Novel function of complement C3d as an autologous helper T-cell target

Abstract

The C3d fragment of complement component C3 has been shown to enhance immune responses to antigens that lack T-cell epitopes such as bacterial polysaccharides. C3d binds to the B-cell complement receptor 2 (CR2 or CD21); this binding serves as a co-activation signal to the B cell when the polysaccharide antigen portion binds simultaneously to the B-cell receptor (surface Ig). Bringing together receptor-associated signal transduction molecules CD19 and Igalpha/beta, respectively, results in a lower threshold of activation. Paradoxically, C3d has also been shown to enhance antibody titers in the CD21 knockout (KO) mouse model as well as increase Th1 and Th2 cytokine secretion, suggesting that that an auxiliary CR2-independent pathway of immune activation may exist. We hypothesized that in addition to its molecular adjuvant property that enhances signal 1 during B-cell activation (co-signal 1), C3d also contains T-cell epitopes that are able to stimulate autoreactive C3d peptide-specific helper T cells which we term 'co-signal 2'. Using the EpiMatrix T-cell epitope-mapping algorithm, we identified 11 putative T-cell epitopes in C3d, a very high epitope density for a 302 amino-acid sequence. Eight of these epitope candidates were synthesized and shown to bind a variety of class II HLA-DR molecules of different haplotypes, and to stimulate C3d peptide-specific T cells to secrete pro-inflammatory cytokines in vitro. Further, we demonstrate a C3d-peptide specific increase in CD4(+) intracellular IFN-gamma(+) T cells in peripheral blood mononuclear cells (PBMCs) exposed to C3d peptides in vitro. We believe that the discovery of these autologous T cells autoreactive for C3d provides evidence supporting the 'co-signal 2' hypothesis and may offer a novel explanation of the CD21 KO paradox.

Full text

OUTSTANDING OBSERVATION
Novel function of complement C3d as an autologous
helper T-cell target
Paul M Knopf
1
, Daniel S Rivera
2
, Si-Han Hai
2
, Julie McMurry
2
, William Martin
2
and Anne S De Groot
2,3
The C3d fragment of complement component C3 has been shown to enhance immune responses to antigens that lack T-cell
epitopes such as bacterial polysaccharides. C3d binds to the B-cell complement receptor 2 (CR2 or CD21); this binding serves
as a co-activation signal to the B cell when the polysaccharide antigen portion binds simultaneously to the B-cell receptor
(surface Ig). Bringing together receptor-associated signal transduction molecules CD19 and Iga/b, respectively, results in a lower
threshold of activation. Paradoxically, C3d has also been shown to enhance antibody titers in the CD21 knockout (KO) mouse
model as well as increase Th1 and Th2 cytokine secretion, suggesting that that an auxiliary CR2-independent pathway of
immune activation may exist. We hypothesized that in addition to its molecular adjuvant property that enhances signal 1 during
B-cell activation (co-signal 1), C3d also contains T-cell epitopes that are able to stimulate autoreactive C3d peptide-specific
helper T cells which we term ‘co-signal 2’. Using the EpiMatrix T-cell epitope-mapping algorithm, we identified 11 putative
T-cell epitopes in C3d, a very high epitope density for a 302 amino-acid sequence. Eight of these epitope candidates were
synthesized and shown to bind a variety of class II HLA-DR molecules of different haplotypes, and to stimulate C3d peptide-
specific T cells to secrete pro-inflammatory cytokines in vitro. Further, we demonstrate a C3d-peptide specific increase in CD4
+
intracellular IFN-c
+
T cells in peripheral blood mononuclear cells (PBMCs) exposed to C3d peptides in vitro. We believe that the
discovery of these autologous T cells autoreactive for C3d provides evidence supporting the ‘co-signal 2’ hypothesis and may
offer a novel explanation of the CD21 KO paradox.
Immunology and Cell Biology (2008) 86, 221–225; doi:10.1038/sj.icb.7100147; published online 8 January 2008
Keywords: C3; C3d; complement; helper T cell; epitope; vaccine
The efficacy of polysaccharide vaccines has been significantly
improved by conjugation with either an immunogenic foreign carrier
protein or with complement component fragment C3d.
1–5
The
adjuvant effect of the carrier protein is attributed to its role in
recruiting cognate T help, augmenting the B-cell response to poly-
saccharide antigens. Polysaccharides usually feature multiple repeats of
their sugar subunit residues, which can cause crosslinking of the
breakpoint cluster regions (BCRs) and the internalization of
the conjugate into the antigen processing compartments of the
B cell. For protein antigens or protein conjugates of polysaccharides,
this processing step also generates carrier-derived peptides that bind
internally to MHC class II molecules and are subsequently displayed
on the B-cell surface. Thus, help (in the form of cytokines and
differentiation factors) is generated by activation of CD4
+
helper T
cells via their T-cell antigen receptors (TCR). The generation of T-cell
dependent (Td) responses may serve to create antipolysaccharide
antibodies that are more robust than T-cell independent (Ti)
responses by promoting affinity maturation and isotype switching.
The prevailing model of the mechanism responsible for the
adjuvant effect of C3d is quite different. In this model, the
simultaneous binding of C3d to complement receptor 2 (CR2 or
CD21) and the polysaccharide antigen to the B-cell receptor have been
shown to provide a co-activation signal to the naive B cell (co-signal
1). However this model does not explain the enhancement of immune
responses in studies with CD21 knockout mice, nor does it provide
insight into C3d augmentation of Th1 and Th2 immune responses.
Moreover, an elegant study comparing the antibody response to a
covalent conjugate of the capsular polysaccharide of serotype 14
Streptococcus pneumoniae (PPS14) with C3d in athymic nude mice
demonstrated that T cells were required for enhancement of the
memory B-cell response after a second injection of PPS14-C3d. We
therefore hypothesized that T-cell epitopes contained within C3d may
be processed and presented by antigen presenting cells (APCs) to
stimulate autoreactive C3d peptide-specific helper T cells. These
activated autologous C3d epitope-specific T cells would secrete pro-
inflammatory cytokines and provide a co-activation signal (co-signal
2) allowing B-cell maturation.
In a previous immunoinformatics study of the human serum
proteome
6
we mapped T-cell epitopes in complement C3 using the
EpiMatrix algorithm and confirmed the presence of multiple HLA–
Received 7 September 2007; revised 1 November 2007; accepted 4 November 2007; published online 8 January 2008
1
Department of Molecular Microbiology and Immunology, Brown University, Providence, RI, USA;
2
EpiVax Inc., Providence, RI, USA and
3
Department of Medicine, Brown
University, Providence, RI, USA
Correspondence: Dr PM Knopf, Department of Molecular Microbiology and Immunology, Brown University, Box G-B6, 146 Clifford Street, Providence, RI 02912, USA.
E-mail: Paul_Knopf@Brown.edu
Immunology and Cell Biology (2008) 86, 221–225
&
2008 Australasian Society for Immunology Inc. All rights reserved 0818-9641/08 $30.00
www.nature.com/icb

DR binding motifs in the protein. We noted that the C3 component
of complement had a significantly higher ‘cluster-score’ (concentra-
tion of HLA binding motifs) than most other serum proteins
analyzed.
7
The high potential for immunogenicity of C3 was
found to contrast sharply with other serum protein components
including albumin, constant domain of immunoglobulin, transferrin
and certain hormones, which scored significantly lower. The C3d
fragment of C3 (aa residues 1002–1303 in C3 or 1–302 in
C3d)
8
scored highest in the subsequent EpiMatrix analysis. The
present studies were conducted to determine whether the previously
observed enhancement of the immune response by C3d might be
due in part to the activation of C3d peptide-specific autoreactive
helper T cells, and whether the unusually high concentration of
T-cell epitopes in C3d reflect an auto-inflammatory role of this
particular protein subunit.
RESULTS
Epitope analysis and peptide synthesis
Candidate T-cell epitopes in C3 were identified as described in
Methods. Eleven clusters that scored greater than 10 units on an
epitope identity scale
9
were identified in the 302 aa fragment C3d.
(Table 1). Compared to other serum proteins, C3d contains a 10- to
100-fold higher T-cell epitope cluster density, with 3 of these 11
clusters having very high scores (420 units). An additional
four clusters have the scores 415. These seven top scoring candidate
epitopes are distributed into 2 regions of C3d—Region 1 (aa 10–120)
has four of the peptides with scores 415 and Region 2 (aa 175–290)
has the remaining three. The amino and carboxyl terminal 10–15
residues and the mid-molecule region aa 120–175 segments were
found to be devoid of epitopes, based on EpiMatrix analysis (Figure 1).
Eight of the 11 peptides that were initially identified by cluster score
using the EpiMatrix algorithm as putative T-cell epitopes were
prepared commercially (at greater than 90% purity on an automated
Rainen Symphony/Protein Tech synthesizer (SynPep, Dublin, CA,
USA and New England Peptide, Gardner, MA, USA). Peptides
32–50, 100–118, 190–209, having three of the lowest four cluster
scores, were not selected for synthesis. Epitopes 73–96 and 176–198
had the highest cluster scores but were difficult to synthesize due to
high hydrophobicity.
HLA binding assays
In order to validate the in silico predictions, HLA binding assays were
performed. All of the peptides were shown to bind to at least one of
the four different HLA alleles tested with high affinity (Table 1),
confirming the EpiMatrix predictions. Peptide 176–198 was shown to
bind very strongly to three alleles and moderately to one allele, peptide
45–65 bound moderately to all four alleles tested. Peptides 223–246
and 73–96 bound to DRB1*0401 with very high affinity as did peptide
62–80 to DRB1*1501.
Stimulation of PBMCs by C3d-derived peptides leads to IFN-c
secretion
To determine if C3d-specific responses could be detected in human
subjects, freshly isolated peripheral blood mononuclear cells (PBMCs)
from four healthy subjects were stimulated with or without a C3d
peptide pool (comprised of 11–26, 45–65, 62–80, 223–246, 248–265
and 269–286) for 7–10 days. PBMCs were then each re-stimulated
with the C3d peptide pool or no peptide. Levels of IFN-g secretion
were determined 3 days post re-stimulation by ELISA or ELISpot
assay. The stimulation of PBMCs with the C3d peptide pool led to
greater than a threefold increase in IFN-g secretion over no peptide
stimulation (Figure 2a). To further characterize the C3d peptide
responses, PBMCs were stimulated with the C3d peptide pool or no
peptide for 7–10 days. PBMCs were then re-stimulated with individual
C3d peptides or no peptide. C3d-specific IFN-g production was
determined by ELISpot. In both subjects tested, dramatic increases
in IFN-g production (greater than threefold) were seen with peptides
223–246 and 269–286 (Figure 2b).
Autologous helper T cells recognize C3d-derived peptides
A key component of our ‘co-signal 2’ hypothesis relies on the
recognition of C3d-derived peptides by autoreactive helper T cells.
Cryopreserved PBMCs from an HLA-DR1 subject were stimulated as
described above and IFN-g secretion was assayed on day 10 by ELISA.
Stimulation with the C3d peptide pool led to a nearly 10-fold increase
of IFN-g over background (Figure 3a). We subsequently evaluated the
phenotype of the IFN-g secreting cells by surface and intracellular
cytokine staining (ICCS). IFN-g+ cells were found to reside exclusively
in the CD4
+
helper T-cell population (Figure 3b).
Table 1 C3d peptide locations, sequences, EpiMatrix cluster scores and in vitro binding affinity
Start and stop
in mature C3d
Equivalent start
and stop in C3
Peptide EpiMatrix
cluster score
IC50 binding affinity (m
M)
DRB1*0101 DRB1*0401 DRB1*0701 DRB1*1501
11–26 1012–1027 EQNMIGMTPTVIAVHY 16.3 7.3 3.5 16.9 Nonbinder
32–50 1033–1051 QWEKFGLEKRQGALELIKK 12.9 Not synthesized
45–65 1046–1066 LELIKKGYTQQLAFRQPSSAF 17.8 23 16.8 260.19 4.4
62–80 1063–1081 SSAFAAFVKRAPSTWLTAY 17.5 36.2 Nonbinder 9.3 0.3
73–96 1074–1097 PSTWLTAYVVKVFSLAVN-
LIAIDS
26.4 Nonbinder 0.3 8.2 Nonbinder
100–118 1101–1119 CGAVKWLILEKQKPDGVFQ 13.3 Not synthesized
176–198 1177–1199 EANYMNLQRSYTVAIAGYA-
LAQM
28.4 0.2 51.8 0.15 0.48
190–209 1191–1210 IAGYALAQMGRLKGPLLNKF 10.4 Not synthesized
223–246 1224–1247 GKQLYNVEATSYAL-
LALLQLKDFD
15.3 Nonbinder 1.1 114.2 23.9
248–265 1249–1266 VPPVVRWLNEQRYYGGGY 11.4 Nonbinder Nonbinder Nonbinder 3.7
269–286 1270–1287 QATFMVFQALAQYQKDAP 23.8 Nonbinder 183.6 25 Nonbinder
C3d as a helper T-cell target
PM Knopf et al
222
Immunology and Cell Biology

DISCUSSION
Initiation of a primary humoral immune response can often be
augmented by the formation of complexes composed of the antigen
covalently linked to complement component C3d.
10,11
This enhanced
response has been attributed to the crosslinking of the antigen
recognition molecules (Ig) and complement-binding molecules
(CD21 also known as CR2), bringing together Iga/b and CD19,
respectively to generate signal 1.
12,13
It has been postulated that this
crosslinking thereby lowers the threshold for generating signal 1 and
for response to signal 1.
14,15
The antigen conjugated with C3d can be
either a protein or a polysaccharide. This adjuvant effect of C3d has
been employed extensively in vaccine design and the engineered
attachment of two or three C3d molecules per antigen has been
shown to be optimal for amplifying humoral response.
3,16
Conjugate
vaccines using Ti polysaccharides and C3d have been shown to be
as effective as Ti polysaccharides covalently linked to Td protein
antigens.
2
Conjugation of polysaccharide antigens with C3d has been
suggested as a means of harnessing the adjuvant potential of the
innate immune system. Published studies have demonstrated that C3d
1
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
HL I VTPSGCGEQNMI GMTPTV I AVHYLDETEQWEKFGLEKRQGALEL I KKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNL I A I DSQVL
100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
CGAVKWL I LEKQKPDGVFQEDAPVI HQEMI GGLRNNNEKDMAL TAFVL I SLQEAKDI CEEQVNSLPGS I TKAGDFLEANYMNLQRSYTVA I AGYALAQMGRLKGPL LNKF
210
215
220
225
230
235
240
245
250
255
260
265
270
275
280
285
290
295
300
LTTAKDKNRWEDPGKQLYNVEATSYALLAL LQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAPDHQELNLDVSLQLPSR
-30
-10
10
30
-20
0
20
40
16.3
12.9
17.8
17.5
26.4
13.3
28.4
10.4
15.3
-40
11.4
23.8
Moderate
High
V. High Highest
Neutral
EpiMatrix Cluster Score Scale
(Density of HLA binding motifs compared to expectation)
C3d Epitope Cluster Map
CD21 binding domain
Figure 1 Map and score of putative C3d T-cell epitopes. Each amino acid within C3d is given a number corresponding to its position (N terminus to C
terminus); position 1 of C3d is the position 1002 of C3. Horizontal bars below the sequence correspond to the location of the putative clusters. The color of
the bar corresponds to its EpiMatrix cluster score on the scale; the scores are broken down into strength of putative immunogenicity as benchmarked by
published epitopes. Scores also shown below bars. Cluster scores of 10 and higher are considered to be potentially immunogenic. The highest scoring
clusters were synthesized as peptides and were analyzed for their ability to bind HLA and activate T cells in vitro.
0
2
4
6
8
10
12
14
16
Expansion
with C3d
peptide pool
Expansion
with No
peptide
Fold Increase Over Background
(Stim Index)
Subject A
Subject B
Subject C
Subject D
0
2
4
6
8
10
12
14
16
p11 p45 p62 p223 p248 p269
Individual C3d Peptides
Fold Increase Over Background
(Stim Index)
Subject A
Subject B
Figure 2 (a) Stimulation with C3d peptide pool leads to increased secretion of IFN-g. Peripheral blood mononuclear cells (PBMCs) were cultured with or
without C3d peptide pool (10 mgml
1
). IFN-g secretion was measured by ELISA upon re-stimulation with C3d peptides; all four subjects had greater than
twofold increases over a background of IFN-g secretion in response to C3d, compared to no peptide. (b) Stimulation with individual C3d peptides leads to
increased secretion of IFN-g. PBMCs were cultured with or without C3d peptide pool (10 mgml
1
). IFN-g secretion was measured by ELISpot upon
re-stimulation with individual C3d peptides. Stimulation with peptides 223–246 and 269–286 led to dramatic increases in IFN-g secretion over background.
C3d as a helper T-cell target
PM Knopf et al
223
Immunology and Cell Biology

conjugated to PPS14 led to increased immunogenicity and isotype
switching from a predominantly (IgM) to an IgG1 response by day 25
following primary immunization. Subsequent immunization with
PPS14-C3d caused a booster response and a further increase in the
ratio of IgG1 to IgM anti-PPS14;
2
this boost effect and isotype
switching is consistent with our ‘co-signal 2’ hypothesis that T cells
specific for C3d peptides are at least partially responsible for the
observations.
In our study, only IFN-g was found to be consistently elevated.
Whether the variability of other cytokine/chemokine responses is
due to the small number of subjects tested, immune state of the
subjects or sensitivity of the assay used remains to be determined.
The C3d-derived peptides described here exhibit different binding
affinities to the HLA molecules. It may be that some haplotypes
are more responsive to C3d stimulation than others. The immune
state of the individual may also influence the observed responses.
Differences in the cytokine profiles of memory and effector helper
T cells have been described previously.
17
The basal frequency of C3d
autoreactive memory T cells would be expected to be low in most
individuals and be tightly regulated. An individual under challenge
with a bacterial antigen may have higher levels of C3d autoreactive
effector T cells and those cells may display a different cyokine/
chemokine profile upon stimulation in vitro, than memory cells.
Finally, the assays and methods used to detect a given cytokine of
chemokine may not be the best for the measurement of others due to
differences in the rates of transcription, translation, secretion, stability
and re-uptake.
In this study we have shown that predicted T-cell epitopes derived
from the complement fragment C3d, bind to a multiple HLA-DR
alleles and stimulate autoreactive helper T cells to produce IFN-g.
These data are consistent with existence of a CD21-independent
pathway by which peripheral helper T cells are activated by self-
peptide–MHCII complexes.
The identification of 223–246 as the most active C3d peptide in the
stimulation of PBMCs deserves additional comment, since the loca-
tion of the 13-residue amino segment of 223–246 overlaps with the
carboxyl segment of P28, the 28-mer peptide containing the major
binding site of C3d for CD21.
18
When conjugated to protein antigens,
P28 has been shown to enhance both humoral (antibody isotype
switching) and cellular (IFN-g, IL-4 secretion) immune responses
almost as well as the whole C3d molecule
19
which is in agreement with
our hypothesis and findings.
Finally, in addition to elucidating a possible adjuvant mechanism of
C3d, this data raises questions relevant to self-tolerance and suggests
the possibility of AIRE-independent differentiation into nonpatho-
genic, self-reactive T cells.
METHODS
Immunoinformatics
The C3d protein was screened for potential immunogenicity using previously
published EpiMatrix System.
20
Briefly, the 302-amino-acid sequence was parsed
into overlapping 9-mer frames where each frame overlaps the last by eight
amino acids. Each frame was then scored for predicted binding to each of eight
common class II HLA alleles (DRB1*0101, DRB1*0301, DRB1*0401,
DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301 and DRB1*1501). Due to
their prevalence and their difference from each other, these eight alleles cover
around 97% of human populations worldwide.
21
EpiMatrix raw binding score
predicted for each 9-mer sequence was normalized with respect to a distribu-
tion of scores derived from a very large set (N410 000) of randomly generated
9-mer sequences. This results in a ‘Z’ score for each analyzed 9-mer. The Z score
determines the position of a 9-mer relative to the distribution of all binding
scores generated for the random 9-mer sequences. Any peptide scoring above
1.64 on the EpiMatrix ‘Z’ scale (approximately the top 5% of the random
peptide set) has a significant chance of binding to the MHC molecule for which
it was predicted. Peptides scoring above 2.32 on the scale (the top 1%) are
extremely likely to bind; most published T-cell epitopes fall within this range of
scores. Therefore, the higher the Z score, the higher is the probability that a
peptide will be presented to T cells by APCs. Previous studies have demon-
strated that EpiMatrix accurately predicts published HLA ligands.
22,23
EpiMatrix has been shown to predict HLA-DR epitopes from a therapeutic
protein and demonstrated that the reactivity to those epitopes correlated with
the strength of the antitherapeutic antibody response.
24
Potential immuno-
genicity is not randomly distributed throughout protein sequences but instead
tends to ‘cluster’ in immunogenic regions. ClustiMer, an ancillary algorithm
used with EpiMatrix, maps MHC motif matches along the length of a protein
and calculates the density of motifs for several HLA. Typical T-cell epitope
‘clusters’ range from 9 to roughly 25 amino acids in length and, considering
their affinity to multiple alleles and across multiple frames, can contain
anywhere from 4 to 40 binding motifs.
Peptide synthesis
Peptides were synthesized (New England Peptide, Gardner, MA, USA) by
9-fluoronylmethoxy-carbonyl (Fmoc) synthesis using an automated Rainen
Symphony-Protein Technologies synthesizer (to a purity of 90% by HPLC).
The chosen peptides (comprised of amino acids 11–26, 45–65, 62–80, 223–246,
248–265 and 269–286) were based on EpiMatrix analysis described above.
0
1000
2000
3000
4000
5000
C3d Influenza HA
Frequency of
IFN-gamma+ CD4+ cells/10^6
0
300
600
900
1200
1500
C3d Negative
IFN-gamma (pg/mL)
Figure 3 (a) Stimulation of peripheral blood mononuclear cells (PBMCs) from normal healthy human donor with pool of C3d peptides led to secretion of
IFN-g. PBMCs were stimulated in culture with C3d peptide pool (10 mgml
1
) or no peptide for 7 days. Cells were then washed and re-stimulated with C3d
peptides or no peptide. Culture supernatants were collected 3 days later and analyzed by IFN-g ELISA. (b) Cells were collected on day 10 and re-stimulated
for 4 h at 37 1C with C3d peptides or flu HA (10 mgml
1
as a negative control) in the presence of GolgiPlug (BD Biosciences). IFN-g helper T cells were
detected in the C3d re-stimulated cells but not in the negative control. No CD8
+
IFN-g
+
cells were detected (data not shown).
C3d as a helper T-cell target
PM Knopf et al
224
Immunology and Cell Biology

HLA binding assays
Class II HLA binding assay was performed as initially described by Kwok and
colleagues
25
and adapted for high throughput by EpiVax. Nonbiotinylated test
peptide over a wide range of concentrations (0.001–400 mM) competes for
binding to purified class II molecules (50 nM) against a biotinylated standard
peptide at a fixed concentration (0.1 m
M) for 24 h in 96-well plates at 37 1C.
Class II molecules are then captured on ELISA plates using pan anti-class II
antibodies (L243, anti-HLA-DR) developed by addition of streptavidin-euro-
pium and read on a time-resolved fluorescence (TRF) plate reader. Nonlinear
regression analysis is performed and an IC50 value is calculated. Binding assays
were performed for four HLA alleles: DRB1*0101, DRB1*0401, DRB1*0701
and DRB1*1501, which provide a broad representation of HLA class II allele
binding pockets.
21
T-cell assay
PBMCs from four healthy human subjects were isolated by ficoll separation.
The purified PBMCs were stimulated with dimethyl sulfoxide (DMSO; negative
solvent control) or with a pool of six C3d-derived peptides at a final total
peptide concentration of 10 mgml
1
in RPMI media supplemented with 20%
human serum and incubated for 7–10 days at 37 1C, 5% CO
2
.PBMCswere
then re-stimulated with the appropriate C3d peptide, an irrelevant peptide, no
peptide or PHA. Cryopreserved PBMCs (Cellular Technology Limited, Shaker
Heights, OH, USA) from an HLA-DRB1*0101 subject were initially stimulated
with no peptide (DMSO negative control) or a pool of six C3d peptides for 7
days. Cells were then re-stimulated with DMSO or a pool of six C3d peptides
for 3 days.
Intracelluar cytokine staining
Cryopreserved PBMCs previously re-stimulated with C3d-derived peptides as
described above were re-stimulated again for 4 h with GolgiPlug (BD Bio-
sciences, San Jose, CA, USA) and the appropriate peptide (C3d pool of six, Flu
HA
306–318
or DMSO solvent control). A total of 110
6
PBMCs were stained
with fluorescently labeled antibodies to surface proteins CD4 and CD8
(eBioscience, San Diego, CA, USA and BD Biosciences) for 30 min on ice in
Flow Staining Buffer (eBioscience) and washed twice with buffer. Following cell
surface staining, cells were fixed and permeabilized (eBioscience) and stained
intracellularly for IFN-g (eBioscience) following the manufacturer’s protocol.
Cells were run on a FACSCalibur (BD Biosciences) and data was analyzed using
the FlowJo software (Treestar).
IFN-c ELISA analysis
Supernatants from stimulated cells were evaluated 3 days after re-stimulation
by human IFN-g ELISA (R&D Systems, Minneapolis, MN, USA).
Multiplex cytokine ELISA analysis
Supernatants from stimulated cells were assayed for levels of multiple cytokines
and chemokines by the SearchLight Multiplex ELISA contract service (Pierce
Biotechnology, Woburn, MA, USA).
ACKNOWLEDGEMENTS
The initial phases of the work were supported by a grant from EpiVax Inc. to
PMK at Brown University. SHH received a summer UTRA fellowship from
Brown University and is an undergraduate student at Brown University. All of
the other authors are employed by EpiVax Inc. PMK is the Charles A & Helen B
Stuart Professor Emeritus of Medical Science, Department of Molecular
Microbiology & Immunology at Brown University. DSR is a postgraduate
student at the University of Edinburgh, College of Medicine and Veterinary
Medicine, UK. ADG is an Adjunct Professor, Brown University School of
Medicine. We thank Claire Rodrı
´
guez and Christine Malboeuf for their work
on this project.
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