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R. W. Johnson Pharmaceutical Research Institute, San Diego, CA 92121
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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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In this study, we have analyzed the extent and quality of the humoral responses in DM-/- mice. We found that after immunization these animals were capable of mounting T cell-dependent Ig production in vivo, although the quality of the responses in terms of kinetics, titers, and affinity maturation were decreased. The responses were improved, but not normalized in DM-/--deficient mice expressing an H2-Ead transgene. To investigate the molecular basis for the T-B interaction resulting in Ab production, we analyzed the presentation of multiple epitopes derived from the two protein Ags used for the immunization studies, OVA and hen egg lysozyme (HEL), and show that presentation by H2-Ab and H2-Ed/b molecules in general is highly dependent on DM, whether B cells internalized Ags by fluid phase or after uptake via membrane IgM receptors. Together the data presented demonstrate an essential role for DM in humoral immune responses including affinity maturation, but confirms that different class II molecules show different sensitivities to the absence of this molecule.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of DM-deficient (DM-/-) mice has been described (11). Mice transgenic for membrane IgM receptors recognizing phosphorylcholine (21) or trinitrophenyl (22) were obtained from Dr. J. Kenny (Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD). Mice transgenic for H2-Ead were obtained from C. Surh (Department of Immunology, The Scripps Research Institute, La Jolla, CA). Such mice predominantly express H2-Ab and H2-Ed/b MHC class II molecules. All transgenic mice were used as heterozygotes at 6–10 wk of age.
Mice were immunized with 100 µg of 4-hydroxy-5-nitophenylacetyl-OVA (NP-OVA) or NP6-HEL (NP-HEL; Biosearch Technologies, Novato, CA) in alum (Sigma-Aldrich, St. Louis, MO) i.p. Mice were eye-bled under anesthetic, sera isolated, and stored at -20°C.
Immunoprecipitations
Splenocytes were labeled with [35S]methionine as indicated before lysis in 1% Triton X-100, PBS, and complete proteinase inhibitor (Roche Diagnostics, Indianapolis, IN). H2-Ed/b was immunoprecipitated with mAb 14-4-4S (23). Immunoprecipitates were harvested with protein A, washed, and resuspended in SDS-PAGE sample buffer with 2% SDS and 5% 2-ME. Samples were boiled for 3 min, then separated on 7.5–12.5% polyacrylamide gels. Gels were fixed, dried, and autoradiographed. Autoradiographs were scanned using an Agfa Arcus II scanner (Ridgefield Park, NJ). Composites were printed on a Kodak XLS 8600 printer (Kodak, Rochester, NY).
T cell hybridomas
DM+/+ mice were immunized in the hind
footpad with 50 µg of OVA (Sigma-Aldrich) or HEL (Calbiochem, La
Jolla, CA). After 7–10 days, draining lymph node (LN) cells were
restimulated in vitro with OVA or HEL (50 µg/ml) and T cell blasts
were expanded in RPMI 1640 with 10% FBS and 5% supernatant
from Con A-stimulated mouse splenocytes. T cells were
restimulated in vitro and fused with BW5147/TCR
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- thymoma cells
(24). Ag-specific T cell hybridomas were selected and
expanded using standard protocols. The exact epitopes have not been
mapped for all OVA-specific hybridomas, but HPLC-fractionated tryptic
digests of OVA define distinct specificities between the individual
hybridomas. HEL-specific T cell hybridomas H30.44 and H46.13 were made
after immunization of DM+/+ mice with peptides
HEL 30–53 and 46–61, respectively. LN cells were stimulated in vitro
with HEL and subsequently fused with BW5147/TCR
-- cells.
OVA-specific hybridoma 426.6 was generated after fusion of activated
CD4+ T cells from OT-2-transgenic mice. T cell
hybridoma BO4 was obtained from A. Rudensky (Department of Immunology
and Howard Hughes Medical Institute, University of Washington School of
Medicine, Seattle, WA).
H2-Ed/b-restricted T cell
hybridomas were generated after immunization of
H2-Ed/b
H2-A-/- mice (generated by breeding
H2-Ead-transgenic mice with
H2-Abb-deficient mice) with HEL or OVA.
Ag presentation assays
Primary B cells (purification described previously (25)) were cultured (4 x 105 cells/well) in triplicate with T cell hybridomas (2 x 105 cells/well) and Ag. Supernatants from overnight cultures were assayed for IL-2 production by ELISA. For Ig receptor-mediated uptake and presentation to T cell hybridomas, B cells were incubated on ice for 30 min, followed by a 30-min pulse with Ag on ice. Non-Ig-bound Ag was removed by centrifugation through a FBS density gradient. Ag-pulsed B cells were split equally between the T cell hybridomas used (giving 4 x 105 B cells/well) and cultured overnight. Data (IL-2, ng/ml) are expressed as mean values of triplicate cultures ± SD. Trinitrophenyl (TNP)-conjugated OVA (TNP-OVA) was obtained from Biosearch Technologies. Phosphorylcholine (PC)-conjugated OVA (PC-OVA) and PC-HEL were prepared as described elsewhere (26).
For recall responses in vitro, mice were immunized in the hind footpad with peptide HEL 20–35 in IFA. After 8 days, draining LN cells were isolated and cultured (2 x 105/well) in triplicate with Ag for 72 h. Cell proliferation was determined by [3H]thymidine incorporation during the final 16 h of culture. Cells were counted using a Topcount (Packard Instrument, Meriden, CT) with liquid scintillation (Microscint; Packard Instrument).
Data are expressed as mean cpm ± SD.
Ab assays
NP-specific Abs were measured by ELISA using plates coated with NP23-BSA or NP2-BSA (1 µg/ml; Biosearch Technologies) in PBS to determine total or high-affinity Abs, respectively. Bound Ig was detected using alkaline phosphatase-conjugated goat anti-mouse IgM or IgG subclass Abs (Southern Biotechnology Associates, Birmingham, AL) and assays were developed using Sigma 104 (Sigma-Aldrich) in diethanolamine substrate buffer (Pierce, Rockford, IL). OD 405 nm was measured and values are expressed as arbitrary units after reference to standard hyperimmune anti-NP sera.
Immunohistochemistry and immunofluorescence
Mice were immunized with 100 µg of NP-OVA in alum (i.p.). Spleens were removed, embedded in OCT Tissue-Tec (Sakura Fine Tek, Torrance, CA) and frozen on dry ice 7 or 14 days after immunization. After acetone fixation, cryosections were incubated with fluoroscein-conjugated peanut agglutinin (FITC-PNA) (Vector Laboratories, Burlingame, CA) or biotinylated NP23-BSA followed by Alexa 594-streptavidin (Molecular Probes, Eugene, OR). For the immunohistochemistry, biotin-PNA or biotin-conjugated primary Abs and an avidin-biotin complex kit with diaminobenzidine were used (Vector Laboratories) along with hematoxylin counterstaining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ab responses have not been extensively studied in DM-deficient mice, but immunized animals have been reported to be able to generate IgG responses to keyhole limpet hemocyanin (KLH), as well as to vesicular stomatitis virus (27). This is somewhat surprising considering that presentation of internalized Ags in the absence of DM is usually poor, and thus we decided to investigate T-dependent Ab responses more extensively.
DM-/- mice or wild-type littermates were
immunized i.p. with a high dose (100 µg) of NP-OVA or NP-HEL in alum.
Mice were bled at the time points indicated in Fig. 1
. After immunization,
DM-/- mice generated a substantial NP-specific
IgG1 response in the NP-OVA immunized mice, although the primary IgG1
response was diminished and delayed compared with that of wild-type
mice (Fig. 1, a and b). At later stages of the
response, the magnitude of the IgG1 response showed a 10- to 20-fold
reduction in DM-/- mice compared with wild-type
mice. This reduction remained also after a booster immunization (data
not shown). The poor serum IgG responses in
DM-/- mice were, at least in part, caused by a
reduced number of plasma cells, since ELISPOT analysis showed a marked
reduction in the number of NP-specific Ab-secreting cells (data not
shown).
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, c and
d), yet the IgM response was normal, showing the
immunization to be successful (data not shown). The ability to produce anti-NP Abs in the NP-OVA-immunized DM-deficient mice suggests that for this Ag sufficient Ag processing occurs to allow productive, although inefficient T-B cell interaction.
Severely impaired affinity maturation correlates with aberrant germinal center formation in DM-/- mice
The delayed and reduced Ab response to NP-OVA prompted us to
investigate whether DM influenced the generation of high-affinity Abs.
Levels of high-affinity NP-specific Abs were determined by binding sera
from NP-OVA-immunized mice to plates coated with a sparsely haptenated
substrate (NP2-BSA) (Fig. 2). Strikingly, high-affinity NP-specific
IgG1 Abs were virtually undetectable throughout the primary response of
DM-/- mice, whereas the characteristic delayed
production of high-affinity Abs during the primary response was
observed in wild-type mice. The almost complete absence of
NP2-binding Abs shows that
DM-/- mice have a severe defect in affinity
maturation, and thus that DM is essential for this process.
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A, a and d, and
B, a and c, analysis of spleen sections from
nonimmunized mice showed a few small germinal centers. After NP-OVA
immunization, large germinal centers were abundant (using PNA staining)
in wild-type spleen both at days 7 and 14 (Fig. 3A,b and c, and Bb). In contrast,
germinal centers were virtually undetectable in splenic sections from
DM-/- mice (Fig. 3A, e and
f, and Bd). Using high magnification, small
numbers of atypically organized PNA-reactive cells were occasionally
detected in the sections from DM-/- mice (Fig. 3Bh). Analysis of Ag-specific B cells at day 7 (using
NP-staining) revealed few Ag-specific cells in sections from
DM-/- mice (Fig. 3B, i and
j), whereas such cells were readily detectable in germinal
centers in the wild-type spleen sections (Fig. 3B, e–g).
Similar results were obtained at day 14 (data not shown). These data
show that both germinal center formation and the resulting maturation
of Ab affinity are significantly reduced in
DM-/- mice.
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Two reports have described H2-E-expressing DM-deficient mice,
either by expression of a H2-Eak transgene
(14) or by deletion of DM in BALB/c stem cells
(15). H2-E molecules from these mice had decreased SDS
stability, as well as an increased content of CLIP peptides when
compared with H2-E molecules from wild-type mice. Pulse-chase analysis
of metabolically labeled splenocytes show that also
H2-Ed/b molecules expressed in
DM-/- mice (generated by breeding with
H2-Ead-transgenic mice) are to a large extent
occupied by CLIP (Fig. 4A). A
large part of the H2-Ed/b
molecules were associated with CLIP after 2 h of chase and the
CLIP content did not substantially decrease after a 24-h chase,
suggesting that CLIP-H2-Ed/b
complexes are very stable under physiological condition. However, the
complexes are not SDS stable even if boiling is omitted before gel
analysis (data not shown).
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B, a and
b, show that the presence of the
H2-Ead transgene resulted in
DM-/- mice mounting IgG1 responses comparable
to those of wild-type mice; both the kinetics and the magnitude of the
primary response were restored to near normal levels. Moreover,
high-affinity IgG1 Abs were readily detectable during the primary
response (Fig. 4B, c–f), although the restoration of
affinity maturation in H2-Ead-transgenic
DM-/- mice was partial rather than
complete.
Germinal center formation in H2-Ead-transgenic
mice was investigated 7 or 14 days after immunization with NP-OVA in
alum (i.p.). Fig. 5 shows that few if any
PNA-positive cells were detectable in the sections from the
DM-/- H2-Ead-transgenic
mice, either at day 7 or day 14.
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Ag presentation by DM-/- B cells
Several reports have demonstrated that normal MHC class II-restricted Ag presentation is dependent on DM (reviewed in Ref. 6), and this is particularly true in the case of H2-Ab, which appears to be exquisitely DM sensitive. However, with a couple of exceptions (14, 28), most studies to date have described the presentation of single epitopes from different Ags, and it is not clear that different epitopes in a particular Ag are equally DM dependent.
The Ab responses to NP-OVA in the DM-deficient
H2-Ab-expressing mice, as well as the
H2-Ead-transgenic animals, led us to investigate
whether some epitopes were presented by H2-Ab or
H2-Ed/b on B cells also in the
absence of DM. To do this, we generated
H2-Ab-restricted T cell hybridomas against
several distinct epitopes of OVA and HEL and several
H2-Ed/b-restricted hybridomas
against OVA. Fig. 6 shows the IL-2
response of OVA- and HEL-specific T cell hybridomas after overnight
culture with intact protein together with primary B cells from
wild-type or DM-/- mice (Fig. 6, a,
c, and e). All of the T cell hybridomas
recognized processed Ag presented by wild-type B cells in a
dose-dependent MHC-restricted manner. In contrast, with one exception,
all H2-Ab-restricted T cell hybridomas examined
completely failed to recognize Ag presented by
DM-/- B cells even at the highest doses,
indicating a strict DM dependency for presentation of different
epitopes by H2-Ab molecules (Fig. 6, a
and c). Surprisingly, we found that a single T cell
hybridoma (Hb1.9) recognized HEL processed and presented by
DM-/- B cells equally well as HEL presented by
wild-type cells, suggesting that this epitope (HEL 20–35) was
presented by H2-Ab independently of DM. Three of
the four H2-Ed/b-restricted
OVA-specific T cell hybridomas examined were essentially unable to
recognize OVA presented by DM-/- B cells, yet
responded well to OVA presented by DM-expressing B cells (Fig. 6e). A small response could be reproducibly elicited from T
cell hybridoma OEb14 at a high Ag doses by
DM-/- B cells, although this response was
greatly reduced compared with the response to
DM+/+ B cells. This result suggests that, similar
to the presentation of OVA by H2-Ab, the
presentation by H2-Ed/b molecules
is predominantly dependent on DM.
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Presentation of exogenous peptides has also been reported to be altered
for DM-defective cells (12, 13, 14, 15, 16). In this study, culturing
synthetic peptides of OVA or HEL with DM-/-
H2-Ab-expressing B cells generally elicited 10-
to 100-fold weaker responses from T cell hybridomas than when the
peptides were presented by wild-type cells (Fig. 6. b and
d). In contrast to other peptides, the presentation of
peptide HEL 20–35 to hybridoma Hb1.9 was similar for both wild-type
and DM-deficient B cells, as was observed for the intact HEL. Dose
titration of peptide HEL 20–35 suggested that Hb1.9 was extremely
sensitive to peptide-loaded H2-Ab molecules.
Together, these data show that DM is essential for the process-ing and presentation of a number of different H2-Ab- and H2-Ed/b-restricted epitopes derived from OVA and HEL. We were unable to find any H2-Ab-restricted OVA epitopes that were presented by the DM-deficient cells and only one H2-Ed/b-restricted epitope that was recognized, although very poorly. In contrast, one H2-Ab-restricted HEL-reactive T cell hybridoma appeared to recognize the relevant epitope in a DM-independent manner.
Poor recognition of processed HEL 20–35 in DM-/- mice
Hybridoma Hb1.9 appeared to recognize an epitope from HEL
independently of DM, yet no IgG response against NP-HEL could be
detected after immunization. If hybridoma 1.9 was representative of HEL
20–35-reactive T cells, then the lack of an Ab response to HEL would
suggest that no HEL 20–35-reactive T cell precursors were present in
the DM-/- mice. To address whether this was the
case, DM+/+ and DM-/-
mice were immunized with peptide HEL 20–35 in IFA. Draining LN cells
were isolated 8 days later and assayed in vitro for recall responses to
HEL. Fig. 7 shows that both primed
DM-/- and DM+/+ LN cells
proliferated vigorously to the immunizing peptide, demonstrating that
HEL 20–35-reactive T cell precursors were present in the
DM-/- mice. In contrast, only a minimal
response by the DM-/- cells was observed after
culture with intact HEL, suggesting that the repertoire of HEL
20–35-specific T cells was essentially unable to recognize
endogenously processed HEL presented by DM-/-
LN cells in vitro. In contrast, wild-type LN cells recognized intact
HEL very well, implying this HEL epitope to be efficiently processed by
DM+/+ LN cells. The lack of reactivity of
the DM-/- T cells in response to intact HEL is
likely to reflect poor presentation by the
DM-/- APCs, suggesting that hybridoma 1.9 is
not representative of HEL 20–35-reactive T cells in
DM-/- mice.
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It has previously been reported that receptor-mediated uptake of
Ag can overcome the HLA-DM dependency of Ag presented by HLA-DR4
molecules (29), and thus we investigated the DM dependence
of presentation after Ig receptor-mediated uptake of Ag by B cells.
Mice bearing transgenes encoding one of two independent membrane IgM
receptors specific for the haptens TNP (22) or PC
(21) were bred together with
DM-/-, H2-Ead-transgenic
DM-/-, or control mice. Transgenic B cells from
the different mouse strains were analyzed for their ability to present
multiple epitopes after Ig-mediated uptake of hapten-conjugated Ags
(Fig. 8). Primary splenic B cells were
pulsed with Ag for 30 min at 4°C, separated from excess non-Ig-bound
Ag, and cultured overnight with T cell hybridomas at 37°C. The
majority of H2-Ab-restricted T cell hybridomas
examined recognized Ag presented by Ig-transgenic wild-type B cells
after Ig-mediated uptake, although variations were observed in the
responses of individual hybridomas (Fig. 8, a–c). None of
the H2-Ab-restricted hybridomas were able to
recognize Ag after Ig-mediated uptake by DM-/-
B cells regardless of the specificity of the Ig-transgenic B cells,
suggesting that internalization of Ag by membrane Ig was unable to
overcome the DM dependency of presentation by
H2-Ab on B cells. This indicates a strict DM
dependency for presentation by H2-Ab molecules
whether Ag is internalized via fluid phase or via membrane Ig
receptors.
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d). All
T cell hybridomas examined recognized OVA presented by
H2-Ed/b molecules after
Ig-mediated Ag uptake. Analogous to the
H2-Ab-restricted hybridomas,
variations in the dose-response curves of individual
H2-Ed/b-restricted hybridomas could be
detected which may reflect differential presentation of individual
epitopes or variations in hybridoma sensitivity. However,
internalization of Ag via the Ig receptor by
DM-/- B cells could not overcome the defect in
presentation to the
H2-Ed/b-restricted hybridomas.
Thus, as was the case for H2-Ab, Ag presentation
on H2-Ed/b molecules showed
strict DM dependency whether Ag was internalized via fluid phase or by
membrane Ig receptors. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The almost complete absence of affinity maturation in the DM-deficient H2-Ab-expressing mice, as well as the absence of normal germinal centers, indicates that the interaction between B cells and T cells which results in the initial production of class-switched IgG1 Ab is less DM dependent than the later interaction which results in the formation of germinal centers. This may simply be a reflection of the amount of processed Ag that is available; immunization with a small dose of NP-OVA (1 µg) is not sufficient to raise an IgG Ab response in DM-deficient animals (data not shown), and the formation of germinal centers (in the absence of T cells) has been shown to require higher Ag doses than extrafollicular responses (34). However, affinity maturation of the Ab response is largely driven by the competition for increasingly scarce Ag and since there is clearly sufficient Ag present to initiate a response after high-dose immunization (100 µg), the lack of affinity maturation and germinal centers was somewhat unexpected. Our data suggest that germinal center formation involves quantitatively (or qualitatively) more stringent T cell-B cell interactions than the interactions resulting in Ig subclass switching. It is unlikely that the IgG production in these mice reflects bystander activation of B cells as a result of T cell priming by dendritic cells, since IgG production after OVA immunization requires Ag presentation by the B cells themselves (35)
The Ab response after immunization prompted us to analyze Ag presentation in more detail. Different epitopes from a single Ag may display different DM requirements (14, 28), and it was possible that one or several epitopes were still presented relatively efficiently by DM-deficient APCs. Nanda and Sant (28) have suggested that dominant epitopes presented by H2-Ad are DM dependent, while cryptic epitopes are not. This hypothesis could not be tested in the case of OVA presentation by H2-Ab, since cryptic and dominant epitopes have not been characterized and, indeed, only few epitopes have been defined (36, 37). However, despite the fact that DM-deficient mice do raise Ab responses to OVA (which does suggest that one or more epitopes are presented to CD4+ T cells), we were unable to find any differences in the presentation of different OVA epitopes; none of the analyzed epitopes were presented at detectable levels by DM-deficient B cells, whether the Ag was internalized by fluid phase uptake or by receptor-mediated uptake (after haptenation). In contrast, although we could not detect any IgG response against HEL, one of the three HEL-reactive hybridomas, Hb1.9, apparently recognized processed HEL equally well whether this was presented by wild-type or DM-deficient cells. However, Hb1.9 may be exceptional rather than representative for T cells recognizing epitope HEL 20–35.
The inability to detect presentation of any OVA epitope presented by H2-Ab may suggest that the assays we have used are not sensitive enough to detect small, but sufficient amounts of epitopes presented in vivo. Alternatively, other OVA epitopes that we have not analyzed may be better presented than these. The T cell hybridomas in this study were all generated using DM-expressing APCs and thus they are likely to be biased toward recognition of epitopes that are favored by the presence of DM. It is possible that DM-deficient APCs present partly or totally different OVA-derived peptides than DM-expressing APCs.
H2-Ek/b molecules expressed in DM-deficient mice
are CLIP-associated to a large extent (14) and we find
that this is true also for
H2-Ed/b (Fig. 4A). The
Ag-presenting capacity of H2-Ed by BALB/c
splenocytes has been reported to be relatively less DM dependent than
the presentation by H2-Ad (15), and
expression of functional H2-Ek/b in
DM-/- mice led to improved positive selection,
resulting in normalized numbers of thymocytes and peripheral
CD4+ T cells. In
H2-Ead-transgenic DM-deficient mice,
CD4+ T cell numbers are also normalized, yet like
CD4+ T cells from DM-deficient mice expressing
only H2-Ab, the CD4+ T
cells from H2-Ed/b-transgenic
mice react vigorously against wild-type APCs (6). As shown
here, this is likely to be a reflection of the poor capacity to present
protein Ags by H2-Ed/b, resulting
in suboptimal negative selection. The ability of DM-deficient
H2-Ed/b-transgenic B cells (and
spleen cells, data not shown) to present Ag to several different
OVA-restricted hybridomas was found to be severely decreased when
compared with wild-type cells, but a weak response could be detected
(Fig. 6e), in contrast to the situation when the
presentation by H2-Ab was analyzed (Fig. 6, a–d).
Considering the apparent abnormalities in the repertoire of selected T cells and the poor ability to present OVA-derived peptides, it was somewhat unexpected to find that the kinetics and amplitude of the primary Ab response to NP-OVA were almost normalized in the DM-deficient H2-Ed/b-transgenic mice. A degree of affinity maturation was also apparent, despite the fact that germinal centers were largely absent in these mice, as in the nontransgenic DM-deficient mice. The correlation between affinity maturation and the presence of germinal centers is not absolute since affinity maturation can occur also in mice lacking germinal centers (38, 39). In addition, affinity maturation can continue long after the germinal center reaction has ceased (40). On the other hand, NP-Ficoll can induce germinal center formation in the absence of T cells after immunization of mice expressing a transgenic B cell receptor reactive with NP (34), despite the fact that somatic affinity maturation does not occur after NP-Ficoll immunization (41). Thus, it is possible that very few germinal centers present in H2-Ead-transgenic DM-/- mice are adequate to support the degree of affinity maturation seen in the transgenic mice, but it is also possible that affinity maturation may occur outside morphologically distinguishable germinal centers.
In conclusion, we were unable to find any consistent signs of Ag presentation in H2-Ab mice lacking DM, yet these mice were able to mount an IgG1 response to at least some protein Ags. The lack of affinity maturation of the Ab response reveals different requirements for Ag presentation for the T cell-B cell interaction resulting in isotype switching, and the subsequent interactions resulting in affinity maturation of the Ab response. Expression of H2-Ed/b molecules improved the Ab response and allowed a degree of affinity maturation, despite the fact that Ag presentation by H2-Ed/b was almost as DM dependent as the presentation by H2-Ab. Thus, T cell-dependent humoral responses to protein Ags may occur even under conditions where Ag presentation is severely restricted to the point of being undetectable.
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Acknowledgments |
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-- cells, J. H. Robinson for
advice with production of T cell hybridomas, and C. Surh for providing
transgenic mice and good advice. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Lars Karlsson, R. W. Johnson Pharmaceutical Research Institute, 3210 Merryfield Row, San Diego, CA 92121. E-mail address: lkarlsso{at}prius.jnj.com
3 Abbreviations used in this paper: Ii, invariant chain; NP, 4-hydroxy-5-nitrophenylacetyl; HEL, hen egg lysozyme; CLIP, class II-associated invariant chain-derived peptide; LN, lymph node; TNP, trinitrophenyl; PC, phosphorylcholine; KLH, keyhole limpet hemocyanin; PNA, peanut agglutinin.
Received for publication June 28, 2001. Accepted for publication October 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dimers and facilitates peptide loading. Cell 82:155.[Medline]
genes encoding antiphosphorylcholine antibodies. J. Exp. Med. 164:627. genes in a transgenic mouse line. Nature 314:330.[Medline]
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