By
From the * Harold C. Simmons Arthritis Research Center and Department of Internal Medicine; Howard Hughes Medical Institute and Department of Biochemistry; § Department of Pediatrics and
Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235; ¶ Department of Chemistry, UMIST, Manchester, United Kingdom; ** Micromass Ltd, Wythenshawe,
Manchester, United Kingdom; and the
Department of Medicine, University of North Carolina,
Chapel Hill, North Carolina 27599
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Abstract |
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Human histocompatibility leukocyte antigen B27 is highly associated with the rheumatic diseases termed spondyloarthropathies, but the mechanism is not known. B27 transgenic rats develop a spontaneous disease resembling the human spondyloarthropathies that includes arthritis
and colitis. To investigate whether this disease requires the binding of specific peptides to B27,
we made a minigene construct in which a peptide from influenza nucleoprotein, NP383-391
(SRYWAIRTR), which binds B27 with high affinity, is targeted directly to the ER by the signal peptide of the adenovirus E3/gp19 protein. Rats transgenic for this minigene, NP1, were made and bred with B27 rats. The production of the NP383-391 peptide in B27+NP1+ rats
was confirmed immunologically and by mass spectrometry. The NP1 product displaced ~90%
of the 3H-Arg-labeled endogenous peptide fraction in B27+NP1+ spleen cells. Male
B27+NP1+ rats had a significantly reduced prevalence of arthritis, compared with B27+NP
males or B27+ males with a control construct, NP2, whereas colitis was not significantly affected by the NP1 transgene. These findings support the hypothesis that B27-related arthritis
requires binding of a specific peptide or set of peptides to B27, and they demonstrate a method
for efficient transgenic targeting of peptides to the ER.
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Introduction |
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HLA-B27 refers to a group of closely related alleles of the HLA-B locus that shows strong genetic predisposition to the rheumatic diseases termed spondyloarthropathies (1, 2). The association of B27 with these disorders has been known for 25 years, but the molecular mechanism remains to be identified. The known physiologic function of class I MHC molecules such as B27 is to present peptide antigens, predominantly of intracellular origin, to the immune system (3, 4), and it has been hypothesized that the role of B27 in disease pathogenesis involves presentation of one or more "arthritogenic" peptides (5). It has been difficult, however, to find strong experimental support for this hypothesis. Most of the T cells isolated from patients with B27-related disease that are reactive with implicated pathogens have been of the MHC class II-restricted CD4 variety (6), although occasional CD8+ B27-restricted clones have been described (7). Results from studies of B27 transgenic rodents that develop spontaneous arthritis have also not established a definite role for CD8+ B27-restricted T cells (8).
In the studies described here, we have specifically addressed the role of HLA-B27 in transgenic rats expressing this molecule. These rats develop a spontaneous multisystem disease that shares several pathologic features with the human spondyloarthropathies, including arthritis and colitis (8, 12). We provide evidence that the specificity of the peptide population bound to HLA-B27 in vivo has a significant effect on at least one important aspect of the disease course in these rats, peripheral arthritis.
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Materials and Methods |
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Rats.
The transgenic lines on a Lewis (LEW)1 background, 21-4H and 21-4L, each expressing HLA-B*2705 and humanProduction of the NP1 and NP2 Minigene Constructs.
A modification of the method described by Anderson et al. (18) was used to make two minigenes as outlined in Fig. 1 A. Overlapping oligonucleotides were synthesized to encode a 27mer peptide containing the 18mer signal sequence, MRYMILGLLALAAVCSAA, of the adenovirus protein E3/gp19K (19) at the NH2 terminus, and the 9mer influenza A nucleoprotein, NP383-391 (SRYWAIRTR) (20), at the COOH terminus. 40 ng each of the oligonucleotides were annealed, made fully double stranded with Klenow enzyme and dNTPs, and then amplified by PCR with oligonucleotide primers containing SalI and BamHI restriction sites at the 5' and 3' ends, respectively. These sites were used to clone the minigene into the expression vector pHSE3' (21) under the control of the H-2Kb promoter, as shown in Fig. 1 B. This construct is hereafter referred to as the NP1 minigene. A control minigene construct, hereafter called the NP2 minigene, was similarly produced, in which the codon for Arg at P2 of the NP383-391 peptide was replaced by Leu to encode the peptide SLYWAIRTR, which does not bind to B27 (22). The DNA sequences of the minigene inserts were confirmed to be correct by dideoxy sequencing. Preliminary transfection experiments in B27+ human and mouse cells lines confirmed expression of mRNA from both constructs and immunologically authentic SRYWAIRTR from NP1 (data not shown). For microinjection into fertilized rat eggs, the NP1 and NP2 inserts were excised from the vector at the flanking XhoI sites.
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Northern Analysis.
Northern blot hybridization of whole cellular RNA was carried out on cell lines and rat tissues as previously described (13). Hybridization specific for the NP1 and NP2 constructs was carried out with a 32P-labeled 1.6-kb BamHI-PstI fragment from the humanFlow Cytometry.
Surface B27 expression on lymphoid cells was detected by flow cytometry with the anti-HLA-B,C mAb B1.23.2, as previously described (13, 15).CTL Generation and Assays.
The expression of antigenic NP1 peptide was assayed in B*2705/hPeptide Analysis.
Metabolic labeling, peptide isolation, fractionation by reverse phase HPLC, and sequence analysis by quadrupole time-of-flight mass spectrometry (QTOF/MS) were carried out as previously described (17, 24).IL-1 Assay.
Clinical Assessment.
Rats were scored twice a week, usually without knowledge of their genotypes, for arthritis, diarrhea, and other clinical manifestations of the B27/hHistopathology.
Formalin-fixed, paraffin-embedded, hematoxylin- and eosin-stained sections of proximal colon and acid-decalcified ankle joints were prepared as previously described (8). Histologic colitis was graded on a semiquantitative scale of 0-4, as previously described (25). All histologic assessment was done without knowledge of the genotypes of the specimen donors.Statistical Analysis.
Comparisons among groups of rats were made by a ![]() |
Results |
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The NP1 and NP2 minigene constructs were used to produce transgenic SD by pronuclear microinjection. Genomic integration of the NP1 and NP2 constructs was observed in nine and six founders, respectively. Two NP1 lines, 293-5 and 300-5, and one NP2 line, 338-2, were established. The relative transgene mRNA levels in spleen in these three lines were 1.0, 1.3, and 3.9, respectively. In an mRNA tissue survey in the 300-5 line, the highest mRNA levels were found in thymus and spleen, with weaker expression in jejunum and liver (data not shown).
Immunologic Identification of the NP1 Transgene Product.To test for the presence of the NP383-391 peptide in the
NP1 rats, the NP1 and NP2 transgene loci were backcrossed to the LEW background and crossed with the B27/
h2m transgenic lines 21-4H, LEW.33-3, and 21-4L. B27
surface expression as detected by flow cytometry was not
significantly affected by the simultaneous expression of either the NP1 or the NP2 transgene (data not shown). Con
A blast LN targets from the single and double transgenic
offspring were tested for lysis by the human CTL line,
Q124, which is specific for B27 and the NP383-391 peptide. As shown in Fig. 2 A, the B27+NP1+ targets were
lysed well by the specific CTL, whereas the B27+NP
and
B27+NP2+ targets were not lysed. Moreover, lysis of the
B27+NP1+ targets was inhibited by B27+NP
cold targets
pulsed with synthetic NP383-391 peptide (see Fig. 2 B),
consistent with the predicted specificity of the transgene product. In a separate experiment, LN and spleen cells
from a B27+NP1+ rat were used to prime an RT1-matched B27+NP
recipient, and subsequently to restimulate the primed LN cells in vitro, which were then tested
for lysis of the mouse lymphoma EL-4 cells transfected
with HLA-B27 and h
2m in the presence or absence of
added NP383-391 peptide. As shown in Fig. 2 C, CTL effectors were generated that were specific for B27 and the
added peptide, indicating that the NP1 transgene product is
presented by B27 in vivo.
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To identify the NP1 product biochemically, B27 molecules were immunoprecipitated from detergent lysates of
B27+NP1+, B27+NP, and B27+NP2+ spleen cells, and
the bound peptides were dissociated in acid and fractionated by reverse phase HPLC. When aliquots of the HPLC fractions were tested for sensitization of B27+ human C1R
targets, all of the immunologic activity was found in fraction 83 from the B27+NP1+ peptides (Fig. 3 A), whereas
no activity was found in any of the B27+NP
(Fig. 3 B) or
B27+NP2+ (data not shown) peptide fractions.
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Electrospray mass spectrometric analysis of the active fraction from NP1+ spleen indicated five peptides with molecular masses 1207.7, 1303.6, 1313.8, 1379.8, and 1473.7 (Fig. 4, top). The most abundant ion, of m/z 403.6, was selected for tandem MS analysis and shown to correspond to the [M+3H]3+ ion of the NP383-391 peptide, SRYWAIRTR (Fig. 4, bottom). Analysis of the other four peptides indicated that they were modifications of the SRYWAIRTR sequence by adducts of 96, 106, 172, and 266 Da. Full characterization of these adducts is in progress. It also remains to be determined whether these modifications to the NP1 transgene product occur in vivo or as a result of the purification procedure.
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To estimate the extent to which surface B27 molecules
were engaged by NP1-encoded peptides, spleen cells from
B27+NP1+, B27+NP2+, and B27+NP rats were metabolically labeled with 3H-Arg, and the peptides bound to
B27 were isolated and separated by RP-HPLC, as described above. As shown in Fig. 5, B and C, the peptides
extracted from both B27+NP
and B27+NP2+ B27 molecules eluted in a broad peak between fractions 30 and 70, containing ~98% of the total eluted 3H. This result was
similar to the pattern that we have previously reported for
endogenous B27-bound peptides in transgenic mouse spleen eluted under the same conditions (24). In contrast,
as shown in Fig. 5 A, the B27-bound peptides from
B27+NP1+ spleen eluted in two peaks. The larger peak
was centered at the same position as the NP1-encoded
peptide product identified immunologically and confirmed
by mass spectrometry (Figs. 3 and 4). This peak represented
~87% of the total eluted radioactivity. The smaller peak,
containing ~9% of the eluted counts, appeared to be an attenuated version of the peak of endogenous peptides found
in the B27+NP
and B27+NP2+ eluates. These results
suggest that a ~10-fold reduction in the usual B27-bound
endogenous peptide population occurs in the B27+NP1+
rats through displacement by NP1-encoded peptide.
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To assess the functional consequences of NP1 expression, Con A blast targets were compared as targets for lysis
by B27-restricted anti-HY CTL and by anti-B27 allogeneic CTL. As shown in Fig. 6 A, male B27+NP1+ targets
were lysed by B27-restricted anti-HY CTL to only ~50% the level of male B27+NP1 targets. Similar results were
seen with anti-B27 allospecific CTL (Fig. 6 B). These findings indicate at least a twofold reduction of the endogenous
peptides antigenic in these respective systems as a result of
expression of the NP1 transgene locus.
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To test the effect of the NP1 and NP2
transgenic products on spontaneous clinical disease in rats,
the 293-5 and 300-5 NP1 lines and 338-2 NP2 line were
backcrossed to the disease-prone LEW lines 21-4H and
LEW.33-3, and the double transgenic B27+NP+ and single
transgenic B27+NP offspring were observed for the disease
manifestations characteristic of the 21-4H line (13, 15). The
clinical data from all of the rats observed to age 6 mo are
shown in Table 1. The rats were of backcross generation
N2-N6 to the LEW background (median generation N4
for all three genotypes, NP1+, NP2+, and NP
; mean ± SD 3.8 ± 1.2, 3.7 ± 1.0, and 3.5 ± 0.5, respectively).
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Almost all of the rats developed diarrhea, and there was
no significant difference among the groups regarding the
age of onset of the diarrhea or its maximum severity (data
not shown). Similarly, there was no difference among the
groups in which the IL-1 content of proximal colon was
measured at sacrifice. Histologic examination of proximal
colon was carried out on a subset of the males. There was a
trend toward more severe disease in the groups lacking
NP1, but this was not statistically significant (data not
shown). WBC measurements at 3 mo of age showed no
difference among the groups (Table 1), and there was similarly no difference in the pattern of WBC elevation upon
serial measurements (data not shown).
The one parameter showing a significant difference
among the groups was the prevalence of arthritis, with only
6 of 25 NP1+ male rats developing arthritis during observation to age 6 mo, compared with 15 of 22 NP and 4 of 7 NP2+ controls (P = 0.005 for NP1+ versus NP1
).
Among the female NP1+ and NP
rats, the prevalence of
arthritis was very low (1 of 20 and 3 of 22, respectively),
whereas 2 of 6 NP2+ females developed arthritis. Among
rats developing arthritis, there was no difference in the age
of onset or severity of the arthritis among the six groups.
Among the NP1+ males, arthritis was observed in 4 of 17 rats backcrossed to 21-4H and 2 of 8 backcrossed to
LEW.33-3. Among the NP
males, arthritis was observed
in 10 of 13 backcrossed to 21-4H and 5 of 9 backcrossed to
LEW.33-3. Among NP2+ males, the corresponding numbers were 2 of 3 and 2 of 4. Among the joints assessed histologically, 26 were from rats that never showed clinically
evident arthritis, and these are listed in Table 1. Only three
of these sections showed any microscopic lesions, two from
NP
females and one from an NP2+ male.
A subsequent cohort of 300-5 (NP1) × 21-4H and 338-2 (NP2) × 21-4H rats, of backcross generations N5-N7 to
LEW, was observed to age 4 to 6 mo for arthritis, which
again was seen almost exclusively in males. Of the B27+
males of this group, arthritis was observed in 1 of 4 NP1+, 3 of 4 NP2+, and 6 of 8 NP rats. Upon adding these results
to the tally shown in Table 1, the prevalence of arthritis was
found to be 7 of 29 in NP1+ rats, and 28 of 41 in NP1
rats
(
2 with Yates' correction = 11.54, P < 0.0006).
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Discussion |
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The prevalence of arthritis was significantly reduced in
male B27/h2m transgenic rats also carrying a transgene
targeting the influenza A NP383-391 peptide to the ER,
compared with B27/h
2m rats lacking the NP1 transgene.
In the B27/h
2m female littermates, the prevalence of arthritis was very low in both the NP1+ and NP
groups.
Arthritis is typically more common in male B27/h
2m rats than in the females (13, 15). The sex difference in these studies was more pronounced than usual, and the low
prevalence of arthritis in the NP
females made it difficult
to draw any conclusions from a comparison of the groups
of female rats. Nonetheless, considering both sexes, of the
rats shown in Table 1, 6 out of 13 rats transgenic for the
control NP2 construct developed arthritis, providing evidence that the suppression of arthritis in the NP1+ rats was
not a nonspecific artifact of the NP1 transgene construct itself. This was further supported by a subsequent cohort, in which 3 of 4 NP2+ males developed arthritis, and overall,
there was a significant difference in arthritis prevalence between NP1+ and NP2+ males (7/29 vs. 7/11). The data
from the males thus suggest that the specificity of the peptides bound to B27 in vivo is a key factor in the pathogenesis of spontaneous arthritis in B27/h
2m transgenic rats.
Although the B27 transgenic lines were produced in inbred rats (15), the NP constructs were introduced into SD
eggs because of the far greater efficiency of the procedure
in this outbred strain compared with inbred rat strains, particularly LEW. It is of some potential concern that the rats
in this study were not completely inbred. However, the
correlation of NP1 with reduced prevalence of arthritis is
statistically quite significant and there was no significant effect of either the backcross generation or the origin of the
B27/h2m transgene locus (21-4H or 33-3) on the prevalence of arthritis in any of the three NP genotypes. The
finding of a lower prevalence of arthritis in NP1+ rats compared with NP2+ and NP
rats in a subsequent, more extensively backcrossed cohort further supports this concept.
Overall, the data support the conclusion of a peptide-specific suppression of arthritis.
Sequence analysis of the peptides in the one immunologically active HPLC fraction of the peptides eluted from B27 molecules of B27+NP1+ rats confirmed that the peptide species in this fraction all derived from the transgene, both the expected NP383-391 peptide and an unexpected series of chemical derivatives of this peptide. Determination of the origin and nature of these derivatives is in progress. However, for the main purpose of this study, the most significant characteristic of these derivatives is that all were bound to B27.
The pattern of 3H-labeled peptides eluted from B27
molecules differed dramatically between the NP1+ and
NP or NP2+ rats, and the findings suggested that ~90%
of the endogenous peptides normally binding to B27 were
displaced by the NP1-encoded peptide. This was supported
by the substantial inhibition of CTL recognition of endogenous B27-presented peptides in B27+NP1+ targets. The
reduction of endogenous antigenic peptide seen in these
experiments was probably greater than twofold, because of the sigmoidal nature of peptide/CTL lysis dose response
curves (17, 26). These results indicate that the strategy to
displace endogenous B27-bound peptides through expression of the transgene construct was successful. To our
knowledge, this is the first report of the targeting of a
highly expressed MHC class I-presented peptide antigen to
the ER via a transgene.
Although arthritis was suppressed in the NP1+ rats, there
was no pronounced effect of this transgene construct on
gut inflammation. This finding is consistent with the possibility that peptide specificity is of less overall relevance in
the development of colitis than of arthritis. However, the
finding does not necessarily preclude the need for peptide
specificity in the pathogenesis of colitis in the B27 transgenic rats. We have previously shown that germ-free B27
transgenic rats do not develop either gut inflammation or arthritis (14, 25). Since luminal bacteria are abundant in the
gastrointestinal tract, it may be that in the gut but not in
the joint the generation of a putative B27-presented, disease-related antigenic peptide from an intracellular bacteria
is quantitatively sufficient to overcome the blockade imposed by the NP1 transgene. Alternatively, class I molecules
are capable of acquiring exogenous antigen (27), and if this
were the mechanism operating in the gut to induce B27-
related colitis, then in this case the nature of the peptides
acquired by B27 molecules in the ER, whether endogenous or arising from the NP1 transgene, would presumably
be relatively unimportant. Although the spondyloarthropathies in humans are strongly associated both with HLA-B27 and with gut inflammation (which can range from
subclinical histologic changes to classic ulcerative colitis or
Crohn's disease), there is no particular association between
HLA-B27 and classic inflammatory bowel disease in the
absence of arthritis (reviewed in reference 28). This is quite
different from the case of the disease-prone lines of the B27
transgenic rats, in which marked colitis has virtually a 100%
prevalence and usually develops before any arthritis is seen
(13). In this respect, the disease in rats most resembles human reactive arthritis that arises after intestinal infection. Based on these observations, as well as on the findings that
both the gut disease and arthritis in the B27 rats are abrograted by the germ-free state (14) and by the DA genetic
background (29 and Taurog, J.D., S.D. Maika, N. Satumtira, M.L. Dorris, I.L. McLean, W.A. Simmons, A.T.
Le, A. Sayad, J.B. Splawski, J.A. Richardson, and R.E.
Hammer, manuscript in preparation), we favor a model in
which arthritis in the rats is dependent upon gut inflammation. This model would not require the B27 transgene
product to play the same role in the pathogenesis of the gut
and joint disease, and the evidence from the current study
at least suggests the possibility that it in fact does not. Consistent with this interpretation is the finding that rats transgenic for a B*2705 gene with a mutation in the B pocket
(67Cys Ser) develop severe gut disease but very little arthritis (Taurog, J.D., S.D. Maika, N. Satumtira, M.L. Dorris, I.L. McLean, W.A. Simmons, A.T. Le, A. Sayad, J.B.
Splawski, J.A. Richardson, and R.E. Hammer, manuscript
in preparation).
Because this disease has only been observed in rats with
high gene copy number and supraphysiologic expression of
the B27 and h2m transgenes (13, 15), it has been of some
concern whether the role of B27 in the rat disease is similar
to that in the human spondyloarthropathies. There is no
evidence that the disease is simply an artifact of high HLA
class I expression, since control rats with equally high expression of HLA-B7 or HLA-Cw6 do not develop this disease and the overwhelming majority of these rats remain
healthy (30 and Taurog, J.D., S.D. Maika, N. Satumtira, M.L. Dorris, I.L. McLean, W.A. Simmons, A.T. Le, A. Sayad, J.B. Splawski, J.A. Richardson, and R.E. Hammer,
manuscript in preparation). Moreover, as already noted, the
rat disease resembles the human spondyloarthropathies in
its relationship to the gut flora, modifying background
genes, and a requirement for T cells, as well as in phenotype. Previous investigation of the cellular pathogenesis has
suggested that both CD4+ and CD8+ can separately transfer disease to athymic B27 transgenic rats (9). However, the
recipients of these transferred cells predominantly exhibited
gut and skin disease, with very little arthritis. Thus, it is not
yet clear which effector cells mediate arthritis in these rats.
Moreover, even if CD4 cells were found to be effectors of
arthritis, this would not preclude the requirement for the antecedent participation of B27-restricted, peptide specific, CD8 T cells, for example through a mechanism involving
epitope spreading (31). Part of the rationale for the present
study was the observation that high B27 expression was required for disease expression. This suggested that an arthritogenic peptide may be presented above a critical threshold
level in the high transgene copy rats. If so, it would be predicted that disease would be prevented or suppressed by reducing the level of presentation of this putative peptide. As
noted above, the present data suggest that distinct cellular
processes and molecular recognition events may be operating in the pathogenesis of the arthritis and gut disease in the
B27 rats, and they provide the best evidence to date that
B27 presents a specific peptide at some stage in the development of arthritis in these animals.
Further work will be needed to gain insight into the
mechanism by which arthritis is suppressed. The effect may
be occurring in the peripheral immune system and/or at
the level of thymic selection, and these possibilities can be
better investigated once the NP lines are sufficiently inbred
to carry out thymus graft and cell transfer experiments. Finally, it remains formally possible that the critical influence
of the NP1 transgene product on disease pathogenesis is not
exerted through displacement of a peptide or set of peptides
that is recognized by conventional peptide-specific T cells,
but rather on some aspect of the metabolism of the B27 molecules in a critical intracellular compartment. This might
make a difference, for example, if the critical role of B27 in
arthritis were to provide a B27-derived peptide presented by
MHC class II, as has been hypothesized (32). However, the similar levels of surface-expressed B27 and of 3H-Arg incorporated into immunoprecipitated B27 in the NP1+, NP2+,
and NP rats tend to weigh against this possibility.
There are 12 known subtypes of HLA-B27. Epidemiologic studies of disease association have been carried out for seven of them, B*2702, -03, -04, -05, -06, -07, and -09; the others are too rare and/or too recently discovered to have been studied or to have yielded susceptibility data (1, 2, 33). Of these seven subtypes, two, HLA-B*2706 and -B*2709, have shown little or no association with the spondyloarthropathies in recent epidemiologic studies (33- 36). These two subtypes differ from the disease-associated subtypes at position 116, in the floor of the F-pocket of the peptide binding groove, and B*2706 also differs at an adjacent floor position, 114. The major effect of these differences would be expected to be exerted on the spectrum of peptides accommodated by the binding groove, particularly at the peptide COOH terminus, and indeed significant differences in this regard have been identified (37, 38). The data from these recent studies of the B27 subtypes and from the experiments in B27 rats reported here thus both support the "arthritogenic peptide" hypothesis. Similar structural correlations with genetic epidemiology have also implicated peptide binding in the pathogenetic role of other disease-associated HLA alleles (reviewed in 39).
Studies of the B27 subtypes have also indicated that the peptides eluted from the disease-prone subtypes B*2702, -04, and -07, unlike those from B*2705 (and also -01, -03, and -10), do not include peptides with positively charged COOH termini. These data suggest that if there is a common arthritogenic peptide bound by all of the disease-prone subtypes, it most likely carries an aliphatic or aromatic COOH terminus (reviewed in references 1, 2). This may explain our previous observation that polymorphism of the MHC-linked peptide transporters in the rat had no significant effect on disease in B*2705 transgenic rats, despite an appreciable influence on peptide presentation by B27 (10), since this polymorphism would not necessarily affect transport of peptides with noncharged COOH termini (24, 40).
In summary, this study in transgenic rats provides evidence consistent with recent findings in humans that the peptide specificity of B27 is critical to its role in enhancing susceptibility to the spondyloarthropathies. Further work in the B27 transgenic rat system may help to identify the relevant peptides and the mechanism by which they induce arthritis. This study also suggests the potential feasibility of gene therapy specifically targeting peptides to particular MHC alleles.
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Footnotes |
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Address correspondence to Joel D. Taurog, M.D., Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8884. Phone: (214) 648-6837; Fax: (214) 648-3783; E-mail: taurog{at}utsw.swmed.edu
Received for publication 10 April 1998 and in revised form 17 June 1998.
The technical assistance of Graham Fox, Lisa Holt, and Julie Vorobiov is gratefully acknowledged. We thank Dr. William Biddison for the Q124 CTL line, and Dr. James Forman for providing the pHSE3' expression vector.Supported by National Institutes of Health (NIH) grant 1 R01 AR38319. W.A. Simmons was supported during part of this work by NIH Training grant CA09082-20.
Abbreviations used in this paper LEW, Lewis; LN, lymph node; SD, Sprague Dawley.
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References |
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