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,
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Veterans Affairs Medical Center, Memphis, TN 38104; Departments of
Medicine and
Pathology, University of Tennessee, Memphis, TN 38163; and
Department of Molecular Immunology, Merck Research Laboratories, Rahway, NJ 07065
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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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Recently, animal models have been developed in which DR alleles associated with susceptibility to RA were established as transgenes for the purpose of studying the DR-restricted immune responses to the proposed Ags of RA. In several of these models, immunization of the DR1 (DRB1*0101) or DR4 (DRB1*0401) transgenic mice with human or bovine CII resulted in the development of an autoimmune arthritis (23, 24, 25). Analysis of the DR-restricted T cell response in these mice indicated that the immunodominant determinants for both alleles were located within the same CII peptide sequence and the determinant cores appeared to be the same, CII(263–270) (23, 24, 25). Thus, these data demonstrated that the autoimmune response to CII observed in RA patients is likely mediated by the DR alleles that are associated with susceptibility to the disease, and that the same CII peptide is likely driving the T cell response mediated by both the DR1 and DR4 alleles.
Based on our observations that both DR1 and DR4 present the same CII immunodominant peptide, we have analyzed the DR-restricted presentation of the CII(259–273) peptide at the molecular level. Given the structural differences that exist between DRB1*0101 and DRB1*0401, and especially the amino acid differences found within the binding pockets comprised by the second polymorphic regions, we sought to determine whether these two DR molecules use the same register and anchor residues in binding of the CII(263–270) determinant. Based on T cell stimulations and peptide binding assays, we have determined that DR1 and DR4 bind this CII peptide in a very similar manner. Although T cells clearly discriminate between these two class II molecules, both DR1 and DR4 use the same two amino acids within CII(259–273) as anchor residues for peptide binding. The binding anchors for this CII peptide, however, are quite different from that described for the binding of other peptides to DR1 and DR4. These data indicate that at least two RA susceptibility alleles are presenting the same peptide derived from a candidate autoantigen in a very similar manner, supporting the hypothesis that determinant selection is playing a role in this HLA-based susceptibility to autoimmunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides with site-specific substitutions were synthesized using the Mimotope cleavable pin technology (Cambridge Research Biochemicals, Wilmington, DE), essentially as described by Geysen et al. (26). Active ester F-moc amino acids were coupled via a diketo-piperizine linkage to solid polyethylene supports. The F-moc protecting group was removed by treatment with 20% piperidine in dimethyl formamide (v/v), and following several washes in methanol, the subsequent F-moc amino acids were added in a step-wise fashion. Upon completion of the synthesis, the side groups were deprotected with trifluoroacetic acid (TFA) and anisole (95:5, v/v), and terminal amino groups were acetylated with acetic anhydride in dimethyl formamide and triethylamine (2:5:1; v/v). The pins were then washed thoroughly to remove residual TFA and scavengers, soaked in ethanol, and the peptides were cleaved from the pins into 96-deep-well microtiter plates by overnight incubation in 750 µl of sterile 50 mM HEPES buffer. Peptide concentrations were determined by HPLC analysis using 0.1% TFA in acetonitrile and were found to range from 2 to 5 µg/µl.
Proliferation assays
Mice were immunized s.c. with 100 µg of human type II collagen (hCII) emulsified in an equal volume of CFA. Ten days after immunization, draining lymph nodes were removed, disassociated, and washed in HL-1 (BioWhitaker, 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-Aldrich, 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]thymidine (New England Nuclear, Boston, MA) was added to each well. Cells were harvested onto glass fiber filters and were counted on a Matrix 96 direct ionization beta-counter (Packard Instrument, Meriden CT). Proliferation assays using Mimotope synthetic peptides were performed at one well per peptide and 20 µg of peptide per well. Results were confirmed by replicate experiments, and all data are expressed as 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 
-/
-
BW5147 thymoma cells (27, 28). Lymph node cells were
obtained from B10.M-DR1 (DRB1*0101) (24) and B10.M-DR4
(DRB1*0401) (29) mice immunized 10 days previously with
hCII/CFA. The recovered T cells were cultured with human 1(II) for 4
days, followed by IL-2 for 3 days before fusion. Resulting hybridomas
were screened for their ability to recognize human 1(II) chains and
the CII(259–273) peptide presented by DR1 or DR4. Ag presentation
experiments were performed in 96-well microtiter plates in a total
volume of 0.3 ml containing 105 APCs or 4 x
105 syngeneic spleen cells and
105 T hybridoma cells. APC used were DRAB
10 cells, MUD45 cells transfected with chimeric DR1 constructs, and
MUM21 cells transfected with chimeric DR4 constructs (29).
Cell cultures were maintained at 37°C in 5% humidified
CO2 for 20–24 h, after which 2-fold serial
dilutions were made for determination of IL-2 titers using the
IL-2-addicted cell line HT-2 (24, 25). HT-2 cell viability
was assessed by cleavage of MTT and quantitation of optical density at
650 nm (30, 31). IL-2 titers were quantified by the
reciprocal of the highest 2-fold serial dilution maintaining HT-2 cell
viability greater than 2-fold over control cultures. Results are
presented as units of IL-2 per milliliter of undiluted supernatant as
described by Kappler et al. (32).
Peptide binding assays
Soluble DR1 and DR4 were purified from culture supernatants of S2 Drosophila cells transfected with DRB1*0101 or DRB1*0401 and DRA1*0101. The cytoplasmic and transmembrane portions of these molecules were deleted from the cDNA using PCR, a new stop codon was inserted immediately before the transmembrane domain, and the resulting cDNA was cloned into the Drosophila expression vector pRmHA-3. S2 cells were transfected with a 10:1 ratio of DRB1 and DRA1 to pUChsNeo using calcium phosphate precipitation. Soluble DR production was induced by 1 mM CuSO4, and 5 days later the culture supernatant was collected and adjusted to 0.05% octyl glucoside (OcG). The soluble DR was purified by passage of the supernatant over an affinity column coupled with the anti-DR Ab LB3.1. The column was washed with 0.05% OcG and 0.15 M NaCl in phosphate buffer, pH 7.5, followed by 0.05% OcG and 0.5 M NaCl in phosphate buffer, pH 7.5. The DR was eluted with 100 mM Tris and 0.5 M NaCl, pH 11.2, and the fractions were immediately neutralized with acetic acid. The DR recovered was concentrated using an Amicon Stirred Cell (Amicon, Beverly, MA) and was quantitated by OD 280 absorption and SDS-PAGE before use.
For binding assays, a 10 nM solution of purified DR1 or DR4 was incubated for 4 h at 37°C with HA (307–319) peptide (0.5 nM) that had been labeled at the NH2 terminus with biotin (29). Various concentrations of collagen peptides were added as competitors to HA peptide binding. Bound peptides were separated from free peptides by immobilizing the DR molecules on microtiter plates coated with the mAb LB3.1 and subsequent washing. The Ab was adhered to the plate by an overnight incubation of a 5 µg/ml solution at 4°C. Bound biotinylated peptides were detected by incubations with streptavidin-europium followed by a chelating enhancement solution. Fluorescence was quantitated using a microplate fluorometer (Delfia model 1234; Pharmacia Biotech, Uppsala, Sweden, and FluoroMark; Bio-Rad, Hercules, CA), and data are expressed as relative fluorescence units measured. Each binding assay was performed in duplicate, and data are representative of at least three experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using mice transgenic for the HLA-DR1 (DRB1*0101) or DR4
(DRB1*0401), we (24, 25) and others (23) have
previously shown that these DR1 and DR4 molecules bind and present the
same immunodominant peptide from human CII, CII(259–273). The
-chains of these two molecules differ by nine amino acids in the
1 domain (90.4% identity), with two of the differences residing in
the first polymorphic region and six in the second polymorphic region
(Table I
). To determine whether the
immunodominant cores of CII(259–273) differed for these two class II
molecules, Ala replacements were performed systematically at each
position in the CII peptide, and the ability of the peptides to
stimulate polyclonal T cells and cloned T cell hybridomas was assessed.
As shown in Fig. 1, using polyclonal
CII-primed lymph node cells, the determinant cores of CII(259–273)
appear to be identical for DR1 and DR4. Substitution with Ala at amino
acids 259–262 and 271–273 had little or no effect on the ability of
the polyclonal T cells to be stimulated by either DR molecule. In
contrast, several substitutions within 263–270 either greatly reduced
or eliminated the T cell response. Although the boundaries of the core
determinant appeared to be identical, the role of some amino acids
within the core differed between DR1 and DR4, especially at residue
268. DR4-restricted T cells were tolerant of an Ala at this position,
whereas DR1-restricted T cells were completely unable to respond to the
presentation of this peptide. Despite the high degree of sequence
identity between DR1 and DR4 and the determination that these two class
II molecules appeared to be presenting the same CII determinant, T cell
recognition of the complexes was clearly distinct (Table II). No cross recognition was observed
between DR1-restricted, CII-specific T cells, and DR4 presentation of
hCII, and vice versa.
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and 3). All of the TCR expressed by these
DR1- and DR4-restricted T cell hybridomas use the TCRBV14 gene segment.
The CDR3 regions of these DR1-restricted BV14 chains are all different
and each T cell hybridoma expresses a different TCRAV chain.
Analysis of a large panel of CII-specific DR1- and DR4-restricted T
cell hybridomas indicated a redundant use of TCRBV14 and BV8 by these
cells. This appeared to be due to selection of these T cells by the
DR-CII complex, as there was no evidence that the TCR repertoire was
significantly skewed toward these BV chains in unimmunized mice
expressing the DR1 or DR4 transgenes.
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), whereas substitutions at 263 (F), 264 (K), 266 (E), 267 (Q),
and 270 (K) prevented the peptide from being able to stimulate all of
the DR4-restricted T cell clones (Fig. 3). In general, the sum of the
responses of the T cell clones for each DR restriction closely
resembled the polyclonal response in Fig. 1. DR1 and DR4 use the same anchor residues
Soluble DR-peptide binding assays were performed to determine
whether these DR molecules were using the same anchor residues for
binding the CII(263–270) determinant and to compare their overall
affinities for the CII(259–273) peptide. As can be seen in Fig. 4, the relative affinity of the DR1
molecule for the CII(259–273) peptide is higher than the DR4 affinity
as measured both by competitive binding (Fig. 4A) and by
direct binding of the CII peptide (Fig. 4B). Based on
competitive binding, an average 3.8-fold difference in
IC50 values was calculated based on multiple
experiments. Similarly, in the direct binding assay, a significantly
greater amount of CII peptide was required to achieve 50% maximal
binding with DR4 in comparison to DR1 using equimolar quantities of DR.
Both of the DR1 and DR4 relative affinities for the CII peptide,
however, are quite low in comparison with the 10 nM
IC50 of the hemagglutinin peptide HA(307–319)
for DR1 (data not shown).
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and 6). The F263 was clearly the
dominant residue in the binding of CII(259–273), as Ala substitution
of this amino acid completely prevented any measurable peptide binding
to both DR alleles. Although the effect of substituting the K264 was
not as dramatic, the IC50 was nearly 10-fold
higher for both DR1 and DR4 in comparison with wild-type peptide
binding (Fig. 6). While Ala substitution of no other amino acids
decreased the binding affinity of the CII(259–273) peptide, several
Ala substitutions significantly increased the relative affinity. In the
case of the DR1 binding of this CII peptide, replacement of E266 and
K270 within the determinant core, and amino acids 271–273 outside of
the core, resulted in peptides with enhanced affinity (Figs. 5 and 6).
In the case of DR4 binding, Ala replacement of four different amino
acids located outside of the core determinant (G262, G271, E272, and
P273) generated peptides with significantly higher affinity.
Additionally, Ala replacement of four amino acids within in the core
(G265, E266, G268, and P269) also generated higher affinity peptides
(Fig. 6).
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). For the DR1 molecule, these
assignments are quite clear. The binding of the CII(259–273) peptide
to DR1 appears to be completely controlled by the F263 and K264
residues (P1 and P2). Based on their high binding affinity and poor
ability to stimulate a polyclonal T cell response, CII amino acids
266–268 and 270 appear to be primarily involved in TCR contact. A
similar scenario is also apparent for DR4 presentation of the CII
peptide, although the data for TCR contact are less striking in
comparison with the DR1 data, a likely result of the lower affinity of
the CII peptide for the DR4 allele. As was observed for DR1, F263 and
K264 are clearly involved in DR4 binding; however, the amino acids G265
(P3) and P269 (P7) appear to play a greater role in DR4-restricted T
cell stimulation than with DR1. The remaining core residues, P4-P6 and
P8, all appear to interact with the TCR. These data indicate that
despite all the apparent similarities in DR1 and DR4 presentation of
CII(259–273), there are clear differences in how these DR-peptide
complexes interact with their associated CII-specific TCR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data for DR4 binding of the CII peptide differs somewhat from that described by Andersson et al. (33). As predicted, binding to both DR molecules involved the interaction of the Phe263 in the CII peptide with the binding pocket 1 (P1) of DR1 and DR4. Based on the crystal structures and functional binding data for both of these molecules (34, 35, 36), these results were predictable. However, the determination that only two CII residues were participating in binding to both DR1 and DR4 and the identification of the Lys (position 264 in CII) as the second binding residue was unexpected. Based on functional and crystallographic data with DR4 and noncollagenous peptides (36, 37), it was expected that the side chain of Glu266 in CII(259–273) would have occupied the P4 pocket in DR4. However, we were unable to find any contribution of the Glu266 to the overall affinity of the CII(259–273) peptide for DR4 or DR1, and our data actually indicated that Glu266 was an important residue for TCR interaction. Although Andersson et al. (33) concluded that their studies supported the hypothesis that Glu266 is occupying the P4 pocket, their data, like ours, indicated that the Lys264 had a greater overall contribution to peptide binding than Glu266. In fact, in our experiments, substitution of the negatively charged Glu266 resulted in a peptide with substantially increased affinity for both the DR1 and DR4 molecules, although TCR recognition was adversely affected. Enhanced binding affinities were a frequent observation in our studies when either negatively charged side chains such as Glu266 and Glu272 or amino acids known to disrupt secondary structure were replaced with Ala (Pro at positions 269 and 273, and Gly at positions 262, 265, 268, and 271). These data may be a reflection of the contiguous anchor residues at 263 and 264 used for binding to DR1 and DR4. The physical constraint of using adjoining amino acids to provide sufficient binding energy may alter the secondary structure of the peptide in the context of the DR molecule in such a manner that the charged side chain at residue 266 orients in a less favorable position for peptide binding, and more favorable for TCR interaction, as indicated by our data.
Although the role of CII in the pathogenesis of RA remains uncertain, there is considerable evidence that autoimmunity to CII exists in RA patients. Although no direct evidence yet exists to clearly demonstrate that the CII(261–273) determinant is also immunodominant in humans, CII-specific Ab can be found in the serum and synovial fluid of a significant proportion of RA patients (17, 20, 38, 39, 40, 41), and both CII-specific B cells (42) and T cells (18, 22, 43) have been found in the affected synovial joints of RA patients. Through the use of HLA-DR1 and DR4 transgenic mice (24, 25, 33), it appears quite certain that this observed autoimmunity to CII is mediated by the DR1 and DR4 alleles that are associated with susceptibility to RA, and that both the DRB1*0401 and DRB1*0101 molecules present the same immunodominant peptide. Nevertheless, these observations are supported by CII peptide binding studies that used a large number of DR alleles in which binding of the CII(259–273) dominant peptide was found to be preferentially associated with RA susceptibility alleles (44). Thus, these data support the hypothesis that determinant selection is playing a role in mediating susceptibility to RA (37), and that CII presentation may be preferentially restricted to RA susceptibility alleles. Whether this determinant selection is operating strictly as an immune response stimulus for T cells in the periphery or also as a factor in thymic education and selection is unknown. For example, it is possible that given the low-affinity interaction of the CII peptide with DR1 and DR4, CII-specific T cells may avoid negative selection in the thymus and thus be allowed to establish their presence in the periphery. Because it was first demonstrated that the function of MHC molecules was to bind peptides, hypotheses have been proposed that autoimmunity was the result of a low-affinity interaction between the associated susceptible MHC molecule and the putative autoantigen.
Why would a peptide derived from CII have a different binding
"motif" for DR1 and DR4 than that described for other peptides? One
possible explanation is that the way in which the CII peptide interacts
with these DR molecules is a direct consequence of the unusual primary
sequence of CII, a continuous repeating Gly-X-Y pattern, where X and Y
are frequently Pro, resulting in a conformational structure of an
extended helix. Thus, due to the lack of a side chain on Gly, the
number of side chains available within the CII protein to occupy
binding pockets in an HLA molecule are reduced by one-third. This point
is clearly evident in the CII (259–273) binding data. Gly
residues are located in both the P6 and P9 positions, thus preventing
the peptide from using these pockets for binding. This, in combination
with the repetitive sequence of the CII molecule, appears to greatly
limit the number of potential peptides within CII capable of binding to
these DR molecules and stimulate a T cell response (24, 25). This was also found to be true in peptide determinant
analysis of CII in the context of a murine class II molecule
(45). Finally, it is interesting to note that in the
identification of CII peptides presented in three models of autoimmune
arthritis, all three immunodominant peptides are located within the
same limited region within CII, residues 260–273 (23, 24, 25, 46). Additionally, subdominant determinants for DR1 and DR4 are
located 18 amino acids to the carboxy terminus of CII(259–273)
(24, 25), and yet another determinant was identified for a
nonarthritogenic response seven residues to the amino terminus of this
sequence (24, 25). Given that the 1 chain of CII is
composed of >1000 amino acids, it was unexpected to find all the
antigenic determinants clustered within one small region of this large
molecule. One possible explanation may be that this region of CII,
based on sequence similarity matrix plots, is "unique" in
comparison with the rest of the CII amino acid sequence. Unlike the
rest of the CII molecule that is composed of frequently repeated,
highly homologous stretches of amino acids, this immunogenic region of
CII appears to be unique onto the rest of the molecule. Therefore, this
region may provide not only the necessary amino acids required for
binding to most class II molecules, but may also provide a "unique"
immunological target for focusing the autoimmune response. In addition,
due to this unusual primary sequence and the triple helical
conformation of CII, processing of this molecule may differ from that
of globular proteins, making it difficult to generate peptides that are
capable of binding to the class II molecules.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Edward F. Rosloniec, Veterans Affairs Medical Center, Research Service (151), 1030 Jefferson Avenue, Memphis, TN 38104. E-mail address: erosloniec{at}utmem.edu
3 Abbreviations used in this paper: RA, rheumatoid arthritis; TFA, trifluoroacetic acid; OcG, octyl glucoside; hCII, human type II collagen.
Received for publication January 23, 2001. Accepted for publication October 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain: a means for graft rejection and immunosuppression. J. Virol. 62:100.1(II)-CB11 in H-2q mice. J. Immunol. 152:3088.[Abstract]
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