-specific monoclonal antibodies. J. Immunol. 163:3286.-ugi. 1995. CD4-independent in vivo priming of murine CTL by optimal MHC class I-restricted peptides derived from intracellular pathogens. Int. Immunol. 7:1205.-helices of the class I molecule are simultaneously recognized by the T cell receptor. Cell 54:47.[Medline]
-ugi. 1996. T cell receptor (TCR) recognition of MHC class I variants: intermolecular second-site reversion provides evidence for peptide/MHC conformational variation. J. Exp. Med. 184:253.2-microglobulin and presentation of a viral peptide. J. Immunol. 155:3481.[Abstract]
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and 
,
Binding of indicated Kb-specific Abs to Kb and
KbW9 transgenic splenocytes, respectively. Results are
shown as percentage of mean fluorescence intensity obtained after Y3
staining (Y3 index). Results are representative of three independent
experiments.
and 
) or P815-KbW9 cells (
and 
), incubated in the absence (
and 
