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*
Center for Cellular and Genetic Therapy, Department of Surgery, and
Department of Cell Biology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The composite function of the ER lumenal chaperones is generally thought to be limited to the structural maturation of nascent polypeptides. However, the observations that ER chaperones such as GRP94 (gp96), GRP78 (BiP), and protein disulfide isomerase (PDI) display peptide-binding activity may portend alternative or additional roles for these proteins in the regulation of peptide trafficking within the ER (10, 11, 12, 13, 14, 15). In support of this hypothesis, it is now established that the ER Hsp90, GRP94, binds peptides suitable for assembly onto class I molecules (11, 12, 16, 17, 18). Whether this activity is indicative of a peptide "sink" function or perhaps reflective of a more substantive role in peptide/class I assembly reactions remains to be determined. The potential functional significance of the peptide-binding activity is evident, though, in the observations that vaccination of mice with GRP94 can elicit a substantial cellular immune response to components of the bound peptide pool (11, 16, 17, 18, 19). Thus, GRP94, when isolated from a variety of host backgrounds, including tumor cells or cells expressing viral or bacterial proteins, was capable of eliciting substantial CD8+ T cell responses to the parent tumors, as measured in tumor-mass regression studies, as well as known viral and bacterial peptide epitopes, as determined by CTL assay (11, 16, 17, 19). The conclusion appears evident, then, that APCs are capable of internalizing GRP94-peptide complexes and that, following internalization, at least a subset of the peptides in association with GRP94 can be released and targeted for assembly onto class I molecules. From these data, it may also be postulated that in all cells, GRP94 associates with ER lumenal peptides, a subfraction of which are suitable for assembly onto class I molecules.
Given the established role for calreticulin in the genesis of class I/peptide complexes and the propensity of ER chaperones to display peptide-binding activity, we investigated whether calreticulin displayed in vivo interactions with peptides. This question was addressed by direct biochemical analysis of acid-extracted, tissue-derived calreticulin and by the capacity of calreticulin purified from B16/F10.9, EL4, and E.G7-OVA tumors to elicit specific CTL responses. Direct chemical evidence supporting the existence of a calreticulin-bound peptide fraction was obtained. In addition, vaccination of mice with dendritic cells (DCs) pulsed with B16/F10.9, EL4, or E.G7-OVA-derived calreticulin, was observed to elicit CTL responses to undefined tumor-derived Ags B16/F10.9, EL4) or the immunodominant OVA peptide epitope, SIINFEKL (E.G7-OVA). Lastly, bone marrow-derived dendritic cells (BMDC) pulsed in vitro with E.G7-OVA-derived calreticulin presented tumor-specific peptides in association with class I molecules and were targeted for lysis by the OVA-specific CTL line, 4G3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice (H-2b, 5–6 wk old) and SCID mice were obtained from the Charles River Laboratories (Wilmington, MA). In conducting the research described in this paper, the investigators adhered to the Guide for the Care and Use of Laboratory Animals as proposed by the committee on care of Laboratory Animal Resources Commission on Life Sciences, National Research Council. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care.
Cell lines
Cell lines used were EL4 (C57BL/6, H-2b, thymoma), E.G7-OVA (EL4 cells transfected with the OVA cDNA), RMA-S cells (Rauscher leukemia virus-induced T cell lymphoma RBL-5 of C57BL/6 (H-2b) origin), and B16/F10.9 (F10.9) melanoma. Cells were maintained in DMEM supplemented with 10% heat-inactivated FCS (Life Technologies, Grand Island, NY), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. E.G7-OVA cells were grown in medium containing 400 µg/ml G418 (Life Technologies). T-cell hybridoma RF3370 (H-2Kb restricted, OVA specific) was maintained in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The OVA-specific CTL line 4G3 (20) (H-2Kb restricted, OVA specific) was carried in RPMI 1640, 10% heat inactivated FCS, 2 mM glutamine, and 30 U/ml IL-2 (Genzyme, Cambridge, MA). Cells were split every 2–4 days and restimulated weekly with irradiated E.G7-OVA cells at a 1:1 ratio. OVA peptide (H-2Kb restricted, SIINFEKL, aa 257–264) and the control mut-1 peptide (H-2Kb restricted, FEQNTAQP) were purchased from Research Genetics (Huntsville, AL).
Chaperone purification
Chaperone proteins were purified from solid tumors as described in Reference 21. Tumors were established in either C57BL/6 (B16/F10.9 melanoma) or SCID (EL4, E.G7-OVA thymoma) mice. Solid tumors were harvested, a microsomal, ER-enriched subfraction was prepared, and the ER chaperones GRP94 and calreticulin were purified to homogeneity from the microsomal fraction by selective detergent release, sequential MonoQ 10/10 anion exchange, Superdex 26/60 gel filtration chromatography (Pharmacia Biotech, Piscataway, NJ), and centrifugal ultrafiltration (Amicon, Beverly, MA), as described in Reference 21. The cytosol fraction obtained upon subfractionation of the tumor homogenate was used to purify Hsp90 and Hsp70. The cytosol fraction was initially subjected to a 50–70% ammonium sulfate fractionation. Protein precipitated at 70% ammonium sulfate was resuspended in buffer A (110 mM KOAc, 20 mM NaCl, 20 mM potassium HEPES, and 0.5 mM PMSF) and centrifuged for 20 min at 4°C (100,000 x g) to remove aggregated material, and the soluble fraction was fractionated by preparative gel filtration on a Superdex 26/60 column equilibrated in buffer A at a flow rate of 1.5 ml/min. Fractions containing Hsp70 or Hsp90 were identified by SDS-PAGE, pooled, and sequentially chromatographed on MonoQ 10/10 and Superdex 26/60 as described in Reference 20. BiP, ERp72, and PDI fractions arising from MonoQ fractionation of lumenal protein extracts, as well as Hsp70 and Hsp90 fractions eluting from the final Superdex 26/60 step, were adjusted to 10 mM sodium phosphate, pH 6.8, loaded onto 2.5-ml hydroxylapatite columns (Bio-Rad HTP, Hercules, CA), and eluted with a 25-ml gradient of 10–300 mM sodium phosphate, pH 6.8. By SDS-PAGE, the purity of the Hsp90 and Hsp70 fractions was determined to be >95% (data not shown). Protein purity was assessed by one- and two-dimensional SDS-PAGE.
Peptide extraction and analysis
Calreticulin-associated peptides were extracted from 1 mg (21.7 nmol) of purified porcine calreticulin by denaturation for 30 min at room temperature in the presence of guanidinium chloride/1% trifluoroacetic acid (TFA). The acid-soluble fraction was separated from intact calreticulin by centrifugal ultrafiltration, using acid-washed Centricon-10 filtration units. The low m.w. calreticulin-derived peptide fraction was subsequently bound to a prewashed Sep-Pak C18 unit, washed extensively with 1% TFA, and eluted in 80% acetonitrile, 0.1% TFA. The acetonitrile eluate was dried by vacuum centrifugation, and fractions were either resuspended in 0.2 M sodium phosphate (pH 7.2) and subjected to reductive methylation with 3H-labeled sodium borohydride as described in Reference 22 or subjected to acid hydrolysis in vacuo, with the amino acid content determined by quantitative amino acid analysis. Quantitative amino acid analysis was performed by the Duke University Medical Center Protein Sequencing Facility, a core facility of the Duke University Comprehensive Cancer Center. As a consequence of acid hydrolysis, tryptophan content cannot be determined, and asparagine and glutamine are hydrolyzed to aspartate and glutamate. In reductive methylation studies, the radiolabeled pool was fractionated on Sephadex G-10 to remove unincorporated isotope, concentrated, and analyzed on a Pharmacia Superdex peptide column. Sample absorbance at 280 nm was continuously monitored. Fractions were collected, and 3H content was determined by liquid scintillation chromatography.
Induction of Ag-specific CTL in vivo
Splenic DC or bone marrow precursor-derived DC were generated as described earlier (23, 24). Day 9 precursor-derived DC or splenic DC were pulsed with heat shock proteins in the presence of the lipid N-[1-(2,3-dioleoxyloxy)propyl]-N,N,N-trimethyl ammonium methylsulfate (DOTAP) (Boehringer Mannheim, Indianapolis, IN) or DMRIE (Vical, San Diego, CA). Heat shock proteins (in 100 µl Opti-MEM) and DMRIE or DOTAP (in 100 µl Opti-MEM) were mixed at room temperature for 15 min. The complex was added to the DC suspension in a total volume of 1 ml and incubated at 37°C in a water bath for 30 min. Alternatively, immature DC (day 7 precursor derived) were pulsed with heat shock proteins in the absence of DMRIE for 48 h. Naive, syngeneic mice were immunized i.v. with 5 x 105 precursor-derived DC or 1 x 106 spleen-derived DC per mouse in 200 µl of PBS.
Splenocytes were harvested after 10 days and depleted of RBCs with
ammonium chloride/Tris buffer. Splenocytes (1.0 x
107) were cultured with 5 x 105
irradiated stimulator cells (E.G7-OVA cells irradiated at 20,000 rad or
F10.9 cells pretreated with IFN-
and irradiated at 7,500 rad) in 5
ml of IMDM with 10% FCS, 1 mM sodium pyruvate, 100 IU/ml penicillin,
100 µg/ml streptomycin, and 5 x 10-5 M 2-ME per
well in a 6-well tissue culture plate. Cells were cultured for 5 days
at 37°C and 5% CO2. Effectors were harvested on day
5 on Histopaque 1083 gradient before use in a CTL assay.
In vitro cytotoxicity assay
Target cells (5–10 x 106) were labeled with europium for 20 min at 4°C. Europium-labeled targets (104) and serial dilutions of effector cells at varying E:T ratios were incubated in 200 µl of complete RPMI 1640. The plates were centrifuged at 500 x g for 3 min and incubated at 37°C for 4 h. Fifty microliters of the supernatant was harvested and europium release was measured by time-resolved fluorescence (24). Specific cytotoxic activity was determined using the following formula: % specific release = [(experimental release - spontaneous release)/(total release - spontaneous release)] x 100. Spontaneous release of the target cells was <25% of total release by detergent in all assays. SE of the means of triplicate cultures was <5%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A highly enriched ER microsome fraction was prepared from tissue
homogenates by differential centrifugation, and the lumenal protein
components were subsequently isolated from the microsomes by partial
detergent extraction (21). Under the described conditions, peripheral
and integral ER membrane proteins remain in association with the
detergent-permeabilized membranes and can thus be efficiently
segregated from the lumenal protein extract by centrifugation. The
supernatant fraction resulting from this step contains five major
polypeptides, GRP94 (gp96), BiP, ERp72, PDI, and calreticulin. In the
final stage of the purification, calreticulin and GRP94 undergo gel
filtration chromatography and centrifugal ultrafiltration (21). These
procedures, in addition to yielding homogeneous preparations of the two
proteins, were performed to eliminate circumstantial interactions
between either of the two chaperone proteins and low m.w. peptide
substrates. To assess the purity of the calreticulin and GRP94 used in
these studies, representative samples were analyzed by two-dimensional
SDS-PAGE. As shown in Fig. 1
A,
both proteins are, by this criterion, homogeneous.
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B,
lane 3, SDS-PAGE analysis of the ultrafiltration retentate
indicates that the conditions used for peptide extraction do not yield
detectable hydrolysis or degradation of calreticulin. Analysis of the
filtrate by SDS-PAGE analysis of the filtrate (lane
4) similarly shows no evidence of degradation products. To assay
for the presence of peptides in the filtered extract, amine-specific
radiolabeling (reductive methylation), followed by analytical gel
filtration, or quantitative amino acid analyses were performed.
Depicted in Fig. 1C is the gel filtration elution profile of
a representative fraction. When monitored at 280 nm, a broad band of
UV-absorbing material was observed at elution volumes corresponding to
m.w 600-1500. This range of elution volumes overlapped with the elution
of the radiolabeled material. For the radiolabeled material, the
leading edge of the initial primary peak encompassed elution volumes
corresponding to m.w. 1000–1900. In a paired analysis, an equivalent
quantity of calreticulin was extracted, and the peptide-enriched
fraction, corresponding to the elution profile depicted in Fig. 1C, was subjected to acid hydrolysis and quantitative amino
acid analysis. Fig. 1D details the amino acid composition of
the eluted material. For comparative purposes and to assess whether the
low m.w. peptide fraction was a general calreticulin degradation
product, the relative amino acid composition of calreticulin is
depicted. The most abundant amino acid present in the calreticulin
eluate was glycine, with the relative enrichment of those amino acids
comprising >20% of the total following the order Gly
>Glu/Gln>Ser>Asp/Asn>Ala>Leu. On the basis of quantitative amino
acid analysis, and with the as yet unsubstantiated assumption of a mean
average peptide m.w. of 1000, approximately 200 pmol of total peptide
was recovered from 10 nmol of calreticulin. Induction of in vivo CTL responses by calreticulin and GRP94
To determine by immunological criteria whether calreticulin copurifies in association with host tissue-specific peptides, the capacity of calreticulin to elicit CTL responses in vivo was investigated in two model systems, the B16/F10.9 melanoma and EL4/E.G7-OVA (24, 25, 26). For experiments using the B16/F10.9 model, the ER chaperones GRP94, BiP, ERp72, PDI, and calreticulin were purified to homogeneity from an F10.9 tumor-derived microsomal fraction. Control proteins were purified from either a normal spleen-derived microsomal fraction or from a porcine pancreas rough ER fraction (21).
Mice were immunized i.v. at 14-day intervals with 10 µg of GRP94 or
calreticulin, isolated from either F10.9 tumors or pig pancreas. A
total of two immunizations were performed. Splenocytes were isolated
from the immunized mice 10 days after the last immunization and were
restimulated in vitro with irradiated IFN--pretreated F10.9 cells,
and CTL activity was assayed subsequently against F10.9
(H2-Kb), EL4 (H2-Kb), or BALB/3T3
(H2-Kd) cells. The results of a representative experiment
are depicted in Fig. 2. Immunization with
F10.9-derived calreticulin or GRP94 elicited a significant CTL
response, and the maximum level of CTL lysis observed was comparable
for both proteins. That the observed CTL response, elicited by
F10.9-derived calreticulin and GRP94, was specific for F10.9 cells was
further substantiated by the fact that the control target cells, EL4
and BALB/3T3, exhibited no lysis (Fig. 2). Furthermore, no CTL
responses were generated in mice immunized with DC pulsed with porcine
calreticulin, porcine GRP94, or PBS. From these data, it is clear that
tumor-derived calreticulin and GRP94 elicit an F10.9-specific CTL
response.
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To further extend the conclusions obtained from the experiments
depicted in Fig. 2, the ability of the different ER chaperones to
elicit a CTL response was investigated. In these experiments, mice were
immunized two times with spleen-derived DC pulsed, in the presence of a
cationic lipid, either with mouse spleen-derived calreticulin, GRP94,
or ERp72, or with F10.9-derived calreticulin, GRP94, ERp72, BiP, or
PDI. Ten days after the final immunization, splenocytes were isolated
and restimulated, and CTL activity was assayed against F10.9 and EL4
cells (Fig. 3). Consistent with the data
depicted in Fig. 2, immunization of mice with F10.9 calreticulin or
GRP94-pulsed precursor-derived DC elicited a significant CTL response.
It is noteworthy that only low levels of CTL were generated by
vaccination with tumor-derived PDI, ERp72, or BiP, although it is well
established that these chaperones display peptide-binding activity (13, 14). In these experiments, immunization with spleen-derived
calreticulin, GRP94, or ERp72 yielded little or no CTL. The control
target EL4 showed no lysis. These results, as with those depicted in
Fig. 2, demonstrate that immunization with calreticulin or GRP94-pulsed
DC is sufficient to elicit a CTL response against Ags derived from the
chaperone-host cell.
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In the F10.9 system, immunization with tumor-derived calreticulin and GRP94 elicits a polyclonal CTL against an undefined set of tumor-associated Ags. Although such results clearly identify these two proteins as immunogenic, the lack of insight into the identity of the relevant tumor Ag(s) hampers further investigation into the molecular basis for this activity. Additional experiments were thus performed to determine whether calreticulin and GRP94 associate with a known MHC class I peptide epitope, as defined by immunological criteria. For these experiments, the EL4/E.G7-OVA system was used. E.G7-OVA cells are a clonal derivative of the EL4 tumor cell line (H-2b haplotype) and were selected for stable transfection with the chicken OVA cDNA (27). In a C57BL/6 (H-2b) mouse background, expression of the chicken OVA gene yields the production of a single immunodominant OVA peptide epitope (aa 257–264) (27). With respect to these studies, the EL4/E.G7-OVA experimental system offers two useful and interesting properties. 1) Chaperone-elicited CTL responses against the OVA epitope can be assayed using OVA-specific clonal CTL lines. 2) The hypothesis that calreticulin and GRP94 bind unique and nonoverlapping arrays of peptide substrates can be directly tested in determinations of shared EL4/E.G7-OVA CTL induction.
To prepare the relevant chaperone proteins, E.G7-OVA and EL4 tumors
were established in SCID mice, a cytosol and microsome fraction
prepared from excised tumors, and calreticulin, GRP94, Hsp90, and Hsp70
isolated from the relevant subcellular fractions. Splenic DC were
pulsed in the presence of the cationic lipid DOTAP with calreticulin,
GRP94, Hsp90, or Hsp70, and mice subjected to a single i.v.
vaccination. Splenocytes were subsequently isolated from the immunized
animals and stimulated with irradiated E.G7-OVA cells, and CTL assays
were performed (Fig. 4). As is evident in
Fig. 4, E.G7-OVA-derived calreticulin and GRP94 elicited robust CTL
responses against E.G7-OVA target cells. A substantial CTL response was
also observed in the case of E.G7-OVA Hsp70 (Fig. 4). The data
regarding GRP94 and Hsp70 are in agreement with previous studies
demonstrating that GRP94, HSP70, and to lesser extent HSP90, when
isolated from appropriate cells, prime Ag-specific CTL in vivo (16, 19). Particularly noteworthy in the data depicted in Fig. 4 is the
observation that immunization with EL4-derived calreticulin elicited
CTLs against the E.G7-OVA target. These data can be explained by the
fact that E.G7-OVA is a clonal derivative of EL4, transfected with the
OVA gene. Because the two cell types are genetically very similar, a
substantial overlap in the spectrum of the immunogenic peptides
expressed by the two cell types would be expected. As chaperones by
nature bind a diverse array of peptides, the only predictable
difference between the peptides bound to calreticulin derived from
E.G7-OVA cell and EL4 cells would be the presence of the OVA peptide,
bound to the E.G7-OVA-derived calreticulin. Thus, when the splenocytes
from immunized mice were restimulated with E.G7-OVA cells, in the
instances in which EL4-derived heat shock proteins were used for
immunization, only the EL4/E.G7-OVA-shared CTL would be restimulated.
Conversely, when the E.G7-OVA-derived heat shock proteins were used for
immunization, both the shared EL4/E.G7-OVA and the OVA-specific CTL
would be restimulated.
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), a similar overall pattern
was observed. Notably however, E.G7-OVA-derived calreticulin and GRP94
elicited a more substantial CTL response to EL4 target cells than that
elicited by the EL4-derived calreticulin or GRP94. It appears that the
E.G7-OVA-derived proteins are of higher relative antigenicity than
those obtained from EL4. The molecular basis for this phenomenon is not
understood and is being further investigated. On the premise that the
relative antigenicity of the chaperones is a direct function of the
complement of bound peptides, these data indicate that the spectrum of
immunogenic peptides present on the EL4 and E.G7-OVA chaperones
displays significant similarities, as both elicit CTL responses against
the related cell line, and significant differences, as the E.G7-OVA
chaperones are more immunogenic.
To alleviate concerns regarding CTL specificity, control experiments
with an unrelated target cell line (F10.9) were performed (Fig. 4).
With F10.9 as the target cell, no significant CTL activity was observed
in splenocyte preparations derived from animals immunized with any of
the chaperone preparations. These data substantiate the conclusion that
the CTL activity observed against E.G7-OVA and EL4 target cells is
specific, and thus, that the observed cross-cell reactivity is, at a
fundamental level, a reflection of shared immunogenic epitopes
copurifying with the different chaperone protein preparations.
Re-presentation of calreticulin associated peptides
To better define the OVA specificity of the observed CTL
responses, the capacity of immature BMDC to present
calreticulin-associated peptides and be recognized for lysis by
OVA-specific CTL was investigated. Immature BMDC, as professional APCs,
process and present exogenous Ags on the class I pathway and are
thought to utilize this pathway for the activation of CD8+
CTL in vivo (28, 29, 30). Immature murine BMDC were pulsed with the
immunodominant OVA peptide (SIINFEKL), a control peptide (mut-1), EL4
calreticulin, or E.G7-OVA calreticulin, and, following maturation,
class I presentation of the OVA epitope was assayed by a CTL assay. In
these assays, peptide- or chaperone-pulsed BMDC served as target cells,
and the OVA-specific CTL line 4G3 as effector cells. As shown in Fig. 5A, immature BMDC pulsed with
E.G7-OVA-derived calreticulin present OVA for recognition and lysis by
4G3 CTLs, whereas no activity was observed for EL4 calreticulin. The
specificity of this response is further supported by the data
demonstrating that sensitization of the E.G7-OVA-calreticulin-pulsed
BMDC to lysis by 4G3 was dependent on the concentration of E.G7-OVA
calreticulin present in the medium. As additional controls, it was
observed that lysis could be elicited by the OVA peptide, whereas a
100-fold excess of the control peptide was without effect.
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B. In this experiment, presentation of the class
I-restricted OVA epitope was assayed using a T cell hybridoma IL-2
secretion assay. In this assay, BMDC were cultured in the presence of
E.G7-OVA or EL4-derived chaperone proteins, harvested, and assayed for
their ability to stimulate IL-2 secretion from a class I-restricted,
OVA-specific T cell hybridoma (23). For comparative purposes, IL-2
secretion elicited in response to RMA-S cells pulsed with either
control or OVA peptide was assayed. As depicted in Fig. 5B,
the OVA specificity of the assay was confirmed by data demonstrating
that neither control peptide nor EL4-derived calreticulin elicited IL-2
secretion, whereas OVA peptide elicited a dose-dependent production of
IL-2 (Fig. 5B). E.G7-OVA-derived calreticulin-pulsed BMDC
exhibited a dose-dependent stimulation of IL-2 secretion. These data
clearly demonstrate that calreticulin-associated OVA peptide, or a
structural precursor(s), was processed by BMDC and presented for
recognition by the OVA-specific, class I-restricted T cell hybridoma. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At present, it is not known how calreticulin or GRP94-associated peptides gain access to the ER of professional APCs and thus whether the elicitation of a class I-restricted immune response, which accompanies transfer of the chaperone-associated peptides to MHC class I molecules, reflects a component part of a ubiquitous, calreticulin- and/or GRP94-dependent pathway for peptide loading onto class I molecules; the existence of a pathway, perhaps unique to professional APCs, that supports the trafficking of chaperone-peptide complexes; or the peptides themselves, from the extracellular space to the ER. It is noteworthy that calreticulin has been demonstrated to perform a critical, although undefined, role in class I/ß2m/peptide assembly in the ER (6, 7, 8, 9). It is premature, though, to invoke a direct peptide loading function for calreticulin, as the detailed molecular pathway for peptide loading onto class I molecules, and the contribution of calreticulin to such a pathway, has yet to be determined.
Further clouding speculation on these matters is the absence of insight into how peptide release from calreticulin and GRP94 is regulated, or, for that matter, if the dissociation of peptides from these proteins is a regulated event. In one scenario, calreticulin and/or GRP94 could interact reversibly with ER-localized peptides and thereby assist in the transfer of peptides, directly or indirectly, onto class I molecules. Alternatively, peptide release might only arise as a consequence of proteolysis, such as may accompany internalization into APCs and degradation in endosomal/lysosomal compartments. In the latter scenario, calreticulin-peptide complexes would be kinetically stable, and thus the antigenic profile of bound peptides would vary as a function of the metabolic half-life of the chaperone protein rather than the steady-state variation in ER peptide levels. With these uncertainties in mind, insights into the physiological role(s) of calreticulin and GRP94 in the peptide-trafficking pathways of the ER and in the elicitation of CD8+ T cell responses will benefit from an understanding of the compartmental specificity, regulation, and kinetics of peptide binding.
The studies of Srivastava and colleagues yielded the surprising and
provocative observation that GRP94 can, through its activity as a
peptide-binding protein, elicit CTL responses in vivo and in vitro (16, 19, 31). We have observed that calreticulin displays activity similar
to that of GRP94. As discussed by Srivastava and colleagues, a
noteworthy, and perhaps unique, aspect of chaperone-based
immunotherapeutics concerns the diversity of chaperone-bound peptides,
a diversity that may be reflective of the antigenic repertoire of the
host cell (31). This conclusion is particularly evident in the data
presented in Fig. 4, in which the capacity of ER lumenal chaperones
derived from EL4 and E.G7-OVA thymoma tumors to elicit CTLs directed
against EL4 and E.G7-OVA was determined. In focusing on the data
obtained with calreticulin and GRP94, it is evident that when derived
from E.G7-OVA, both proteins were observed to elicit a substantial CTL
response to both the parent (E.G7-OVA) and the precursor (EL4) cell
lines. Although the shared peptide Ags remain to be identified, these
data clearly illustrate that both calreticulin and GRP94 copurify with
an array of antigenic peptides capable of assembly on class I molecules
and reaffirm the potential immunotherapeutic utility of these proteins,
that being their capacity to elicit a CTL response against the parent,
and genetically related, cell hosts, in the absence of the
identification of specific peptide Ags (31). That the E.G7-OVA-derived
proteins display a substantially higher antigenicity than those
obtained from EL4 may be most simply explained by assuming that the
spectrum of immunogenic peptides associated with E.G7-OVA chaperones
differs significantly from those in association with the EL4-derived
proteins. Thus, although there must indeed be shared immunogenic
epitopes capable of eliciting a CTL response against the two related
cell lines, there must exist differences in either the relative
abundance or structure of these as yet unknown immunogenic epitopes. An
alternative, and admittedly speculative, hypothesis is that the
lytic activity elicited by E.G7-OVA-derived, but not EL4-derived,
calreticulin, GRP94, or HSP70 on EL4 targets may reflect a "helper"
effect of the strong OVA epitope, which serves to alter "tolerance"
against the weaker tumor epitopes expressed by EL4, by a mechanism
similar to that previously observed upon expression of foreign Ags in
tumor cells (32).
An appreciation of the fundamental cell biological significance of chaperone-elicited CTL responses will accompany insights into the structural characteristics of the peptide substrates bound by these molecules and the cellular trafficking pathways accessed by the proteins and their peptide substrates in APCs. With respect to the issue of protein trafficking, it will be of particular interest to determine whether calreticulin and/or GR94 function in vivo as physiologically relevant components of the immunological pathway(s) supporting cross-priming (29, 30, 31, 33, 34).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christopher Nicchitta, Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710. E-mail address:
3 Abbreviations used in this paper: ER, endoplasmic reticulum; TAP, transporter associated with Ag presentation; ß2m, ß2-microglobulin; DC, dendritic cell; DOTAP, N-[1-(2,3-dioleoxyloxy)propyl]-N,N,N-trimethyl ammonium methylsulfate.
Received for publication November 18, 1998. Accepted for publication March 16, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J.-E. Park, J. Facciponte, X. Chen, I. MacDonald, E. A. Repasky, M. H. Manjili, X.-Y. Wang, and J. R. Subjeck Chaperoning Function of Stress Protein grp170, a Member of the hsp70 Superfamily, Is Responsible for its Immunoadjuvant Activity Cancer Res., January 15, 2006; 66(2): 1161 - 1168. [Abstract] [Full Text] [PDF]
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 C. P. Wanamaker and W. N. Green N-Linked Glycosylation Is Required for Nicotinic Receptor Assembly but Not for Subunit Associations with Calnexin J. Biol. Chem., October 7, 2005; 280(40): 33800 - 33810. [Abstract] [Full Text] [PDF]
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A. A. R. Tobian, C. V. Harding, and D. H. Canaday Mycobacterium tuberculosis Heat Shock Fusion Protein Enhances Class I MHC Cross-Processing and -Presentation by B Lymphocytes J. Immunol., May 1, 2005; 174(9): 5209 - 5214. [Abstract] [Full Text] [PDF]
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 Y. Zeng, M. W. Graner, S. Thompson, M. Marron, and E. Katsanis Induction of BCR-ABL-specific immunity following vaccination with chaperone-rich cell lysates derived from BCR-ABL+ tumor cells Blood, March 1, 2005; 105(5): 2016 - 2022. [Abstract] [Full Text] [PDF]
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B. Berwin, Y. Delneste, R. V. Lovingood, S. R. Post, and S. V. Pizzo SREC-I, a Type F Scavenger Receptor, Is an Endocytic Receptor for Calreticulin J. Biol. Chem., December 3, 2004; 279(49): 51250 - 51257. [Abstract] [Full Text] [PDF]
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A. A. R. Tobian, D. H. Canaday, and C. V. Harding Bacterial Heat Shock Proteins Enhance Class II MHC Antigen Processing and Presentation of Chaperoned Peptides to CD4+ T Cells J. Immunol., October 15, 2004; 173(8): 5130 - 5137. [Abstract] [Full Text] [PDF]
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A. A. R. Tobian, D. H. Canaday, W. H. Boom, and C. V. Harding Bacterial Heat Shock Proteins Promote CD91-Dependent Class I MHC Cross-Presentation of Chaperoned Peptide to CD8+ T Cells by Cytosolic Mechanisms in Dendritic Cells versus Vacuolar Mechanisms in Macrophages J. Immunol., May 1, 2004; 172(9): 5277 - 5286. [Abstract] [Full Text] [PDF]
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R. C. Reed, B. Berwin, J. P. Baker, and C. V. Nicchitta GRP94/gp96 Elicits ERK Activation in Murine Macrophages: A ROLE FOR ENDOTOXIN CONTAMINATION IN NF-{kappa}B ACTIVATION AND NITRIC OXIDE PRODUCTION J. Biol. Chem., August 22, 2003; 278(34): 31853 - 31860. [Abstract] [Full Text] [PDF]
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Y. Zeng, H. Feng, M. W. Graner, and E. Katsanis Tumor-derived, chaperone-rich cell lysate activates dendritic cells and elicits potent antitumor immunity Blood, June 1, 2003; 101(11): 4485 - 4491. [Abstract] [Full Text] [PDF]
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X.-Y. Wang, X. Chen, M. H. Manjili, E. Repasky, R. Henderson, and J. R. Subjeck Targeted Immunotherapy Using Reconstituted Chaperone Complexes of Heat Shock Protein 110 and Melanoma-associated Antigen gp100 Cancer Res., May 15, 2003; 63(10): 2553 - 2560. [Abstract] [Full Text] [PDF]
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S. Vogen, T. Gidalevitz, C. Biswas, B. B. Simen, E. Stein, F. Gulmen, and Y. Argon Radicicol-sensitive Peptide Binding to the N-terminal Portion of GRP94 J. Biol. Chem., October 18, 2002; 277(43): 40742 - 40750. [Abstract] [Full Text] [PDF]
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 B. Liu, A. M. DeFilippo, and Z. Li Overcoming Immune Tolerance to Cancer by Heat Shock Protein Vaccines Mol. Cancer Ther., October 1, 2002; 1(12): 1147 - 1151. [Abstract] [Full Text] [PDF]
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M. R. Leach, M. F. Cohen-Doyle, D. Y. Thomas, and D. B. Williams Localization of the Lectin, ERp57 Binding, and Polypeptide Binding Sites of Calnexin and Calreticulin J. Biol. Chem., August 9, 2002; 277(33): 29686 - 29697. [Abstract] [Full Text] [PDF]
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D. Golgher, F. Korangy, B. Gao, K. Gorski, E. Jaffee, M. Edidin, D. M. Pardoll, and T. Elliott An Immunodominant MHC Class II-Restricted Tumor Antigen Is Conformation Dependent and Binds to the Endoplasmic Reticulum Chaperone, Calreticulin J. Immunol., July 1, 2001; 167(1): 147 - 155. [Abstract] [Full Text] [PDF]
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X.-Y. Wang, L. Kazim, E. A. Repasky, and J. R. Subjeck Characterization of Heat Shock Protein 110 and Glucose-Regulated Protein 170 as Cancer Vaccines and the Effect of Fever-Range Hyperthermia on Vaccine Activity J. Immunol., January 1, 2001; 166(1): 490 - 497. [Abstract] [Full Text] [PDF]
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U. Kumaraguru, R. J. D. Rouse, S. K. Nair, B. D. Bruce, and B. T. Rouse Involvement of an ATP-Dependent Peptide Chaperone in Cross-Presentation After DNA Immunization J. Immunol., July 15, 2000; 165(2): 750 - 759. [Abstract] [Full Text] [PDF]
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 M. Graner, A. Raymond, D. Romney, L. He, L. Whitesell, and E. Katsanis Immunoprotective Activities of Multiple Chaperone Proteins Isolated from Murine B-Cell Leukemia/Lymphoma Clin. Cancer Res., March 1, 2000; 6(3): 909 - 915. [Abstract] [Full Text]
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 Q. Huang, J. F.L. Richmond, K. Suzue, H. N. Eisen, and R. A. Young In Vivo Cytotoxic T Lymphocyte Elicitation by Mycobacterial Heat Shock Protein 70 Fusion Proteins Maps to a Discrete Domain and Is Cd4+ T Cell Independent J. Exp. Med., January 17, 2000; 191(2): 403 - 408. [Abstract] [Full Text] [PDF]
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J. Wassenberg, C Dezfulian, and C. Nicchitta Receptor mediated and fluid phase pathways for internalization of the ER Hsp90 chaperone GRP94 in murine macrophages J. Cell Sci., January 7, 1999; 112(13): 2167 - 2175. [Abstract] [PDF]
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J. J. Wassenberg, R. C. Reed, and C. V. Nicchitta Ligand Interactions in the Adenosine Nucleotide-binding Domain of the Hsp90 Chaperone, GRP94. II. LIGAND-MEDIATED ACTIVATION OF GRP94 MOLECULAR CHAPERONE AND PEPTIDE BINDING ACTIVITY J. Biol. Chem., July 21, 2000; 275(30): 22806 - 22814. [Abstract] [Full Text] [PDF]
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M. F. N. Rosser and C. V. Nicchitta Ligand Interactions in the Adenosine Nucleotide-binding Domain of the Hsp90 Chaperone, GRP94. I. EVIDENCE FOR ALLOSTERIC REGULATION OF LIGAND BINDING J. Biol. Chem., July 21, 2000; 275(30): 22798 - 22805. [Abstract] [Full Text] [PDF]
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F. Le Naour, L. Hohenkirk, A. Grolleau, D. E. Misek, P. Lescure, J. D. Geiger, S. Hanash, and L. Beretta Profiling Changes in Gene Expression during Differentiation and Maturation of Monocyte-derived Dendritic Cells Using Both Oligonucleotide Microarrays and Proteomics J. Biol. Chem., May 18, 2001; 276(21): 17920 - 17931. [Abstract] [Full Text] [PDF]
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U. G. Danilczyk and D. B. Williams The Lectin Chaperone Calnexin Utilizes Polypeptide-based Interactions to Associate with Many of Its Substrates in Vivo J. Biol. Chem., June 29, 2001; 276(27): 25532 - 25540. [Abstract] [Full Text] [PDF]
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B. Berwin, R. C. Reed, and C. V. Nicchitta Virally Induced Lytic Cell Death Elicits the Release of Immunogenic GRP94/gp96 J. Biol. Chem., June 8, 2001; 276(24): 21083 - 21088. [Abstract] [Full Text] [PDF]
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