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,§,§
,§,§
*
Department of Molecular Immunology, Merck Research Laboratories, Rahway, NJ 07065; Departments of
Medicine and
Pediatrics, University of Tennessee, Memphis, TN 38163; and
§
Veterans Affairs Medical Center, Memphis, TN 38104
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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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One approach to the problem of determining the functional role of HLA class II molecules associated with autoimmune diseases has been to establish them as transgenes in animal models. By this means, hypotheses can be tested experimentally in ways previously not possible. Since HLA class II interacts poorly with murine CD4 (14), optimal function of these transgenes in the mouse has required that either human CD4 be coexpressed or a chimeric HLA/I-E molecule in which the HLA second domains are exchanged for I-E, thus enabling murine CD4 to interact, be present (15). We recently used this latter approach in studying the function of the RA susceptibility allele, HLA-DR1 (DRB1*0101) (16). Using the model Ag CII, we found that transgenic expression of this DR1 allele conferred susceptibility to an autoimmune arthritis induced by immunization with human CII (hCII), and that the autoimmune response was mediated by DR1-restricted, hCII-specific T cells (16). In the studies presented here we extend these observations to a second RA susceptibility allele, HLA-DR4 (DRB1*0401). We demonstrate that this DR4 susceptibility allele also binds and presents peptides derived from hCII and, as a transgene, confers susceptibility to hCII and bovine CII (bCII)-induced autoimmune arthritis in an otherwise nonsusceptible mouse strain. The autoimmune arthritis was accompanied by a strong T cell and B cell response that reacted with both the autoantigen, murine CII (mCII), and cross-reacted with other heterologous CII. The autoimmune T cell response was mediated by DR4-restricted presentation of the hCII peptides CII263–270 and CII285–300, the same hCII peptides presented by the RA susceptibility allele HLA-DR1 (*0101) (16). These data support the hypothesis that an autoimmune response to CII plays a role in the pathogenesis of RA and indicate that if the immunopathogenesis of RA is initiated via the Ag presentation function of these HLA alleles, it may be possible to immunotherapeutically target these RA susceptibility alleles as a group.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.M-DR4+/- (DRB1*0401) transgenic mice and
B10.M (H-2f) nontransgenic littermates were raised in our
animal facility at the Veterans Administration Medical Center (Memphis,
TN) in a specific pathogen-free environment. Sentinel mice were tested
routinely for the presence of mouse hepatitis and Sendai viruses. The
DR4 transgene was constructed as a chimeric molecule composed of DR4
1 and ß1 domains and I-E 2 and ß2 domains. The production of
the chimeric genes and the B10.M-DR4 transgenic mouse has been
previously described (15). The B10.M-DR4 mice were maintained as
heterozygotes for the DR4 transgene, since homozygous B10.M-DR4 mice
from this founder do not survive.
Collagen preparation
Native bCII was solubilized from articular cartilage; native mCII was solubilized from sternal cartilage of young mice; native hCII was solubilized from sternal cartilage harvested from donors younger than 20 yr of age. Each CII was extracted by limited proteolysis with pepsin and purified by repeated differential salt precipitation as previously described (17, 18).
Immunizations
Six- to eight-week-old mice were immunized with hCII or bCII for the induction of arthritis. The CII was dissolved in cold 10 mM acetic acid by stirring overnight at 4°C and was emulsified at a 1:1 (v/v) ratio with CFA (Life Technologies, Gaithersburg, MD), as previously described (19). Mice were immunized s.c. at the base of the tail with 100 µg of CII. For some experiments mice were boosted 2 wk later with 100 µg of CII in IFA. Each paw was evaluated and scored for the degree of inflammation on a scale of 0 to 4 (18, 20).
Peptide synthesis
Peptides were synthesized by F-moc chemistry using an Applied Biosystems (model 430, Foster City, CA) automated peptide synthesizer or manually using the mimotope cleavable pin technology (Chiron Mimotopes, Victoria, Australia) as previously described (16, 21, 22). Mimotope peptides representing the entire length of hCII (>1000 amino acids) were synthesized as 15-mers with a 12-amino acid overlap. Peptides were cleaved from the mimotope pins by an overnight incubation in 750 µl of 50 mM HEPES buffer. Select peptides were analyzed and quantitated by reverse phase HPLC, and all were >80% pure and contained 2 to 4 µg/µl of peptide.
Proliferation assays
Ten days after immunization, draining lymph nodes were removed, disassociated, and washed in HL-1 (BioWhittaker, Walkersville, MD). Lymphocytes were cultured in 96-well plates at 4.5 x 105/well in 300 µl of HL-1 medium supplemented with 50 µM 2-ME and 0.1% BSA (fraction V, IgG free, low endotoxin, Sigma Chemical Co., St. Louis, MO) at 37°C in 5% humidified CO2 for 4 days. Eighteen hours before the termination of the cultures, 1 µCi of [3H]TdR (New England Nuclear, Boston, MA) was added to each well. Cells were harvested onto glass-fiber filters and counted on a Matrix 96 direct ionization beta counter (Packard Instrument Co., Meriden, CT). Proliferation assays using mimotope synthetic peptides were performed at one well/peptide and 10 µl of peptide/well. Results were confirmed by replicate experiments, and all data are expressed as disintegrations per minute(dpm).
T cell hybridomas and Ag presentation assays
T cell hybridomas were established by polyethylene glycol
(Boehringer Mannheim, Indianapolis, IN)-induced fusion of lymph node
cells with TCR -/ß- BW5147 thymoma
cells (23, 24). Lymph node cells were obtained from B10.M-DR4 mice
immunized 10 days previously with hCII/CFA. The recovered T cells were
cultured with human 1(II) for 5 days, followed by IL-2 for 3 days
before fusion. Resulting hybridomas were screened for their ability to
recognize human 1(II) chains presented by DR4 and I-Af.
Ag presentation experiments were performed in 96-well microtiter plates
in a total volume of 0.3 ml containing 105 APC or 4 x
105 syngeneic spleen cells and 105 T hybridoma
cells. The following APC were used: L243.6, L cells (L66) transfected
with wild-type DRA1*0101 and DRB1*0401 (25); MUM21, cells transfected
with chimeric DR4 constructs (15); and 43.2.1, a B cell hybridoma that
expresses I-Af and I-Ad. Cell cultures were
maintained at 37°C in 5% humidified CO2 for 20 to
24 h, after which twofold serial dilutions were made for
determination of IL-2 titers. Four thousand HT-2 cells were added to
each supernatant dilution, and after 16 to 20 h, HT-2 cell
viability was assessed by visual inspection and cleavage of MTT (26, 27). IL-2 titers were quantified by the reciprocal of the highest
twofold serial dilution maintaining 90% viability of the HT-2 cells.
Results are presented as units of IL-2 per milliliter of undiluted
supernatant as described by Kappler et al. (28).
ELISA
Ab titers specific for hCII and mCII were determined using a solid phase ELISA as previously described (29). Briefly, microtiter plates were coated with 500 ng of either hCII or mCII at 4°C overnight. After extensive washing with 0.15 M saline/0.05% Tween-20, dilutions of sera ranging from 1/4,000 to 1/24,000 in 2% normal goat sera were added to each well and incubated overnight at 4°C. After washing with saline and Tween-20, a goat anti-mouse Ig (1/5,000) was added for 2 h. The plates were then washed and developed by the addition of o-phenylenediamine (Sigma). After stopping the reaction with 2.5 N H2SO4, the degree of color development was measured at 490 nm with background absorbance of 650 nm subtracted. The quantity of specific Ab was measured for each animal, and data are expressed as mean relative units of activity based on a standard anti-hCII serum.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A chimeric HLA-DR4 (DRB1*0401) molecule in which the second
domains of the molecule were replaced with murine I-E domains was
established as a transgene in B10.M mice to enable functional studies
of the DR4 molecule (15). The DR4 molecule was rendered chimeric with
murine I-E to enable murine CD4 interaction with the HLA molecule, thus
enhancing its ability to stimulate T cells. We used this transgenic
model to address the question of whether HLA-DR4 is capable of
mediating the induction of autoimmune arthritis following immunization
with CII. As shown in Figure 1
, B10.M-DR4
mice are susceptible to autoimmune arthritis induced by immunization
with both hCII (Fig. 1, A and B) and bCII
(Fig. 1B). An incidence of arthritis as high as 70%
was achieved in the B10.M-DR4 mice when a booster immunization was
given, whereas a 40 to 50% incidence was routinely observed for mice
receiving a single immunization (Fig. 1A). Mice
developing arthritis did so with a severity similar to that seen in
DBA/1 mice immunized with bCII (data not shown), and ankylosis of
affected joints was often observed among the B10.M-DR4 arthritic limbs
as it is in DBA/1 arthritic limbs. Histologically, no differences were
observed between the collagen-induced arthritis in B10.M-DR4 mice and
what was previously described for DBA/1 mice (data not shown). In
comparison, B10.M nontransgenic littermates were highly resistant to
developing autoimmune arthritis. Of >50 B10.M (H-2f) mice
tested to date, only one developed arthritis following immunization
with hCII.
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) and a B cell (Fig. 3) immune response. The T cell response
to native hCII was characterized by a significant cross-reaction with
both heterologous chick CII (cCII) and bCII as well as with the
autoantigen, mCII (Fig. 2). The T cell response of the nontransgenic
B10.M littermates to hCII was poor, but clearly present (Fig. 4) (16). Correspondingly, as shown in
Figure 3, Ab responses to both immunogens, hCII and bCII, and the
autoantigen mCII were significantly higher in the B10.M-DR4 mice than
in the B10.M litter mate controls. These data indicate that hCII is a
strong immunogen in the HLA-DR4 transgenic mice, but stimulates only a
weak, but measurable, T cell and B cell response in the nontransgenic
B10.M mice.
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Antigenic determinants derived from hCII and presented by the DR4
molecule were identified using an extensive panel of mimotope peptides
(Fig. 4
). These peptides, 15-mers overlapping by 12 residues and
spanning the entire length of the 1(II) chain of hCII, were tested
for their ability to stimulate hCII-primed T cells from B10.M-DR4 and
B10.M mice. As shown in Figure 4, a minimum of three antigenic
determinants were clearly present within the B10.M-DR4 proliferative
response to hCII, and the sequences of these peptides are listed in
Table I. The immunodominant determinant,
represented by three consecutive stimulatory peptides in Figure 4A (peptides 256, 259, and 262), was detected only by the T
cell response of B10.M-DR4 mice. Since this determinant was present
within three consecutive peptides, a core determinant of CII263–271
can be deduced by alignment of the overlapping peptides (Table I). In
addition to this dominant determinant, two others were present in the
B10.M-DR4 response, one within peptide CII250–264 and the other within
peptide CII286–300. The hCII-primed T cells from B10.M
(H-2f) mice responded to only a single mimotope peptide
(peptide 250; Fig. 4B), identifying a weak hCII
determinant presented by the I-Af molecule, CII250–264.
This single peptide was consistently, weakly stimulatory (
10,000
dpm) in all B10.M Mimotope proliferation assays performed in both these
studies and others (16). In addition, it corresponds precisely with the
weak determinant present in the B10.M-DR4 Mimotope proliferative assay
(Fig. 4A), an observation consistent with the fact
that the B10.M-DR4 mice express both HLA-DR4 and I-Af.
In all, these data imply that the antigenic determinants
within CII263–270 and CII286–300 are presented by the DR4
molecule.
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The fact that B10.M-DR4 mice express both DR4 and
I-Af allows for the possibility that a mixed isotype
molecule may be involved in the presentation of the dominant and
subdominant peptides identified in Figure 4. To address this issue, T
cell hybridomas specific for the hCII263–270 determinant were produced
from hCII-immunized B10.M-DR4 mice, and a representative sample of >40
hybridomas is shown in Figure 5. Using
B10.M-DR4 spleen cells as APC, these T cell hybridomas recognized the
same mimotope peptides as those identified in Figure 4A as
containing the dominant determinant. When B10.M (H-2f)
spleen cells or a B cell hybridoma expressing I-Af was used
as APC, none of the hybridomas recognized the hCII263–270 antigenic
determinant (Table II). In contrast, all
the T cell hybridomas recognized this determinant when transfected cell
lines expressing the chimeric DR4 or wild-type DR4 were used as APC,
including the transfected L cells (L243) which do not express any other
class II molecules. We have also examined >100 T cell hybridomas from
B10.M mice immunized with hCII, and none of these hybridomas recognized
the hCII263–270 determinant. Thus, these data clearly demonstrate that
TCR recognition of the hCII263–270 immunodominant determinant is
restricted by the HLA-DR4 (*0401) molecule.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of hypotheses have been proposed for the disease-related function of the RA-associated HLA-DR molecules (5, 6, 8, 36). These hypotheses range from sequence-specific functions of the DR molecules controlling the presentation of pathogenic peptides or stimulating pathogenic T cells (8) to the DR alleles acting as a peptide donor influencing the function of an HLA-DQ molecule (5, 6). The data described here clearly demonstrate that, like the DR1 molecule, DR4 has the capacity to mediate an autoimmune response to hCII. Indeed, not only do both these DR molecules bind peptides derived from hCII, they appear to bind and present the exact same hCII peptides. Alignment of Mimotope hCII peptides that stimulate DR4-restricted T cell proliferation indicates that the determinant cores are identical for DR1 and DR4, although it has yet to be demonstrated that they use identical anchor residues in the binding of these peptides. Using the sequence of bCII and an algorithmic prediction, Fugger et al. have described a peptide sequence, bCII261–273 that is immunogenic in an HLA-DR4 (*0401) human CD4 transgenic mouse (37). The amino acid sequence of this bCII peptide and that of the human immunodominant determinant described here, hCII263–270, are identical. Indeed, we have found that there is a significant amount of T cell cross-reactivity among bCII, cCII, and hCII, and that immunization with bCII induces autoimmune arthritis in the chimeric B10.M-DR4 mouse described here.
Although transgenic expression of both DR4 and DR1 (16) confers susceptibility to hCII-induced autoimmune arthritis, the incidence of arthritis is clearly different between these two transgenic strains. Despite the fact that these class II molecules appear to bind and present the same antigenic determinants from hCII, B10.M-DR1 mice develop autoimmune arthritis at nearly a 100% incidence and at an accelerated rate (16) compared with the B10.M-DR4 mice, in which arthritis develops somewhat slower, and the incidence ranges from 40 to 70% with booster immunizations. There are several possible explanations for these differences. First, the expression level of the DR4 transgene is approximately 10-fold less than that of the B10.M-DR1 (15, 16), a small part of which is due to the heterozygous state of the DR4 transgene. Homozygous DR4 mice from this founder do not survive after birth, probably an unfortunate result of the transgene inserting itself into a critical gene. Second, although they bind the same peptides, the affinity of DR4-hCII peptide binding may be less than that of DR1, an observation supported by the consistently lower T cell proliferative responses to hCII by DR4 T cells compared with the responses of DR1 T cells (16). We are currently in the process of measuring the relative affinities of DR1 and DR4 for the CII263–270 immunodominant determinant. Even though these DR molecules bind and present the same hCII peptide, the DR-restricted T cells fully discriminate between DR1-hCII and DR4-hCII ligands. No cross- or allorecognition has been observed between DR1 T cells and DR4-hCII ligands or between DR4 T cells and DR1-hCII ligands.
Chimeric transgenes of DR4 and DR1 were constructed for the purpose of enabling murine CD4 interactions with these HLA molecules. Murine CD4 interacts poorly with HLA-DR, and it is clear from the work of others that the lack of CD4 interaction reduces the efficacy of class II-mediated T cell stimulation (14, 38). Although the chimeric DR molecules enabled CD4 interaction, surprisingly none of the >30 DR4- or DR1-restricted, hCII-specific, T cell hybridomas we have established to date requires the chimeric molecule for stimulation. Each recognizes the CII263–270 determinant in the context of wild-type DR1 or DR4 as efficiently as in the context of their chimeric counterparts, although different transfected cell types and potential differences in expression levels of the transfected DR molecules may contribute to this. Whether CD4 interaction is only critical at the level of primary stimulation of hCII-specific T cells or only high affinity T cells are selected during hCII immunization is unclear. In addition, CD4 interaction may only be required for a T cell during its early stages of development, such as during positive selection or its primary stimulation, and is less essential during its subsequent interaction with class II and Ag in the periphery.
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Footnotes |
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2 Abbreviations used in this paper: RA, rheumatoid arthritis; hCII, human type II collagen; bCII, bovine type II collagen; mCII, murine type II collagen; cCII, chick type II collagen.
Received for publication September 11, 1997. Accepted for publication November 14, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains. Biochemistry 10:1652.[Medline]
1(II)-CB11 in H-2q mice. J. Immunol. 152:3088.[Abstract]
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; mice were boosted 2 wk after primary immunization
with hCII/IFA), n = 9 for B10.M-DR4 mice (
; not
boosted), n = 20 for the B10.M group (•; boosted)).B, Mice were immunized with bCII (no booster immunization;n = 6 for the DBA/1 group (
), n = 11
for the B10.M-DR4 group (
). Data are
representative of three experiments and are expressed as dpm with
background subtracted (
dpm). Background = 5525 dpm.
