Assessment of Roles for Calreticulin in the Cross-
Presentation of Soluble and Bead-Associated Antigens
Natasha Del Cid1,2, Lianjun Shen3, Janice BelleIsle3, Malini Raghavan1,2*
1Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America, 2Department of Microbiology and
Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America, 3Department of Pathology, University of Massachusetts Medical
School, Worcester, Massachusetts, United States of America
Antigen cross-presentation involves the uptake and processing of exogenously derived antigens and their assembly with
major histocompatibility complex (MHC) class I molecules. Antigen presenting cells (APC) load peptides derived from the
exogenous antigens onto MHC class I molecules for presentation to CD8 T cells. Calreticulin has been suggested to mediate
and enhance antigen cross-presentation of soluble and cell-derived antigens. In this study, we examined roles for
calreticulin in cross-presentation of ovalbumin using a number of models. Our findings indicate that calreticulin does not
enhance in vitro cross-presentation of an ovalbumin-derived peptide, or of fused or bead-associated ovalbumin.
Additionally, in vivo, calreticulin fusion or co-conjugation does not enhance the efficiency of CD8 T cell activation by soluble
or bead-associated ovalbumin either in wild type mice or in mice lacking Toll-like receptor 4 (TLR4). Furthermore, we detect
no significant differences in cross-presentation efficiencies of glycosylated vs. non-glycosylated forms of ovalbumin.
Together, these results point to the redundancies in pathways for uptake of soluble and bead-associated antigens.
Citation: Del Cid N, Shen L, BelleIsle J, Raghavan M (2012) Assessment of Roles for Calreticulin in the Cross-Presentation of Soluble and Bead-Associated
Antigens. PLoS ONE 7(7): e41727. doi:10.1371/journal.pone.0041727
Editor: Rachel Louise Allen, University of London, St George’s, United Kingdom
Received May 25, 2012; Accepted June 27, 2012; Published July 27, 2012
Copyright: ? 2012 Del Cid et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is funded by the National Institutes of Health (grant number AI066131). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Antigen presenting cells present peptides bound to MHC class I
or class II molecules to T cells; this process facilitates T cell
development, homeostasis, peripheral tolerance and activation of
antigen specific T cells. Typically, MHC class I molecules present
peptides derived from endogenous antigens to CD8 T cells.
However, exogenous antigens can also be presented by MHC class
I molecules of professional APCs such as dendritic cells (DC) by a
process termed cross-presentation. APCs internalize extracellular
soluble or cell-associated antigens and traffic the material to
intracellular compartments that facilitate MHC class I presenta-
tion of the exogenously derived antigens (reviewed in ). Cross-
presentation is suggested to be critical for the maintenance of CD8
T cell peripheral tolerance and for generation of cytotoxic T cell
responses against intracellular pathogens and tumor cells (reviewed
Calreticulin is an endoplasmic reticulum (ER)-localized chap-
erone that aids in the intracellular assembly of nascent MHC class
I molecules and other newly synthesized glycoproteins (reviewed in
). Previous studies have also shown that calreticulin purified
from tumor cells can elicit tumor-specific protective immunity
[3,4]. Calreticulin is a protein chaperone with glycoprotein and
polypeptide-specific binding sites (reviewed in ). The immuno-
genic properties of purified tumor cell-derived calreticulin [3,4]
can be explained by co-purification with calreticulin of various
tumor-derived peptides or proteins. Anti-tumor immunity con-
ferred by purified calreticulin could derive from calreticulin-
dependent delivery of intracellular antigens to relevant APCs.
Alternatively or additionally, calreticulin-specific receptors could
confer quantitative cross-presentation advantages. The latter
possibility is suggested by findings that calreticulin cross-presents
associated peptides more efficiently compared to the peptides
alone in vitro and in vivo [3,5].
CD91, scavenger receptor A (SRA), and scavenger receptor
expressed by endothelial cell-I (SREC-1) are suggested to function
as receptors for extracellular or cell-surface forms of calreticulin
[5,6,7,8]. These receptors are not exclusive to calreticulin as they
bind other heat shock proteins such as gp96, HSP90 and HSP70
[5,6,9]. A particular receptor could quantitatively impact cross-
presentation by specific enhancement in antigen uptake  or by
directing antigen into distinct compartments favorable for cross-
presentation . Recent studies indicate that SRA deficiency
enhances cross-presentation of cell-associated antigens and anti-
tumor immunity [12,13]. However, it is unknown whether other
receptors implicated in binding and uptake of calreticulin (CD91,
SREC-1 [5,7,8], or other uncharacterized receptors) can enhance
cross-presentation efficiency of associated antigens. To address the
contributions of any receptor system that may be relevant to
uptake and cross-presentation of calreticulin-associated antigens,
in this study, we examined relative cross-presentation efficiencies
of a calreticulin-antigen fusion compared to antigen alone. For
these studies, we used the model antigen ovalbumin expressed and
purified from E. coli. The mannose receptor is a C-type lectin
receptor suggested to be important for uptake and cross-
presentation of ovalbumin [14,15]. As the mannose receptor is
expected to recognize glycan components of ovalbumin (reviewed
PLoS ONE | www.plosone.org1 July 2012 | Volume 7 | Issue 7 | e41727
in ), it was also of relevance to this study to compare cross-
presentation efficiencies of the non-glycosylated E. coli-derived
ovalbumin with that of glycosylated ovalbumin derived from a
natural source, which was undertaken.
Calreticulin does not enhance the cross-presentation of a
Calreticulin-peptide complexes form when peptides and calre-
ticulin are mixed and heat shocked to 50uC . To examine
whether calreticulin-specific receptors enhance the cross-presen-
tation of a calreticulin-associated peptide, we heated calreticulin or
bovine serum albumin (BSA) with a FITC labeled peptide derived
from the model antigen OVA (amino acids 255–267: QLESIIN-
FEKLTE-FITC) at 50uC for 1 hour. Free peptide was removed
using a centrifugal filter device, and the amount of peptide bound
to calreticulin and BSA was examined. We observed that the
peptide bound equally to both calreticulin and BSA (Figure 1A,
left panel). We next incubated bone marrow-derived dendritic cells
(BMDC) with the peptide complexes or peptide alone, and then
added to the culture a T cell hybridoma line (B3Z) whose T-cell
receptor ligand is the OVA258–265epitope (SIINFEKL) bound to
the murine MHC class I allele H2-Kb. We observed equal IL-
2 levels in the culture supernatants when comparing responses to
calreticulin-associated or BSA-associated peptide (Figure 1A, right
panel) and equal IL-2 levels when comparing calreticulin-
associated peptide or free peptide (Figure 1B). We also incubated
the calreticulin- or BSA-peptide complexes with bone marrow-
derived macrophages (BM Mw), as Mw express a different set of
receptors than dendritic cells. Similar to the results seen with
BMDC, calreticulin did not enhance the cross-presentation of a
peptide-associated antigen compared to BSA when BM Mw were
used as the APC (data not shown). Thus, the calreticulin-specific
receptors that have been reported [5,6,7] are not sufficient to
enhance the cross-presentation of the peptide used in this study.
Calreticulin does not enhance the cross-presentation of a
fused protein antigen
The extended peptide used in Figure 1 does not have high
specificity for calreticulin binding, as similar amounts of peptide
were recovered following heat shock with calreticulin or BSA
(Figure 1A, left panel). The peptide-binding site of calreticulin and
specificity of peptides that bind to calreticulin are poorly
understood and defined. Hence, we generated a fusion protein
between full-length OVA and calreticulin that linked OVA to the
N-terminus of calreticulin (OVA-CRT). Both OVA-CRT and
OVA were expressed in E. coli and contained a histidine tag for
purification. Proteins were first purified over a nickel column and
then further purified and analyzed on a size-exclusion column.
OVA-CRT and OVA were both isolated predominantly as single
peaks, results indicative of the homogeneity and stability of both
proteins. The major peak of both proteins was isolated and used
for subsequent experiments (Figure 2A).
To assess the cross-presentation efficiency of OVA-CRT
compared to OVA, OVA-CRT or OVA were incubated with
BMDC and CFSE labeled OT-I T cells (obtained from a
transgenic mouse whose CD8 T cells express a T cell receptor
that recognizes the OVA257–264epitope [SIINFEKL] bound to the
murine MHC class I allele H2-Kb). Levels of IL-2 in the
supernatant were measured after 24 hours and OT-I T cell
proliferation was measured after 3 days. No significant differences
were observed in levels of IL-2 produced by the OT-I T cells in
response to OVA or OVA-CRT, and proliferation of the OT-I T
cells was found to be very similar (Figure 2B). To assess binding of
OVA and OVA-CRT to BMDC, both proteins were labeled with
allophycocyanin. Higher level of fluorescence incorporation was
observed for OVA-CRT compared to OVA, and correspondingly,
binding to BMDC was slightly enhanced for OVA-CRT
compared to OVA (Figure 2C and 2D). Due to the difficulty in
achieving equivalent labeling of OVA-CRT and OVA, it remains
unclear whether calreticulin fusion to OVA confers a specific
BMDC binding advantage to OVA.
To evaluate the in vivo responses to the soluble proteins, CFSE
labeled OT-I T cells were injected intravenously (i.v.) into wild-
type (WT) recipient mice. Twenty-four hours later, mice were
immunized subcutaneously (s.c.) with equimolar amounts of OVA
or OVA-CRT. Three days after the immunization, proliferation of
the OT-I T cells from the draining lymph node was assessed.
Percentages of proliferating OT-I T cells (Figure 3A and 3C, left
panel) and percentages of OT-I T cells recovered (Figure 3B and
3C, right panel) were similar in response to OVA, whether or not
OVA was fused to calreticulin.
In the experiments described thus far, lipopolysaccharide (LPS)
contamination was of concern because the recombinant proteins
used in these experiments are of E. coli origin. As LPS is an agonist
for TLR4 , it was conceivable that LPS contamination could
mask potentiating effects of calreticulin. To address this concern,
TLR42/2recipient mice  were used, previously characterized
for their inability to respond to LPS. OVA-CRT did not induce a
greater CD8 T cell response compared to OVA alone even when
TLR42/2mice were used as recipients (Figure 3A, left panel and
3B). We concluded that there was no specific advantage for OT-I
T cell proliferation when OVA was fused to calreticulin compared
to OVA alone, even in the absence of LPS signaling. It is however
Figure 1. Cross-presentation of a peptide antigen. (A) 10 mM
calreticulin (CRT) or bovine serum albumin (BSA) were incubated with
10 mM peptide (QLESIINFEKLTE-FITC). Free peptide was removed using
a centrifugal filter device at 4uC, and peptide still in complex with CRT
or BSA was measured (left panel). CRT- or BSA-peptide complexes were
incubated with BMDC and B3Z cells. IL-2 production was determined by
ELISA of the supernatants after 24 hours (right). Peptide concentration
is indicated; CRT or BSA were present at a final concentration of 1 mM.
(B) Cross-presentation of free peptide or CRT-peptide complexes was
measured as in A. Data are representative of two independent analyses
for both A (right panel) and B. Mean 6 s.e.m. are shown in A and B.
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noteworthy that percentages of OT-I T cells recovered in response
to OVA and OVA-CRT were reduced in TLR42/2mice
compared to WT mice (Figure 3B). This reduction was not
surprising as TLR4 engagement on APCs induces APC matura-
tion and migration to lymph nodes, resulting in augmented T cell
responses (reviewed in ). These results are also consistent with
previous findings that TLR signaling enhances in vitro cross-
presentation of soluble antigen , and that LPS induces cross-
presentation of ovalbumin, following intramuscular immunization
Calreticulin does not enhance the cross-presentation of a
We next hypothesized that calreticulin may induce cross-
presentation of a particulate antigen, as calreticulin has been
shown to function as an ‘‘eat-me’’ signal when present on the
surface of cells [8,24,25,26]. OVA or an equimolar mixture of
OVA and calreticulin (OVA+CRT) were conjugated to 1.5 mm
iron oxide beads, and cross-presentation efficiencies were assessed
in vitro and in vivo. Levels of OVA conjugated to beads were
quantified with a fluorescent Ab. Comparable amounts of OVA
were conjugated in the OVA and OVA+CRT bead preparations
(Figure 4A). Similar to Figure 2B, OVA or OVA+CRT beads
were next incubated with BMDC and CFSE labeled OT-I T cells.
Again, there was no difference in the levels of IL-2 generated or in
the proliferation of the OT-I T cells induced in response to OVA
or OVA+CRT beads (Figure 4B). Similar results were obtained in
vivo with WT or TLR2/42/2recipient mice  (Figure 4C and
4D). As noted above, percentages of OT-I T cells recovered in
response to OVA and OVA+CRT beads were reduced in TLR2/
42/2mice compared to WT mice (Figure 4D). Both sets of results
echoed our findings using the soluble OVA-CRT fusion protein.
Taken together, we concluded that extracellular calreticulin, in a
soluble or particulate context, does not enhance the cross-
presentation of associated antigen.
Glycosylated and non-glycosylated OVA are cross-
presented with similar efficiencies
Cross-presentation of soluble OVA is suggested to be mediated
by the mannose receptor . OVA purified from E. coli is not
glycosylated, as E. coli lack eukaryotic glycosylation machinery.
However, we found that non-glycosylated OVA isolated from E.
coli was cross-presented in vivo (Figure 3), indicating that the
mannose receptor may be non-essential for the cross-presentation
of soluble OVA. This result prompted us to compare the in vivo
cross-presentation efficiencies of OVA purified from E. coli and
OVA obtained from Sigma [OVA (Sigma)], which is glycosylated
as it is isolated from chicken eggs. This comparison would allow us
to further determine the importance of OVA uptake through the
mannose receptor or alternative pathways upon the proliferation
of OT-I T cells.
Figure 2. In vitro cross-presentation of a calreticulin-fused soluble antigen. (A) Gel-filtration chromatogram of E. coli-derived OVA or the
OVA-calreticulin (OVA- CRT) fusion protein (left). SDS-PAGE analysis of pooled fractions from left panel; proteins were loaded in equimolar amounts
(right) and coomassie stained. (B) Indicated proteins were incubated with BMDC for 3 hours. BMDC were fixed and CFSE labeled OT-I T cells were
added. IL-2 levels in supernatants were determined by ELISA (left panel; 24 hour time point). OT-I T cell proliferation was measured at 72 hours in
response to 44 mM OVA or OVA-CRT. The solid grey profile indicates the condition where no antigen (no Ag) was added. Data are representative of
two independent analyses. (C, D) OVA-CRT and OVA were labeled with allophycocyanin. (C) Labeling intensity was determined by fluorescence
imaging of the proteins after separation by SDS-PAGE (inset). Fluorescence intensity was quantified for the indicated proteins. (D) Binding of
fluorescent proteins to BMDC was assessed by flow cytometry. BMDC were incubated with labeled proteins on ice before being analyzed by flow
cytometry. BMDC not incubated with proteins are depicted as a grey filled. Representative of two independent experiments performed with the same
labeled proteins. Mean 6 s.e.m. are shown in B.
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We assessed the abilities of s.c. delivered OVA (E. coli) and OVA
(Sigma) to induce proliferation of CFSE labeled OT-I T cells in
vivo. Using WT mice as recipients, the percentages of proliferating
OT-I T cells were similar in response to OVA (Sigma) compared
to OVA (E. coli) (Figure 5, left and middle panels; p=0.5247). The
percentage of OT-I T cells recovered was also similar in response
to OVA (Sigma) compared to OVA (E. coli) (Figure 5, right panel;
p=0.1785), although there was a trend towards a stronger
response with OVA (Sigma). We concluded that glycosylated
OVA does not have a significant cross-presentation advantage
compared to non-glycosylated OVA in vivo. Taken together, the
data in Figure 5 suggest that the mannose receptor is non-essential
for the cross-presentation of soluble OVA in vivo.
One previous study showed enhanced cross-presentation of a
soluble peptide by Mw following heat-shock in the presence of
calreticulin, compared to peptide alone . Using an extended
version of the OVA-derived SIINFEKL peptide, we were unable
to demonstrate enhanced calreticulin-dependent cross-presenta-
tion by BMDC or BM Mw (data not shown). The discrepancies
could reflect differences between antigens used in the two studies,
and it was possible that the extended SIINFEKL peptide lacked
adequate specificity for calreticulin. Rules for high affinity binding
of particular peptide sequences to calreticulin are not well
understood. By linking full-length OVA to calreticulin, we by-
passed the need to identify a peptide with a high affinity for
calreticulin. We were unable to observe enhanced CD8 T cell
proliferation in vitro or in vivo in response to extracellular OVA-
CRT compared to OVA alone (Figures 2 and 3). Previous studies
indicate a significant but non-essential role for the mannose
receptor in the uptake of ovalbumin [14,15], although other
studies suggest the requirement for mannose receptor may be
APC-dependent . The ovalbumin proteins used in Figures 2
and 3 were recombinant proteins of E. coli origin, and are thus not
expected to be internalized via the mannose receptor due the
absence of glycan modifications. Nonetheless, significant activation
of the OT-I T cell response was induced in vivo following OVA or
OVA-CRT s.c. immunizations at antigen doses as low as 1 mg/
mouse (Figure 3C and replicate analyses). In the analyses of
Figures 2 and 3, uptake of the OVA and OVA-CRT proteins may
occur via receptor-mediated endocytosis that involves mannose
receptor-independent uptake pathways, or via pinocytosis. This
possibility is further supported by the analyses shown in Figure 5,
where no significant differences were measurable between cross-
presentation efficiencies of ovalbumin derived from E.coli or
chicken eggs, which are expected to be non-glycosylated and
glycosylated respectively. Altogether, the studies described in
Figures 2, 3, and 5 taken together with previous studies [14,15,28]
indicate that multiple uptake pathways can contribute to the cross-
presentation of ovalbumin. Regardless of the precise uptake
pathway, it is noteworthy that ovalbumin delivered as a
calreticulin fusion does get cross-presented, although a specific
cross-presentation advantage that results from the calreticulin
fusion is not apparent. Thus, if calreticulin-specific receptors
Figure 3. In vivo cross-presentation of a calreticulin-fused soluble antigen. (A, B) WT or TLR42/2recipient mice were injected i.v. with CFSE
labeled OT-I T cells. Twenty-four hours later, mice received s.c. injections of the indicated antigen (100 ml of a 220 nM solution). OT-I T cell
proliferation was measured 3 days later in the dLN (inguinal). (A) The % of proliferating OT-I T cells averaged from the mice of one experiment is
shown in the left panel. Two to three mice were used in all groups. The right panel depicts proliferation in WT recipient mice. (B) Quantification of the
% of OT-I T cells of all CD8 T cells recovered in A. Data for A and B are representative of three out of four independent analyses for WT recipients and a
single analysis with TLR42/2recipients. Similar results were obtained in comparisons of OVA and OVA-CRT-induced OT-I proliferation in WT and TLR2/
42/2recipient mice (data not shown). (C) Compilation of the % of proliferating OT-I T cells (left panel) and of the % of OT-I T cells as a function of all
CD8 T cells (right panel) from 4 independent experiments performed with WT recipient mice. Two experiments contained 2 doses of antigen and two
experiments contained 1 antigen dose. Antigen doses ranged from 0.22 mM–22 mM, using 100 ml. Each point represents the mean of 2–3 mice for
that condition. Mean 6 s.e.m. are shown in A and B. A two-tailed pair-wise student t-test was used for statistical analyses in C.
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contribute to increased uptake of the OVA-CRT fusion in vitro or
in vivo, such uptake does not result in increased cross-presentation.
Taken together, our studies suggest that the immunogenic
properties of calreticulin purified from tumor cells [3,4] must
result from co-purification and subsequent cross-presentation of
one or more tumor-derived antigens, rather than a calreticulin-
dependent influence on the cross-presentation pathway per se. By
binding to antigen within its substrate binding site(s), calreticulin
could protect antigen from complete proteolytic degradation, thus
preserving antigen in a form that is competent for subsequent
cross-presentation. General uptake pathways for soluble antigen
may be operative during the cross-presentation of calreticulin-
antigen complexes, and it is possible that calreticulin binding can
confer a kinetic advantage for cross-presentation over complete
degradation, at least for some antigens. The latter mechanism
Figure 4. Cross-presentation of a particulate antigen. (A) OVA alone or with calreticulin (OVA+CRT) were conjugated to iron-oxide beads.
Levels of OVA conjugated to the beads were quantified using a fluorescence-based assay. (B) The indicated beads were incubated with BMDC for
3 hours. BMDC were fixed and CFSE labeled OT-I T cells were added. Left and middle panels are representative of 1 independent experiment. IL-2
production was determined by ELISA of the supernatants at 24 hours (left panel). OT-I T cell proliferation was measured after 72 hours in response to
10 ml OVA or OVA+CRT beads. The solid grey profile indicates the condition where no antigen (no Ag) was added (middle panel). A compilation of the
% of proliferating OT-I T cells from 3 independent experiments is depicted in the right panel in response to 1–5 or 10 ml beads. (C, D) OT-I T cell
proliferation and recovery were measured in WT or TLR2/42/2mice in response to 50 ml beads on day 3. (C) Average proliferation values for 2–3 mice
per group (except PBS, where 1 mouse was used) are shown in the left panel. A representative proliferation prolife from WT recipients in response to
OVA beads, OVA+CRT beads or PBS (filled in grey) is shown on the right panel. (D) Quantification of the % of OT-I T cells of all CD8 T cells recovered in
C. Data are representative of two independent analyses for C. Mean 6 s.e.m. are shown in A–D. A two-tailed pair-wise student t-test was used for
statistical analysis in B.
Figure 5. In vivo cross-presentation of glycosylated and non-glycosylated OVA. WT recipient mice were injected i.v. with CFSE labeled OT-I
T cells. Twenty-four hours later, mice received s.c. injections of the indicated antigen (2.5–100 mg). OT-I T cell proliferation was measured 3–5 days
later in the dLN (inguinal). Left panel: A representative proliferation profile is presented in response to 2.5 mg OVA. Middle and right panels: Two to
three mice are averaged to generate each data point, which represents the % of proliferating OT-I T cells (middle) or the % of OT-I T cells as a function
of all CD8 T cells recovered (right). Three independent experiments are represented. In one experiment, 2 different OVA (E. coli) preps were used in 2
different groups of mice. In another experiment, two doses (10 mg and 100 mg) of OVA were used in 2 different groups of mice. A two-tailed pair-wise
student t-test was used for statistical analysis.
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might explain previous findings of the potentiating activity of
calreticulin during cross-presentation of elongated peptides .
It remains possible that the soluble OVA-CRT construct may
not have been able to bind calreticulin-specific receptors with a
high enough avidity to impact the cross-presentation of OVA.
OVA may have masked the calreticulin-receptor binding site on
calreticulin. To address this issue, calreticulin and OVA or OVA
alone were conjugated to iron oxide beads and cross-presentation
efficiencies were assessed. OVA and calreticulin are not fused in
this system. Thus, calreticulin-receptor interactions should not be
inhibited. We show that calreticulin was not able to enhance the
cross-presentation of the bead-associated OVA compared to beads
with OVA alone, both in vitro and in vivo (Figure 4). Calreticulin has
been reported to be an ‘‘eat me’’ signal on the surface of apoptotic
cells [8,25]. The findings of Figure 4 suggest that calreticulin does
not work independently in a phagocytic context, but rather might
work in conjunction with other ‘‘eat me’’ signals such as
phosphatidylserine. Hence, calreticulin in isolation is not sufficient
to enhance cross-presentation of a particulate antigen. However, it
is also possible that phagocytic uptake of the iron oxide beads is
intrinsically high, even in the absence of calreticulin.
In summary, we have examined whether calreticulin can
influence CD8 T cell proliferation against peptide, soluble and
bead-associated antigen. We show that ovalbumin is cross-
presented with similar efficiency when delivered alone compared
to delivery with calreticulin as a soluble fusion, or co-conjugated
on beads. Additionally, glycosylated and non-glycosylated forms of
ovalbumin are cross-presented with similar efficiencies. TLR4
signaling induces efficiency of cross-presentation of subcutaneously
delivered soluble and bead-associated antigens. Further studies are
needed to understand roles for calreticulin and relevant receptors
in phagocytosis and cross-presentation in the context of cell-
associated antigens and subcutaneous immunizations.
Materials and Methods
All mice were maintained in specific pathogen-free conditions at
the University of Michigan or the University of Massachusetts
Medical School (UMMS) mouse facilities. All experiments
involving mice were approved by and performed in accordance
with guidelines set forth by the University Committee on Use and
Care of Animals (UCUCA) at the University of Michigan or the
UMMS Department of Animal Medicine and the Institutional
Animal Care and Use Committee. C57BL/6J (WT or B6 in text;
CD45.2), B6.SJL-PtprcaPepcb/BoyJ (WT or B6 in text; CD45.1),
and C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I in text) mice were
purchased from The Jackson Laboratory. OT-I transgenic mice
were used directly (CD45.2) or bred with B6.SJL-Ptprca Pep3b/
Boy mice (The Jackson Laboratory) to yield CD45.1 T cells.
TLR22/2and TLR42/2mice [20,27] were provided by Dr. S.
Akira at the Laboratory of Host Defense, Osaka University,
Osaka, Japan. TLR2/42/2mice were generated by crossing
TLR22/2with TLR42/2mice. TLR42/2mice were back-
crossed onto the B6 background six times before being bred with
Cell culture, purification and labeling
The B3Z hybridoma T cell line  was
maintained in RPMI+ [RPMI medium 1640 (Invitrogen) supple-
mented with 10% (v/v) fetal bovine serum, 100 mg/ml strepto-
mycin, and 100 units/ml penicillin (Invitrogen)]. Cells were
maintained in an incubator kept at 37uC with 5% CO2.
with RPMI+. The red blood cells were lysed using red cell lysis
buffer (Sigma), and the cells were re-suspended in RPMI medium
supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 mg/
ml streptomycin, 100 units/ml penicillin (Invitrogen), 1 mM
HEPES (Gibco), 0.1 mM MEM Non-Essential Amino Acids
(Gibco), 1 mM Sodium pyruvate, 50 mM b-mercaptoethanol and
granulocyte Mw colony-stimulating factor (GM-CSF). The bone
marrow obtained from one mouse was plated into two 24-well
plates (Corning). The medium was replaced on days 2 and 4, and
the cells were harvested for in vitro experiments on day 5 or 6.
Purification of splenic CD8+ + T cells.
extracted from OT-I transgenic mice. The red blood cells were
lysed using red cell lysis buffer (Sigma), and the CD8+cells were
isolated by positive selection using anti-CD8a (Ly-2) microbeads
(MACS, Miltenyi Biotec), respectively, following the manufactur-
er’s suggested protocol.
MACS purified CD8+ T cells from OT-I
transgenic mice were labeled with CFSE for proliferation analyses.
CD8+ T cells were washed once with PBS, centrifuged, and re-
suspended in PBS + 5 mM CFSE. The cells were incubated at
37uC for 10 min. The cells were washed once with medium, re-
suspended in RPMI+ and incubated at 37uC, 5% CO2for 1 hour.
The cells were then centrifuged and re-suspended in an
appropriate volume of RPMI+ for in vitro experiments or PBS
for in vivo experiments. On average, 3–56106live, CFSE labeled,
CD8+ OT-I T cells were recovered from one spleen, where trypan
blue staining assessed viability.
Bone marrow was flushed from the femur and tibia
Expression of calreticulin, OVA, and OVA-CRT in
BC003453) was amplified from the pCMV-SPORT6 (ATCC,
MGC-6209) vector using primers that allowed for subsequent
ligation-independent cloning (LIC) into the pMCSG7 vector .
The following primers were used: forward, 59 TAC TTC CAA
TCC AAT GCT GCC GCA CAT CCT TGG CTT 39 and
reverse, 59 TTA TCC ACT TCC AAT GTT ACA GCT CAT
CCT TGG CTT 39. Underlined bases represent those that are
complementary to the sequence encoding calreticulin, and
additional 59 sequences were introduced for LIC.
Chicken egg OVA (accession number V00383) was amplified
for LIC. The following primers were used: forward: 59 TAC TTC
CAA TCC AAT GCT ATG GGC TCC ATC GGC G 39 and
reverse, 59 TTA TCC ACT TCC AAT GTT AAG GGG AAA
CAC ATC TGC 39. Underlined bases represent those that are
complementary to the sequence encoding OVA, and additional 59
sequences were introduced for LIC.
The OVA-CRT fusion protein was constructed using a 2-step
amplification process resulting in a full length OVA molecule fused
to the N-terminus of full length calreticulin by a flexible linker (gly-
gly-ser-gly-gly). The reverse OVA primer was complementary to
the forward calreticulin primer, allowing for the fusion of the two
PCR products in a second PCR reaction. OVA was amplified
using the following primers: 59 TAC TTC CAA TCC AAT GCT
ATG GGC TCC ATC GGC G 39 and reverse, 59 GGC AGG GTC
TGC GGC TCC TCC TGA TCC ACC AGG GGA AAC ACA
TCT 39. Calreticulin was amplified using the following primers:
forward, 59 AGA TGT GTT TCC CCT GGT GGA TCA GGA
GGA GCC GCA GAC CCT GCC 39 and reverse, 59 TTA TCC
ACT TCC AAT GTT ACA GCT CAT CCT TGG CTT 39.
Underlined bases represent those that are complementary to the
sequence encoding OVA, bold bases represent the introduced
linker sequences (gly-gly-ser-gly-gly), italicized bases represent
Redundancies in Cross-Presentation Pathways
PLoS ONE | www.plosone.org6July 2012 | Volume 7 | Issue 7 | e41727
those that are complementary to calreticulin, and additional
sequences were introduced for LIC. Both PCR products were run
on a 0.8% agarose gel and gel-purified (Qiagen). Both products
were then used together as templates in a second PCR reaction
using the forward OVA primer and reverse calreticulin primer,
which allowed for subsequent LIC of the OVA-CRT fusion into
the pMCSG7 vector.
LIC was performed to introduce calreticulin, OVA, and OVA-
CRT sequences into the pMCSG7 vector as previously described
DNA constructs were sequenced and transformed into BL21
(DE3) cells for protein expression. All bacterially expressed
constructs lacked their signal sequence and contained an N-
quence for nickel affinity chromatography.
Glycerol stocks of BL21 (DE3) cells expressing calreticulin,
OVA, or OVA-CRT were inoculated into a 25-ml terrific broth
culture (with 50 mg/ml ampicillin) and incubated at 37uC
overnight. The starter culture was added to 1 liter of terrific
broth with 50 mg/ml ampicillin and incubated at 37uC until cell
density measured by A600 was 0.8–0.9. Cultures were then
incubated at room temperature for 1 h before inducing calreticu-
lin protein expression with 200 mM isopropyl 1-thio-ß-D-galacto-
pyranoside. Bacterial cultures were incubated at room tempera-
ture for 16–20 h before harvesting cells by centrifugation. Cell
pellets were resuspended in 50 ml of 50 mM Tris with 0.33 mg/
ml lysozyme and EDTA-free complete protease inhibitors (Roche
Applied Science). Cells were lysed by sonication. Subsequently,
10 mg/ml DNase, 1% Triton X-100, 10 mM MgCl2, and 1 mM
CaCl2were added, and the cell lysis suspension was incubated at
room temperature for 30 min. Cell debris was removed from
samples by centrifugation, followed by vacuum filtration of the
supernatant with a Steriflip having a 0.22-mm pore membrane
(Millipore). The filtrate was then incubated with nickel-nitrilo-
triacetic acid-agarose beads (Qiagen) for 2–4 h at 4uC. The beads
were washed with 10 mM imidazole in wash buffer (50 mM Tris,
150 mM NaCl, 1 mM CaCl2, pH 7.5). Protein was eluted from
beads with 75 and 100 mM imidazole in wash buffer. Protein was
concentrated to 0.5–4.0 ml, by centrifugation using Centriplus
centrifugal filter devices (Millipore) with molecular weight cut-offs
of 10 or 30 kDa, and analyzed by gel-filtration chromatography.
Purified concentrated protein was analyzed by gel filtration at
4uC using a Superdex 200 10/300 GL column or Highload 16/60
Superdex 200 (Amersham Biosciences). Buffer used was 20 mM
Hepes, 150 mM NaCl, 10% glycerol, 1 mM CaCl2, pH 7.5.
Following gel filtration, fractions were pooled and concentrated by
centrifugation using Centricon centrifugal filter devices (Millipore)
with molecular weight cut-offs of 10 or 30 kDa. Protein
concentration was determined measuring absorbance at 280 nm.
Extinction coefficients were calculated from the protein amino
acid sequence using ProtParam (www.expasy.ch) and are as
follows (units are in M21cm21): Calreticulin, 82,975; OVA,
33,265; OVA-CRT, 114,750.
Generation of calreticulin-peptide and BSA-peptide
10 mM calreticulin or BSA were incubated with 10 or 100 mM
peptide (QLESIINFEKLTE-FITC; University of Michigan Pro-
tein Structure Facility) for 1 hour at 50uC. Free peptide was
removed by centrifugal filter (Centricon-30, Millipore). Peptide
bound to calreticulin or BSA was measured using a VICTOR
plate reader (fluorimeter; PerkinElmer) against a peptide standard.
OVA and OVA-CRT allophycocyanin conjugation
Allophycocyanin was conjugated to OVA and OVA-CRT
following manufacturer’s instructions at a 1:1 molar ratio
(Lightning-Link Conjugation Kit, Innova Biosciences). To mea-
sure fluorescence intensity, OVA and OVA-CRT were separated
by SDS-PAGE and fluorescence was measured using a fluores-
cence imager (Typhoon TRIO, GE Healthcare). Quantification
was performed using ImageQuant5.2 software.
In vitro antigen cross-presentation assays
Assays using peptide antigen.
36104B3Z cells were plated in a 96 well plate. Calreticulin-
peptide complexes, BSA-peptide complexes, or peptide alone were
added to the wells. At 24 hours, supernatant IL-2 levels were
measured by ELISA.
Assays using soluble or bead-associated antigen.
56105BMDC were plated in a 96 well plate. Soluble proteins
were then added to the wells. The cells were incubated at 37uC for
3 hours. BMDC were fixed with 1% formaldehyde for 7 min at
room temperature. The wells were then washed two times with
200 ml PBS and once with RPMI+. Medium was aspirated from
the wells, and a suspension of 4–56105B3Z or 1–36105CFSE
labeled OT-I T cells in 250 ml RPMI+ was added to the wells. At
24 hours, supernatant IL-2 levels were measured by ELISA, and
proliferation of OT-I T cells was measured at 72 hours by flow
cytometry. OVA (Sigma) was obtained from Sigma (A5503).
3–56104BMDC and 2–
In vivo antigen cross-presentation assay
CD8+ OT-I T cells were MACS purified from CD45.1 or
CD45.2 mice and transferred i.v. into CD45.2 or CD45.1
recipient mice, respectively. One day later, mice were immunized
s.c. in either the left or right flank with the indicated concentra-
tion/amount of soluble or bead-associated antigen in 100 mL PBS.
Three to four days later, draining lymph nodes (inguinal) were
harvested and analyzed by flow cytometry.
OVA and OVA-CRT binding to BMDC.
were incubated with 1.3 mg OVA-CRT or 0.6 mg OVA that were
allophycocyanin labeled in a total reaction volume of 100 ml for
30 minutes on ice. Cells were washed twice with flow cytometry
buffer and analyzed by flow cytometry.
Detection of proliferating OT-I T cells.
nodes (inguinal) were harvested; a single cell suspension was
obtained and plated in a 96-well plate. Cells were lysed in red cell
lysis buffer (Sigma). These cells were washed once with flow
cytometry buffer (2% FBS in PBS) and then incubated for 10 min
with unconjugated murine IgG (Jackson ImmunoResearch) to
bind and block Fcreceptors. Cells from draining lymph nodes or
from in vitro cell cultures were stained with a rat anti-mouse CD8a
Ab conjugated to allophycocyanin (BD Pharmingen, 1:400) or
anti-CD8a Ab conjugated to PerCP (eBioscience) and mouse anti-
mouse CD45.1 or CD45.2 Ab conjugated to PE or allophycocya-
nin for in vivo experiments (BD Pharmingen, 1:300 and
eBioscience). Either CD45.1 or CD45.2 OT-I mice were utilized
for in vivo adoptive transfers into WT strains (CD45.2 or CD45.1,
respectively); the appropriate antibody was used to discriminate
between donor and recipient cells. The cells were incubated with
the Ab for 20 min, washed twice with flow cytometry buffer and
then analyzed by flow cytometry. CFSE labeled OT-I T cells were
analyzed in the FITC channel. All centrifugations in the 96-well
plate were performed for 1 minute at 2,000 rpm.
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PLoS ONE | www.plosone.org7July 2012 | Volume 7 | Issue 7 | e41727
96-well plates were coated with purified anti-mouse IL-2 Ab
(BD Pharmingen, catalog # 554424) overnight at room temper-
ature. After blocking with 10% calf serum for 3 hours, cell culture
supernatants were incubated in the plate for 1 hour at 37uC. The
plates were washed 3 times with 0.05% Tween-20 in PBS and a
biotinylated anti-mouse IL-2 Ab (BD Pharmingen, catalog #
554426) was added for an overnight incubation at room
temperature. The plates were washed 3 times, and streptavidin
conjugated to HRP (BD Pharmingen, catalog # 554066) was
added to the plates for 20 min at room temperature. The plates
were again washed 3 times, and the assay was developed using a
TMB substrate reagent set (BD OptEIA, catalog # 555214).
We thank Dr. Kenneth Rock for various mouse resources that were
provided, Dr. Nilabh Shastri for the B3Z cell line, Drs. Yasmina Laouar,
Yang Liu and Philip Lapinski for various experimental suggestions and
suggestions on data analyses.
Performed the experiments: NDC LS JB. Analyzed the data: NDC MR
LS. Wrote the paper: NDC MR.
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