Mapping Immune Responses to mRBP-3 1-16 Peptide
with Altered Peptide Ligands
Carly J. Guyver,1David A. Copland,2Claudia J. Calder,1Alessandro Sette,3John Sidney,3
Andrew D. Dick,2and Lindsay B. Nicholson1,2
PURPOSE. Experimental autoimmune uveoretinitis (EAU) can be
induced in C57BL/6 mice (I-Ab) using human retinoid-binding
protein-3 (hRBP-3, previously IRBP) residues 1-20. This study of
a truncated murine peptide (mRBP-3 1-16) was conducted to
determine its pathogenic potential and to characterize partially
its interaction with specific T cells.
METHODS. After immunization with mRBP-3 1-16 or hRBP-3
1-20, EAU was assessed by immunohistochemistry. The im-
mune response was assessed by tritiated thymidine incorpora-
tion and cytokine production analyzed by enzyme-linked im-
munosorbent assay (ELISA). T-cell receptor (TCR)- and major
histocompatibility complex (MHC)-binding of mRBP-3 1-16
was studied by modeling and by using altered peptide ligands
(APLs) and T-cell clones.
RESULTS. mRBP-3 1-16 induced EAU in C57BL/6 mice, with
severity and kinetics comparable to that after immunization
with hRBP-3 1-20. T cells taken from mice immunized with
mRBP-3 1-16 had a Th1 phenotype and proliferated in response
to reactivation with mRBP-3 1-16, hRBP-3 1-20, or mRBP-3 1-16
APLs. mRBP-3 1-16 APLs elicited at least five distinct patterns of
reactivity when tested with the mRBP-3 1-16-reactive T-cell
CONCLUSIONS. mRBP-3 1-16 immunizes and causes EAU in
C57BL/6 mice. The studies using T-cell clones and APLs dem-
onstrate that the immune response to mRBP-3 1-16 is drawn
from a diverse population of antigen-specific T cells with a Th1
phenotype. Modeling and analysis of clones indicate that non-
pathogenic T cells of an mRBP-3 1-16-reactive T-cell line rec-
ognize the peptide in a single register. (Invest Ophthalmol Vis
Sci. 2006;47:2027–2035) DOI:10.1167/iovs.05-0984
mans, which is believed in many cases to have an autoimmune
etiology. The histopathology of EAU is characterized by inflam-
matory infiltration in the posterior segment of the eye,1which
varies, depending on species, strain, dose, and route of immu-
nization. In mice, immunization leads to a CD4?Th1 T-cell–
xperimental autoimmune uveoretinitis (EAU) serves as a
model for noninfective, intraocular inflammation in hu-
mediated disease,2and the most frequently used antigen is
retinoid-binding protein (RBP)-3 (previously known as inter-
photoreceptor retinoid-binding protein [IRBP]). This is a 140-
kDa extracellular matrix protein that transports vitamin A de-
rivatives between the photoreceptor cells and the retinal
(h)RBP-3 peptide 1-20 is known to induce EAU in C57BL/6
(I-Ab) mice, and removal of residues 1-5 generates a peptide
that is poorly immunogenic and unable to induce EAU.4Con-
sistent with other models of autoimmune disease, it has been
shown that the level of RBP-3 expression in the thymus affects
the susceptibility of mice to the induction of EAU with this
protein,5and that deleting RBP-3 reveals T-cell reactivity that is
not detected in wild-type mice.6More detailed characterization
of the immune response to RBP-3 is therefore relevant to
studies of EAU.
Although CD4?T cells are critical to the induction of EAU,
the infiltrating myeloid cells (macrophages) also play a key role
in both tissue destruction and tissue repair during EAU.7Fur-
thermore, studies of uveoretinitis and other organ-specific au-
toimmune diseases show that susceptibility is governed by
many genes. Thus, these are complex conditions influenced by
both genes and environment.
To penetrate this complexity, it is important to have a
complete understanding of the autoantigen-specific responses
involved. We have therefore extended the analysis of the T-cell
response to RBP-3 in C57BL/6 mice. We noted that the se-
quence of hRBP-3 1-20 differs at position 17 (valine) from that
of murine (m)RBP-3 (isoleucine at position 17) and that the
four carboxyl-terminal amino acids are predominantly hydro-
phobic. We therefore synthesized mRBP-3 1-16 and studied its
pathogenicity. In this report, we show that mRBP-3 1-16 in-
duced EAU equivalent to hRBP-3 1-20 and that having an amino
or carboxyl terminus did not alter the ability of mRBP-3 1-16 to
induce disease. Analysis of the T-cell response shows that it is
diverse and includes nonpathogenic T cells whose receptors
we have mapped by using altered peptide ligands (APLs). Using
a combination of activation of mRBP-3 1-16-reactive T-cell
clones with APLs, a binding matrix to analyze interactions with
major histocompatibility complex (MHC) class II, and classic
studies of MHC-peptide binding, we have identified residues
within mRBP-3 1-16 that are important T-cell receptor (TCR)
MATERIALS AND METHODS
C57BL/6 mice were originally obtained from Harlan UK Limited (Ox-
ford, UK) and were housed in specific pathogen-free conditions with
continuously available water and food. Mice immunized for disease
induction were aged between 6 and 8 weeks. Treatment of the animals
conformed to the ARVO Statement for the Use of Animals in Ophthal-
mic and Vision Research.
hRBP-3 peptide 1-20 (GPTHLFQPSLVLDMAKVLLD), the truncated
mRBP-3 peptide 1-16 (GPTHLFQPSLVLDMAK) (with either an amino or
From the1Department of Cellular and Molecular Medicine, School
of Medical Sciences and2Unit of Ophthalmology, Department of Clin-
ical Sciences South Bristol, University of Bristol, Bristol, United King-
dom; and3La Jolla Institute for Allergy and Immunology, San Diego,
Supported in part by a grant from the National Eye Research
Centre. CJG is funded by a University of Bristol studentship.
Submitted for publication July 27, 2005; revised January 11, and
February 1, 2006; accepted March 22, 2006.
Disclosure: C.J. Guyver, None; D.A. Copland, None; C.J.
Calder, None; A. Sette, None; J. Sidney, None; A.D. Dick, None; L.B.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Lindsay B. Nicholson, Department of Cellular
and Molecular Medicine, School of Medical Sciences, University of Bristol,
University Walk, Bristol BS8 1TD, UK; firstname.lastname@example.org.
Investigative Ophthalmology & Visual Science, May 2006, Vol. 47, No. 5
Copyright © Association for Research in Vision and Ophthalmology
carboxyl terminus), and the nine altered peptide ligands (APLs) of
mRPB-3 1-16, F6Y (GPTHLYQPSLVLDMAK), Q7R (GPTHLFRPSLVLD-
MAK), Q7S (GPTHLFSPSLVLDMAK), P8A (GPTHLFQASLVLDMAK),
S9Q (GPTHLFQPQLVLDMAK), L10Q (GPTHLFQPSQVLDMAK), L10W
(GPTHLFQPSWVLDMAK), L12N (GPTHLFQPSLVNDMAK), and D13I
(GPTHLFQPSLVLIMAK) were obtained from Sigma-Genosys Ltd.
(Poole, UK). (Bold letters indicate the single amino acid changes in the
sequence.) The control peptide, murine hepatitis virus (KVIAKW-
LAVNVL) was synthesized by Quality Controlled Biochemicals, Inc.
(Hopkinton, MA). The peptide purity was determined by HPLC. Pep-
tide preparations were aliquotted and stored at ?80°C. Culture me-
dium, fetal calf serum (FCS), and supplements were supplied by In-
vitrogen (Paisley, UK), unless otherwise stated.
Lymph Node Cell Analysis
Mice were immunized subcutaneously in both flanks and the scruff of
the neck with 100 ?g/mouse of peptide diluted in PBS (100 ?L/site) in
emulsion with CFA (1 mg/mL, 1:1 vol/vol; Invitrogen). Draining lymph
nodes were removed 10 days after immunization, and single cell
suspensions were prepared. Primed lymph node cells (LNCs) were
seeded at 5 ? 105per 200 ?L of DMEM supplemented with 10% FCS,
100 U/mL penicillin-streptomycin, 100 ?g/mL gentamicin, 2 mM L-
glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids,
1? MEM vitamin mixture, 0.1 mM asparagine (Sigma-Aldrich, Dorset,
UK), and 5 ? 10?5M 2-mercaptoethanol (culture medium). Each
condition was plated in 200 ?L per well in 96-well, round-bottomed
plates (Corning-Costar, Corning, NY).
Primed LNCs were stimulated with peptide and incubated for 2 days at
37°C in 10% CO2in a humidified atmosphere. Supernatants were
aspirated at 48 hours for cytokine assays and frozen at ?80°C, and the
plates were pulsed with 18.5 kBq tritiated thymidine (GE Healthcare,
Bucks, UK) per well for the last 18 hours of incubation. Cells were
harvested with a 96-well harvester (Tomtec, Hamden, CT), and thymi-
dine uptake (measured in counts per minute [cpm]) was determined
by liquid scintillation with a microbeta liquid scintillation counter
(Wallac 1450; PerkinElmer Life Sciences, Cambridge, UK).
Cytokine production (TNF-?, IFN-?, IL-2, IL-4, and IL-10) in culture
supernatants was assayed by capture enzyme-linked immunosorbent
assay (ELISA). Briefly, on day 0, capture antibody (in carbonate buffer,
pH 9.6) was applied to flat-bottomed, 96-well plates (Nunc Immuno
Plate; Fisher Scientific, Leicestershire, UK) and left overnight at 4°C.
On day 1, nonspecific binding sites were blocked using 1% bovine
serum albumin (BSA; Sigma-Aldrich) in PBS (1% PBSA) for 1 hour at
37°C. The supernatants were then added for 1 hour at 37°C, and the
plates were washed with 0.5% Tween 20 (Sigma-Aldrich) in PBS. The
appropriate detection antibody was added for 1 hour at room temper-
ature, and plates were washed as just described. Extra-avidin peroxi-
dase (Sigma-Aldrich) was applied to the plates for 30 minutes at room
temperature, plates were washed, and the chromogen substrate (3,3?,
5,5?-tetramethylbenzidine (TMB) and hydrogen peroxide [H2O2]; BD
Biosciences, Oxford, UK) were added. The reaction was stopped with
2 N sulfuric acid (H2SO4) (Sigma-Aldrich). The monoclonal antibody
pairs for capture and detection were obtained from PharMingen, along
with the TNF-? ELISA kit. Recombinant cytokines (PharMingen) were
used as standards, with curves generated from doubling dilutions used
to calculate concentrations of cytokine in the test sample. All data
shown were obtained from supernatants diluted 1:2 with culture
medium, and values are therefore 0.5 of the final cytokine concentra-
T-Cell Lines and Clones
Mice were immunized as described earlier, and lymph nodes were
removed 10 days later. Single-cell suspensions were prepared, and 5 ?
106cells/mL were seeded at 1 mL/well in 24-well plates (Corning-
Costar). Cells were stimulated with 20 ?g/mL mRBP-3 1-16 peptide on
day 0 and then with 20 ?g/mL mRBP-3 1-16 and 1 ? 105irradiated,
syngeneic spleen cells/well on days 7, 14, and 21, and once per month
thereafter to generate the mRBP-3 1-16-reactive T-cell line. The T-cell
line (derived from the lymph nodes of four C57BL/6 mice) was in
culture for at least 2 months before the experiments. Single-cell T-cell
clones were obtained from the T-cell line by plating at limiting dilu-
tions, followed by expansion as described earlier.
EAU Induction and Scoring
Mice were immunized subcutaneously in one flank with 230 nano-
moles peptide in PBS, in emulsion with CFA (1 mg/mL; 1:1 vol/vol)
supplemented with 1.5 mg/mL Mycobacterium tuberculosis complete
H37 Ra (BD Biosciences), and also 1.5 ?g Bordetella pertussis toxin
(Sigma-Aldrich) intraperitoneally (IP). At various time points after im-
munization, eyes were collected and carefully snap frozen, oriented in
optimal cutting temperature (OCT) compound (R. Lamb Ltd., East
Sussex, UK). After they were made and stored at ?80°C, serial 8-?m
sections were thawed at room temperature and fixed in acetone for 10
minutes. They were stained with rat anti-mouse monoclonal anti-CD45
antibody or anti-F4/80, or rat anti-human CD3? (Serotec, Oxford, UK)
and counterstained with hematoxylin (ThermoShandon, Pittsburgh,
PA). Sections were scored for inflammatory infiltrate (presence of
CD45-positive cells) and structural disease (disruption of morphology),
as described previously.8
Analysis with the I-AbScoring Matrix
Using their scoring matrix, we analyzed the set of unique I-Abbinding
peptide sequences reported by Zhu et al.9and compared them with
mRBP-3 1-16. Briefly, each peptide was scored in every possible regis-
ter throughout its length, using a program written in FORTRAN. For
each peptide tested, the highest value was taken to indicate the most
likely binding register. The mean highest value was 86.6, and maxi-
mum scores for all the known I-Abbinding peptides ranged from 43 to
The mouse B-cell lymphoma LB27.4 was used as the source of murine
I-Abmolecules. LB27.4 cells were maintained, and I-Abmolecules
purified by affinity chromatography using the anti-I-Ab,s,umonoclonal
antibody Y3JP,10as previously described.11Quantitative peptide-I-Ab
binding assays were based on the inhibition of binding of radiolabeled
ROIV peptide (sequence YAHAAHAAHAAHAAHAA)12,13to purified
I-Abmolecules. Assays were performed at pH 7.0 in PBS containing
0.7% digitonin, and in the presence of a protease inhibitor cocktail.11
MHC binding of the radiolabeled peptide was determined by capturing
MHC/peptide complexes on Y3JP antibody–coated plates (Lumitrac
600; Greiner Bio-one, Frickenhausen, Germany) and measuring bound
counter. The average IC50of ROIV was 28 nM. Any change less than
threefold was regarded as insignificant, and peptides with affinities
?1000 were not considered as binders.
Effect of mRBP-3 1-16 Immunization
EAU can be induced in C57BL/6 mice with the human RBP-3
1-20 (hRBP-3 1-20) peptide. Amino terminal truncation of this
peptide (residues 1-5) produces an antigen that does not im-
munize or produce disease.4To characterize the uveitogenic
epitope further, we synthesized a peptide truncated at the
carboxyl terminus, to produce mRPB-3 1-16 (mRBP-3 1-16; Fig.
1A) and tested whether this peptide was immunogenic. Mice
were immunized with mRBP-3 1-16 or hRBP-3 1-20 peptide,
and draining lymph node cells (LNCs) were prepared 10 days
2028Guyver et al.
IOVS, May 2006, Vol. 47, No. 5
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