Synthetic peptides from the N-terminal regions of CD200 and CD200R1 modulate immunosuppressive and anti-inflammatory effects of CD200–CD200R1 interaction

Dang-Xiao Chen, Hao He and R. M. Gorczynski

Transplant Research Division, Toronto Hospital, University Health Network, 200 Elizabeth Street, NU-G001, Toronto, Ontario M5G2C4, Canada; Department of Surgery, 100 College Street, University of Toronto, Toronto, Ontario M5G 1L5, Canada; and Department of Immunology, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Correspondence to: R. M. Gorczynski; E-mail: reg.gorczynski{at}utoronto.ca


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A series of 15-mer peptides were synthesized defining continuous sequences of the extracellular region of the murine and human CD200 molecule. In addition, peptides mapping to the presumptive CDR1, CDR2 and CDR3 of the human and mouse CD200R1 molecules were synthesized. The ability of these various molecules to block the interaction of CD200 with CD200R1 was studied in a competitive ELISA using plate-bound CD200R1Fc and biotinylated CD200Fc, and by FACS using FITC-conjugated CD200Fc binding to 24-h LPS-activated adherent cells. Results from these data were compared with the functional ability of the same peptides to suppress the inhibition of generation of allo-specific CTL in vitro following inclusion of CD200Fc in mixed leukocyte culture reactions. Peptides defining discrete regions in the N terminal regions of CD200 and CD200R1 were functionally active in these different assays. Moreover, infused in vivo, the same mouse-specific peptides suppressed protection from graft rejection afforded by injection of soluble immunosuppressive CD200Fc. Used alone in vitro, these peptides enhanced alloimmunity.

Keywords: Ig superfamily, immunosuppression, inflammation, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The desire to develop reagents to prevent and/or treat autoimmune disease and transplant rejection has focused attention on approaches to induce and maintain T cell tolerance (18). Many of these protocols for inducing tolerance involve extrinsic blockade of positive T cell co-stimulation. Though these have often proven to be effective in prolonging graft survival, it is now generally thought that these strategies alone might not be universally sufficient to achieve true tolerance, and indeed, it is becoming appreciated that exploiting the intrinsic molecular and cellular mechanisms that act physiologically to turn off immune responses may facilitate the current protocols used to establish T cell tolerance. A variety of such negative co-stimulation pathways have been described, including PD-1–PD-L, GITR–GITRL, BTLA–B7x and CTLA4–CD86 interactions (5, 911). These data have suggested a new principle for curtailing pathogenic T cell responses, in which blockade of positive T cell co-stimulation is reinforced by concurrent engagement of a negative co-stimulation machinery.

Our own laboratory has been at the forefront of characterization of another ligand–receptor pair, CD200–CD200R, which is implicated in the direct delivery of immunosuppressive responses after antigen challenge (12, 13). Treatment of animals receiving foreign allo- or xenografts or immunized with bovine collagen, with a soluble form of CD200 (CD200Fc) or with a cross-linking anti-CD200R, prevents graft rejection or the development of collagen-induced arthritis (CIA), respectively (14, 15). Supporting evidence for a role for CD200–CD200R interactions in the regulation of inflammation and/or autoimmunity comes from the work of Hoek et al., assessing the immunologic properties of CD200 KO mice (16). These animals showed an increased susceptibility to both CIA and experimental allergic encephalomyelitis, an animal model of multiple sclerosis, along with evidence for increased proliferation of CD200R+ cells. Taken together, these data implicate a physiological role for CD200 in the regulation of activation of CD200R+ cells of the monocyte/myeloid lineage responsible for inflammation in these conditions (17). A unique isoform of CD200R, CD200R1, is expressed also on cells of T lymphocyte origin (12, 18, 19), and thus, CD200 can exert both direct and indirect roles in T cell activation (17, 20).

Molecular modeling of CD200 and CD200R, both members of the Ig-supergene family, revealed domain structures (CDR1, CDR2 and CDR3) typical of all family members (19, 2123). We have recently described a variant form of CD200 in both mouse and man (truncated CD200, CD200tr), lacking the NH2-terminal 18aa of CD200, which seems to have antagonistic effects on the functional (suppressive) properties of the full-length form (D. X. Chen and R. M. Gorczynski, in preparation). Together, these data are consistent with structural predictions made by Preston et al. (21) suggesting that the N-terminal region of both CD200 and CD200R was important in their mutual interaction. In order to explore the interaction of CD200–CD200R in more detail, we have synthesized a consecutive series of 15-mer peptides for both mouse and human CD200. Because (see above) we know that CD200R1 is the CD200R isoform implicated in direct modulation of T cell immunity (being expressed both on activated T cells and dendritic cells) (12, 1820), we also synthesized peptides predicted to overlap the CDR1, CDR2 and CDR3 regions of both human and mouse CD200R1 (18, 19). The functional activities of all these peptides were examined in the following assays: ELISA binding assays, binding to cells measured by FACS and functional inhibition of the immunosuppression induced in cell populations in vitro following CD200–CD200R1 interactions. Finally, we have asked whether peptides that are active when used in these in vitro assays retained modulatory activity (for CD200–CD200R interactions) in vivo in a skin graft rejection model (13). Our data characterize unique regions of the CD200 and CD200R1 molecules associated with the transmission of immunosuppressive function.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female C3H/HEJ and C57BL/6 mice were purchased from the Jackson Laboratories, Bar Harbor, ME, USA. Mice were housed five per cage and allowed food and water ad libitum. All mice were used at 8–12 weeks of age.

Purification of CD200Fc and CD200R1Fc from transfected CHO cells
Human and mouse CD200Fc (and CD200R1Fc) were cloned into CHO cells from a PIRES neo vector (Clontech, Palo Alto, CA, USA). For murine reagents, the Fc construct used was an IgG2a, obtained from T. Strom (24). Human fusion proteins were constructed using an IgGFc construct we produced, with deliberate mutations introduced at positions to delete Fc-binding regions. Rat mAbs to both human and mouse CD200Fc and CD200R1Fc were isolated and tested as described in previous reports (25).

Transfected cells were selected in G418 and serially passaged into medium (GIBCO, Burlington, ON, Canada) with decreasing FCS concentration at weekly intervals. After ~10 weeks, all transfected cells were adapted to serum-free conditions, with specific protein expression in the culture supernatant of ~1 µg ml–1. Material was purified from affinity columns, eluted with Pierce buffer (Fisher Scientific, Mississauga, ON, Canada), concentrated and stored at 4°C at a concentration of 200 µg ml–1. The molecular mass of the recombinant products was within the range of that anticipated for the naturally (cell surface) expressed glycosylated molecules, i.e. ~66 kDa for CD200Fc and ~85 kDa for CD200R1Fc. An aliquot (150 µg) of both human and mouse CD200Fc was biotinylated for subsequent use in ELISA (see below) using D-biotinoyl-{varepsilon}-aminocaproic acid N-hydroxysuccinimide ester, as per the manufacturer's instructions (Roche Diagnostics Corp., Indianapolis, IN, USA).

Peptide synthesis and ELISA assays
A continuous series (from the NH2 terminus) of thirteen 15-mer peptides was synthesized (HSC protein sequence facility, Toronto, ON, Canada) corresponding to the protein sequence information for the extracellular regions of both human and mouse CD200. In addition, a series of peptides defining the predicted CDR regions in the NH2 terminus of both CD200 and CD200R1 was synthesized by the same facility. All peptides were stored at a concentration of 1 mg ml–1. Figure 1a shows a schematic illustrating the location of the CD200 peptides used, while Fig. 1b shows the location of CD200R1 peptides.



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Fig. 1. Identification of peptides used for inhibition of CD200–CD200R1 binding. Data in panel a show amino acid (AA) sequences for murine and human CD200, with the location of the important structural domains, and the sequence of the peptides indicated above the full-length sequence. CDR regions for both human/mouse are indicated in italics. Panel b represents equivalent features for the human and mouse CD200R1 molecules. Also shown is the location of a ‘framework’ region peptide synthesized (s). The subscript bars predict the beta strand regions characterizing the important Ig folds implicated (BIAcore) in CD200–CD200R binding (23).

 
In ELISA assays, plates were pre-coated with CD200R1Fc (100 ng per well), biotinylated CD200Fc was added (200 ng per well in 50 µl PBS) and competition for binding was performed by inclusion of either excess CD200Fc (from 1–3 µg per well) or peptides (at 1–30 µg per well). The assays were developed using streptavidin-coupled HRP followed by appropriate substrate (both obtained from Cedarlane Laboratories, Hornby, ON, Canada). Data shown in subsequent figures used peptides at 30 µg ml–1.

Preparation of cells
Single-cell suspensions from different tissues of C57BL/6 mice were prepared aseptically by incubation of teased tissue in collagenase for 30 min at 37°C, and after centrifugation, cells were re-suspended in {alpha}-MEM supplemented with 2-mercaptoethanol and 10% FCS ({alpha}F10). LPS-stimulated splenic macrophages (Mphs) for use in FACS were obtained by overnight stimulation (LPS at 1 µg ml–1) of plastic adherent spleen cells (two times at 37°C for 45 min).

Human peripheral blood leukocytes (PBL) were isolated from healthy normal donors by centrifugation over Ficoll-Hypaque, with cells re-suspended in {alpha}F10 containing in addition 10% pooled human AB serum ({alpha}Hu10).

In FACS assays designed to perturb CD200–CD200R1 interactions, CD200R1+ cells (overnight LPS-activated adherent spleen Mphs or PBL) were incubated with FITC-labeled CD200Fc (0.5 µg ml–1) in the presence/absence of different peptide concentrations. Cells were washed three times and analyzed. Data show suppression of FACS staining (Figs 4 and 5) with peptide concentrations of 60 µg ml–1.



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Fig. 4. As for Fig. 3, except for the inhibition of FACS staining used FITC-mouse CD200Fc, binding to LPS-stimulated adherent mouse splenocytes and peptides synthesized for mouse CD200/CD200R1. Staining (no inhibition) was 36 ± 5.8 (mean ± SD of three studies).

 


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Fig. 5. Blockade of inhibition of CTL induction in human MLCs by CD200Fc using soluble peptides for CD200/CD200R1 as described in Figs 1–3GoGo. Culture groups (in triplicate) contained a 1 : 1 mixture of allogeneic responder and mitomycin-C-treated stimulator PBL (1.25 x 106 of each) in 300 µl medium, and all except a control contained human CD200Fc (1.5 µg ml–1). Experimental groups contained putative blocking peptides at a final concentration of 50 µg ml–1. Data show arithmetic means (±SD) from four independent studies. Again, data to the far left show results with titration of peptide c (see Fig. 2).

 
Cytotoxicity and cytokine assays
In allogeneic mixed leukocyte cultures (MLCs) used to assess cytokine production and generation of CTL, responder mouse spleen (or Hu PBL) cells were stimulated with equal numbers of mitomycin-C-treated (45 min at 37°C) stimulator cells in triplicate in {alpha}F10 ({alpha}Hu10). In the studies described in the text, CD200Fc (and/or putative antagonist peptides) were added into the MLCs at the concentrations indicated. Supernatants were pooled at 40 h from replicate wells and assayed in triplicate in ELISA assays for tumor necrosis factor-{alpha} (TNF{alpha}) production.

Biotinylated detection mAbs for ELISA were obtained from Pharmingen (San Diego, CA, USA), namely, anti-mouse TNF{alpha} (G281-2626) and anti-human TNF{alpha} (MAb11). Capture goat heteroantibodies to TNF{alpha} for use in ELISA, along with streptavidin–HRP, were purchased from Cedarlane Laboratories. Recombinant cytokines were purchased from Pharmingen. Varying volumes of supernatant were bound in triplicate at 4°C to plates pre-coated with 100 ng ml–1 mAb, washed three times and biotinylated detection antibody added. After washing, plates were incubated with streptavidin–HRP (Cedarlane Laboratories) and developed with appropriate substrate and OD405 was determined using an ELISA plate reader. Recombinant cytokines for standardization were obtained from Pharmingen. All assays showed sensitivity in the range 40–4000 pg ml–1.

Where cytotoxicity was assayed, cells were harvested from MLCs at 6 days and titrated at different effector:target ratios for killing (6 h at 37°C) of 51Cr-labeled 72-h Con A-activated target cells.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Suppression of binding of CD200Fc to CD200R1 in ELISA and FACS by different peptides
CD200 and CD200R1 are molecules of the IgSF, and typical V-region and C-region domains have been mapped for both human and mouse molecules. Previous work has documented that the NH2-terminal domain of CD200 was important for CD200R1 binding (21), and more recently, Hatherley and Barclay have used point mutational analysis of CD200R to provide supportive evidence that four IgSF domains account for most of the important interactions between CD200 and CD200R1 as measured by BIAcore (23). These domains map to the GFCC' face of the NH2-terminal domain of CD200R1 (Fig. 1), consistent with data suggesting that a similar region of CD200 was important in binding (21, 26).

We have used an alternate approach to assessing the structural regions of interest for binding, initially using competition ELISA studies, followed by exploration of binding of CD200 to its native receptor on CD200R1+ cells (FACS analysis). Data in Fig. 2 (human reagents only) show inhibition of binding of 200 ng ml–1 biotinylated CD200Fc to ELISA plates coated with 100 ng ml–1 CD200R1Fc by a variety of peptides (see Fig. 1 for definition of regions encoded by different peptides), each used at a final concentration of 30 µg ml–1. Data to the right in this figure represent sample titration data for one of these peptides (Fig. 2c: range of concentrations 3–30 µg ml–1). Equivalent ELISA data were seen using murine reagents (not shown).



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Fig. 2. Inhibition of binding in ELISA of human biotinylated CD200Fc protein to plates coated with human CD200R1Fc protein by a series of peptides found in human CD200/CD200R1 (see Fig. 1). All groups were set up in triplicate (data show mean ± SD over three separate studies). Control ELISA wells were inhibited using whole CD200R1Fc or two doses of CD200Fc (see left of figure: values in parentheses indicate inhibitory concentrations, in µg ml–1). Data to the right show titrated concentrations of peptide c.

 
The control groups (far left) indicated that the ELISA assay was capable of registering suppression using full-length CD200R1Fc or CD200Fc protein. Furthermore, using peptides encompassing the entire V-region and C-region domains of human CD200Fc (Fig. 1a), we documented optimal inhibition with peptides located at the C–C'–C'' and the F–G face (human peptides c, d, f and g; mouse peptides d' and g'), consistent with data from other groups using a more biochemical approach (21, 23). The d (d') and g (g') peptides are located in the CDR2 and CDR3 regions, respectively, of the CD200 V-domain (Fig. 1). Interestingly, a peptide defining the CDR1 region (b) and located in the B face of CD200 also blocked ELISA binding. Peptides located throughout the C-region domain (h–n) were ineffectual in inhibition.

When peptides were prepared based upon the CD200R1 sequence (Fig. 1b), again functional inhibitory activity was seen for those encompassing the F–G loop (the CDR3 region; peptides r and r') and the C'–C'' face (approximating the CDR2 region; peptides q and q'). No significant inhibition was seen for peptides in the CDR1 region or in the C-domain (p, p' and t, t', respectively), though interestingly significant inhibition was seen for a human CD200R1 peptide located in the A–B face (peptide s).

Data in Figs 3 and 4 recapitulate those shown in Fig. 2, in this case, analyzing suppression of FITC–CD200Fc binding by peptides to the native cell-bound CD200R1 receptor data for both mouse and human cells/reagents are shown. The conclusions reached from ELISA for the important role of peptide sequences in both CD200 and CD200R1 located in the C–C'–C'' region, and the F–G loop, for CD200–CD200R1 interactions were borne out by FACS analysis.



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Fig. 3. As for Fig. 2, except that the inhibition shown is for binding of FITC-human CD200Fc to LPS-stimulated adherent human PBL, as assayed by FACS. Staining (no inhibition) in controls was 19.8 ± 2.8 (mean ± SD over four studies).

 
Suppression of induction of CTL and TNF{alpha} in MLCs using peptides defining interacting regions of CD200–CD200R1
In order to test whether the peptides defined in the ELISA and FACS assays described above as characterizing important regions for CD200–CD200R interactions also delineated functionally important regions contributing to a previously documented suppressive interaction following CD200–CD200R1 binding (12), we performed the following assays. MLCs were initiated with equal number of responder and stimulator cells, along with an immunosuppressive concentration of human (or mouse) CD200Fc. Experimental cultures also contained aliquots of the peptides described in earlier figures. A control set of cultures contained no CD200Fc or peptide. All groups were set up in triplicate. Supernatants were harvested from all wells at 48 h and assayed by ELISA for TNF{alpha} production. All cultures were maintained until day 6, at which time, CTL was assayed using 51Cr-labeled 72-h Con A-activated cells of stimulator origin. Data for these assays are shown in Figs 5 and 6 (human data shown only—equivalent results were seen using mouse cells/reagents).



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Fig. 6. Blockade of inhibition of TNF{alpha} induction in human MLCs by CD200Fc using soluble peptides for CD200/CD200R1, as described in Fig. 5. Data show arithmetic means (±SD) from four independent studies. Control groups (with/without CD200Fc alone are shown to the left in the figure).

 
Data from both the CTL and cytokine assays confirm the results obtained from ELISA and FACS staining for ability of the different peptides to characterize important CD200–CD200R1 interaction regions. Once again, the same set of peptides (contributing to the C–C'–C'' and F–G loop) for both CD200 and CD200R1 were effective in blocking the suppression mediated by CD200Fc in these assays. Data in Fig. 7 represent data similar to those in Fig. 5, using murine cells/peptides, but in addition assaying the ability of peptides to modulate MLC reactivity in the absence of exogenous CD200Fc-mediated suppression. In this case, we noted that peptides with optimal blocking ability (of CD200Fc suppression), when used alone, augmented development of CTL in vitro (and TNF{alpha} activity-not shown). We interpret this to imply that in this case, the peptides used block ‘natural’ modulation of CTL induction in vitro by endogenously expressed CD200 (25).



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Fig. 7. Modulation of murine CTL induction using soluble peptides for CD200/CD200R1. Culture groups (in triplicate) in panel a contained a 1 : 1 mixture of C3H responder and mitomycin-C-treated C57BL/6 stimulator spleen cells (1.25 x 106 of each) in 300 µl medium along with peptides at a final concentration of 50 µg ml–1. An independent set of cultures (b) contained murine CD200Fc (1.5 µg ml–1) and peptides. Data show arithmetic means (±SD) from four independent studies.

 
Use of CD200/CD200R1 peptides in vivo to block suppression of skin allograft rejection by CD200Fc
In a final study, we asked whether the most potent of the CD200/CD200R1 peptides identified in the assays described above as characterizing regions of importance for mouse CD200–CD200R1 interaction would function in vivo in modulating this interaction. For this, we used a skin graft rejection model (C3H mice receiving C57BL/6 skin allografts), and infused 20 µg per mouse CD200Fc intravenously at 60-h intervals five times to suppress graft rejection (24). Experimental groups received in addition 500 µg per mouse soluble CD200 peptide (g') or CD200R1 peptide (r') on each occasion of infusion of CD200Fc. A control group received CD200Fc and an inactive peptide (t'—see Fig. 1 and 4). All groups consisted of six mice, and graft rejection was recorded daily by an observer blinded to the respective groups, beginning on day 9 post-transplantation. Data in Fig. 8 are pooled from two independent studies.



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Fig. 8. CDR3 peptides for mouse CD200 (g') and CD200R1 (r') block suppression of graft rejection in vivo. See text for more details. Data show graft survival/group summed over two independent studies (six mice per group in each experiment). Control C3H groups received C57BL/6 skin grafts only. Experimental groups received CD200Fc (20 mg per mouse) with/without 500 mg per mouse of the different peptides shown. Note that peptide t' is derived from a C-region domain of mouse CD200R1 (see Figs. 1b, 4 and 7). Closed circle indicates different from control group (Mann–Whitney U test, P < 0.05).

 
It is apparent from these data, and consistent with data in previous figures, that the two peptides documented previously as being most critical in defining the CD200–CD200R1 interaction regions [both mapping approximately within the CDR3 region, and representing the F–G IgSF face as per previous studies (21, 23)], namely g' and r' (for CD200 and CD200R1, respectively), blocked the suppression mediated in vivo by CD200Fc. This in turn reversed the increased graft survival afforded by CD200Fc infusion (24).


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our interest in CD200–CD200R1 interactions stems from previous studies from this laboratory which documented a role for increased expression of the molecule CD200 in immunosuppression in general (24), including regulation of CIA and allograft rejection. Immunoregulation induced by a solubilized form of CD200, CD200Fc, was correlated with a polarization in cytokine production away from type-1 cytokines and toward type-2 cytokines, and decreased induction of graft-specific CTL. We reported independently that these effects were mediated following engagement of an ‘immunosuppressive-signaling’ receptor (CD200R1) expressed on Mphs/activated T cells (12, 17, 18), a feature supported by other groups (16, 19).

Genes encoding proteins with IgSF domains are common in the mammalian genome (27). Many of these encode proteins implicated in either innate or acquired immune responses, which in turn are often found expressed as membrane proteins. The simplest structure for these, containing two major domains (like CD200/CD200R1), is also the one most commonly found (27). A characteristic feature of such IgSF molecules is their interaction through their N-terminal domains (28), and again independent evidence suggests this is true also for CD200 and CD200R1 (19, 21). Interestingly, pairs of interacting IgSF members such as CD200–CD200R1 (and CD2–CD48, CD2–CD58 and CD15–CD150) are also located close to one another on the chromosome, and seem to have arisen by gene duplication (19). Moreover, these molecules are in general heavily glycosylated, a feature potentially important in prevention of undesired cis interactions (29).

In a recent publication, Hatherley and Barclay present evidence based on BIAcore studies indicating that human CD200R1 and CD200 interact through the GFCC' faces on their NH2-terminal domains, in a fashion similar to that described for CD2–CD58 interactions (23). This same region in the CD200 molecule has been documented to be critical for molecular interaction with its receptor (21). We have used a series of synthetic peptides, encompassing the complete V-region and C-region domains of human CD200, as well as a number of peptides overlapping the C–C'–C'' and F–G faces of both CD200 and CD200R1, to explore perturbation both of the molecular interactions of CD200R1 with CD200 as well as the functional effects of those interactions. Our data support earlier studies implicating an important role for these two regions, which are located in the N-terminal domains of both molecules, in a functionally important interaction between CD200 and CD200R1. Peptides located outside of these regions were in general ineffective in perturbing either the molecular interactions [analyzed by ELISA, using soluble CD200R1 (Fig. 2), or by FACS, with cell-bound CD200R1 (Figs 3 and 4)] or the functional (immunosuppressive) effects of those interactions (Figs 5–8GoGoGo). Interestingly, using peptides alone in MLCs, we showed that those molecules with optimal activity (for inhibition of suppression mediated by CD200Fc) augmented reactivity in vitro (Fig. 7). We take this to reflect a block of suppression mediated by endogenously expressed CD200 in such cultures (25), which in turn implies that such peptides, used alone in vivo, might produce effects parallel to those seen in CD200 KO mice (16)—this remains to be investigated. However, we have shown, in a limited study in a graft rejection model in vivo, that these peptides can modulate CD200Fc-mediated suppression of graft rejection (Fig. 8). Figure 9 is a modification of a model proposed by Hatherley and Barclay (23), showing the approximate location of the two most relevant CD200R1 peptides we used for modulation of CD200–CD200R1 interactions in the experiments reported above.



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Fig. 9. Model showing location of major faces in IgSF CD200R1 molecule [modified from Hatherley and Barclay (23)] to demonstrate also the location of major inhibitory CD200R1 peptides (see previous figures). Assignation of critical amino acids (and moderately important/unimportant amino acids) for CD200–CD200R1 interaction follows data reported by Hatherley and Barclay (23).

 
It is important to note that the studies shown here, and those discussed by others (23), deal only with interactions between CD200 and CD200R1. Several groups, including our own, have now described evidence for the existence of a number of other members of the CD200R family (including in mouse, CD200R2, R3 and R4, and in man CD200R2) (18, 19, 22, 30). We have provided evidence that these isoforms can bind CD200 (18), but we suggested that the functional consequences of CD200 interaction with CD200R1 are not the same as following interaction with other isoforms (18, 20, 22). The possibility that unique peptides could be designed to inhibit selectively the interaction of CD200 with distinct CD200R isoforms, and thus selectively interrupt modulation afforded by unique isoforms, remains under investigation.


    Acknowledgements
 
This work was supported by a Medical Research Council grant to R.M.G. (#MT-14678)


    Abbreviations
 
CDR   complementarity determining region
CIA   collagen-induced arthritis
IgSF   immunoglobulin supergene family
MLC   mixed leukocyte culture
Mph   macrophages
PBL   peripheral blood leukocytes
TNF{alpha}   tumor necrosis factor-{alpha}

    Notes
 
transmitting editor: M. Feldman

Received 25 August 2004, accepted 17 December 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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