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*
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
CRC Medical Oncology Unit, Southampton General Hospital, Southampton, United Kingdom
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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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10-fold
(10). TAP is not the sole mechanism for potential class I ligands to gain entry to the ER. Peptides within ER targeting sequences of secreted or membrane proteins can be presented relatively efficiently by TAP-deficient cells (11, 12, 13). More generally, class I-binding peptides immediately COOH terminal to ER targeting sequences are efficiently presented in a TAP-independent manner (11, 14), and such minigene products are highly immunogenic in vivo (15, 16). Positioning peptides at the COOH terminus of secreted or type II membrane-anchored proteins also enables their TAP-independent presentation (termed the C-end rule), but usually at much lower efficiencies in the absence of a specific ER-associated proteolytic cleavage event (17). More sporadically, peptides can be liberated from internal sequences of ER-targeted proteins, and in some cases this can be influenced by N-linked glycosylation (18). The contribution of proteases in the secretory compartment to Ag presentation is based entirely on in vitro studies, a situation that we rectify in this report.
It is generally assumed that viral Ags are presented to naive TCD8+ in vivo by professional APCs (pAPCs). An important question is under what circumstances viral Ags are presented by direct priming (infected pAPCs presenting endogenous viral gene products) vs cross-priming (uninfected pAPCs presenting gene products synthesized by another host cell) (19). In the event of cross-priming, the rules governing TAP dependence of presentation of Ag in vitro may be discarded in vivo. The rules governing the priming of naive TCD8+ are currently based on in vitro studies of Ag presentation. In the present study, we use TAP-/- mice to gain insight into the mechanisms of Ag presentation to naive TCD8+.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TAP1-/- mice (10), a generous gift from Dr. Luc Van Kaer (Vanderbilt University School of Medicine, Nashville, TN) were bred onto a B6 background. OT-1 mice (20) were a generous gift from Dr Kristin Hogquist (University of Minnesota). C57BL/6, B6 TAP1-/-, OT-1, and F5 (21) mice were all bred at Taconic Farms (Germantown; NY). Bm1 mice were obtained from The Jackson Laboratory (Bar Harbor, ME).
Recombinant vaccinia viruses (rVVs)
rVVs expressing antigenic peptides from vesicular stomatitis
virus (VSV) nucleocapsid (N), Sendai virus nucleoprotein (NP), OVA, and
influenza NP as outlined in Table I
have
been previously described. PR8 NP was directed to the secretory pathway
using the signal sequence from IFN-
(22).
Minigene constructs, with the exception of NT60 NP constructs, were
targeted to the secretory pathway using the signal sequence from the
adenovirus E3/19K protein (14). All NT60 NP constructs
were targeted to the ER using the signal sequence from the influenza
hemagglutinin (HA) protein (23).
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2-microglobulin (2m)
and Kb plus 2m
(25). S represents a signal/leader sequence that
targets the peptide to the ER. The full-length murine TAP1 was inserted
into the thymidine kinase locus of vaccinia viruses (VVs) by homologous
recombination as described using the pSC11 plasmid to express foreign
proteins under the control of the VV p7.5 early/late promoter
(26). The coding sequence was verified by sequencing
PCR-amplified copies of the full-length TAP1 gene isolated from
the rVV. Cell lines and cultures
All media were purchased from Life Technologies (Gaithersburg, MD). 1E12 cells were maintained in DMEM containing 10% FBS (D-10). CTL culture was in RPMI 1640 containing 10% FBS, 5 x 10-5 M 2-mercaptoethanol, antibiotics (penicillin and streptomycin), nonessential amino acids, sodium pyruvate (1 mM), and 2 mM glutamine.
TAP1-/- 1E12 cells were generated as follows.
Kidneys were harvested from B6 TAP1-/- mice and
then dissected, and a single-cell suspension was produced. Cells were
resuspended in 20 ml D-10, to which 2 x 107
PFU SV40 were added. After 3–4 wk of culture, cells had grown out,
and a clonal population of TAP1-/- cells was
isolated by limiting dilution culture. TAP1-/-
1E12 cells exhibited much diminished cell surface expression of
H2-Kb and Db molecules when
examined by cytofluorography using conformation-specific Abs (Y3 and
B22, respectively).
Bone marrow-derived dendritic cells (DC) were obtained as previously described (27). Briefly, bone marrow was flushed from femurs, and cells were grown in Iscove’s modified medium containing 20% FBS and 10% supernatant from X63 cells transfected with murine GM-CSF. After removal of nonadherent cells on days 2 and 4 of culture, the majority of the nonadherent cells harvested on day 7 were CD11c+, MHC class II+ DC.
CTL priming in vivo
Female C57BL/6 mice (8–10 wk old) or B6
TAP1-/- mice (male or female, sex and age
matched in each experiment) were injected with 5 x
106 –1 x 107 PFU rVV
in 0.5 ml balanced salt solution-BSA i.v. To generate
TCD8+, splenocytes from mice immunized with
viruses 7 days (TAP1-/- mice) or 2–6 wk
(C57BL/6) previously were stimulated in vitro for 6 days with antigenic
peptides at 1 µg/ml. After culture, live cells were recovered via a
Ficoll-Hypaque gradient. Purified TCD8+ were
isolated from cultures derived from TAP1-/-
mice by positive selection over MACS columns, using the protocol
provided by the manufacturer. Briefly, live cells were incubated at
5–8°C with anti-CD8 microbeads (Miltenyi Biotech, Auburn, CA)
for 20 min before isolation over RS+-positive
selection columns held in an OctoMACS apparatus. After removal from the
magnet, cells were eluted to give a cell population that was 70–90%
CD8+.
Splenocytes (from C57BL/6 mice) or purified TCD8+ T cells (from TAP1-/- mice) were used as effectors in microcytotoxicity assays. Generally, 106 target cells were labeled with 100 µCi Na51CrO4 (Amersham, Arlington Heights, IL) in a minimum volume of medium at 37°C for 60 min. When the activity of effectors from TAP1-/- mice was assayed, TAP2-/- RMA/S cells, pulsed with a specific or irrelevant Kb or Db binding peptide, were used as targets. After two washes, 104 cells were aliquoted into round-bottom 96-well plates containing serial dilutions of effector TCD8+. The radioactivity in supernatants collected after 4–6 h incubation at 37°C was determined using a gamma counter. The percent specific release was then determined as: % specific release = [(CTL-induced release - spontaneous release)/(release by detergent - spontaneous release)] x 100.
Purification and adoptive transfer of TCR-transgenic (Tg) T cells
Two days before transfer of TCR Tg T cells, recipient mice were irradiated with 800 rad. T cell-enriched populations were obtained from TCR Tg mice as follows. Lymph nodes (popliteal, inguinal, brachial, axillary, and superficial cervical) and spleen were taken and a single-cell suspension of cells was generated. Live cells were isolated via a Ficoll-Hypaque gradient and cells were incubated with anti-CD90 (Thy-1.2) microbeads for 20 min at 5–8°C. T cells were isolated by passing over VS+ selection columns to yield a population of cells that was typically 80–90% CD3+ by flow cytometric analysis. Cells were washed in PBS and then incubated with 5 µM CFSE (Molecular Probes, Eugene, OR) for 20 min at 37°C. After washing in D-10, 5–10 x 106 CFSE-labeled cells were injected into each recipient via the tail vein. Two hours after injection of T cells, mice were immunized with 5 x 106 PFU rVV i.v. Two days later, spleens were removed and analyzed as outlined below.
Flow cytometric analysis
For analysis of cell division in vivo, spleens were harvested
from two mice per group and homogenized, and the cells were pooled.
Mononuclear cells were isolated by centrifuging over lymphocyte
separation medium (BioWhittaker, Walkersville, MD) and harvesting the
cells at the lymphocyte separation medium-medium interface. Cells were
incubated in 2.4G2 supernatant, 20% normal mouse serum for 20 min on
ice to block Fc receptor-mediated uptake of Ab and then stained with
either anti-V
2-PE (clone B20.1; PharMingen, San Diego, CA) or
anti-V11-PE (clone RR3-15; PharMingen) Abs for 40 min on ice.
Cells were washed five times; then data were captured using a FACScan
(Becton Dickinson, San Jose, CA). Only V2 (for OT-1)- or V11 (for
F5)-positive cells were analyzed for CFSE staining, and data were
analyzed using FlowJo software ((Tree Star, San Carlos, CA). Similarly,
for analysis of cell surface staining of bone marrow-derived DCs, cells
were harvested, FC receptors blocked, and then stained with
FITC-conjugated 25.D1.16 (specific for
H2-Kb-OVA257–264 complex)
Ab at a 1:10 dilution for 30 min at 0°C before washing extensively in
ice-cold PBS and analyzing as above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our strategy to study in vivo processing of viral proteins
entailed comparing the capacity of rVVs expressing various forms of Ags
to elicit TCD8+ responses in
TAP1-/- mice. To facilitate direct comparisons
between presentation in vitro and in vivo, it was necessary to generate
a permanent cell line from TAP1-/- mice. (The
only TAP-deficient mouse cell line available, RMA/S, lacks TAP2 and has
a number of additional mutations resulting from the chemical mutagen
used for its generation. RMA/S cells also suffer from being poorly
infected by rVV virus.) By using SV40 (28) to transform
kidney cells ex vivo, we cloned a TAP1-/- cell
line (1E12 cells). 1E12 cells were infected with rVVs encoding a number
of well-characterized H-2b-restricted antigenic
peptides in different protein contexts (Table I
). Infected cells were
examined for recognition by TCD8+ specific for
the corresponding determinant using a standard
51Cr microcytotoxicity assay.
These findings are summarized in Table I because they largely mirror
previous in vitro findings with the same rVVs used in human
TAP-deficient cells (29, 30). As in prior studies
(14), the highest levels of lysis were achieved by
infecting cells with an ER-targeted minigene. Expressing the
full-length gene product that is the source of the peptide resulted in
the lowest levels of lysis (usually slightly above or at levels of
lysis observed with control rVVs). Infection with rVVs expressing the
peptide at the COOH terminus of a secreted protein resulted in
intermediate levels of lysis, as did targeting influenza NP residues
328–498 to the ER using the leader sequence of influenza HA.
Unexpectedly, three of the four peptides tested (all but
NP366–374) were presented at intermediate
levels as cytosolic minigenes (scored as TAP independent in Table I).
Given the vast number of peptides that are usually produced from
cytosolic minigenes (25, 31), we attribute the modest
levels of presentation to "leakiness," i.e., a low efficiency
TAP-independent alternate means of entering the ER. (Although it is
formally possible that TAP2 forms a low efficiency channel, we have
never seen supporting evidence in comparing presentation of TAP1 and
TAP2-deficient cells with TAP2-expressing cells.)
The cytotoxicity assay is an extremely blunt tool for quantitating levels of peptide-class I complexes; once the threshold number of complexes is reached on a given cell for triggering TCD8+ lysis, further increases are not registered by the assay (a cell can die but once). To more accurately quantitate complex expression, we examined the increase in expression of conformed class I molecules on the surface of TAP1-/- cells after infection with rVVs by staining with the conformation-dependent Abs to H2-Kb (Y3) or H2-Db (B22). We were unable to detect increases in the expression of class I molecules on 1E12 cells after infection with rVVs encoding either TAP or appropriate ER-targeted peptides, probably owing to relatively low levels of rVV gene expression in combination with low levels of class I synthesis (not shown).
We therefore turned to DC prepared from the bone marrow of
TAP1-/- mice by short term culture in
GM-CSF-containing medium. Infection of TAP1-/-
DC with rVVs expressing either mouse or human TAP1 resulted in a slight
(1.5-fold) but significant increase in Kb cell
surface expression (not shown). Curiously, infection with rVV
expressing both human TAP subunits resulted in a greater enhancement of
Kb expression (3-fold). A similar enhancement was
observed after infection with ER-targeted
OVA257–264
(VV-S-OVA257–264). To test whether this
up-regulation of Kb expression on the cell
surface was due to stabilization by the
OVA257–264 peptide, cells were stained with an
Ab specific for the
H2-Kb-OVA257–264 complex
(25). As shown in Fig. 1, infection with ER-targeted OVA257–264
(VV-S-OVA257–264) induced an approximate 3-fold
up-regulation of
Kb-OVA257–264 complexes on
the cell surface. By contrast, neither Kb
expression nor expression of the specific
Kb-OVA257–264 complex was
changed after infection with rVVs expressing full-length OVA (VV-OVA),
cytosolic minimal peptide determinant
(VV-OVA257–264), or the minimal peptide
determinant OVA257–264 attached to the COOH
terminus of ER-targeted influenza NP
(VV-S-NP-OVA257–264). This confirms that
ER-targeting results in the generation of far greater numbers of
Kb-OVA257–264 complexes in
TAP1-/- cells than are generated from other
constructs.
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We next turned our attention to the main question: how does in
vitro Ag presentation compare with in vivo CTL priming? We infected
TAP1-/- mice with the rVVs described above and
measured lytic activities after secondary stimulation of splenocytes in
vitro with the appropriate synthetic peptide.
TAP1-/- mice possess only 10% as many
TCD8+ as in normal mice (10).
Despite this, Sandberg et al. (32) have shown that
TAP1-/- mice maintain the ability to respond to
selected determinants after immunization with synthetic peptides in
adjuvant, including VSV N52–59 and SEN
NP324–332, but not NT60
NP366–374 or OVA257–264.
Indeed, after infection with rVVs expressing the corresponding
ER-targeted versions of these peptides, TCD8+
responses to VSV N52–59 and
NP324–332 were easily detected (Fig. 2). Like Sandberg et al., we did not
detect responses to S-OVA257–264 or NT60
NP366–374 (not shown), supporting the conclusion
that appropriate TCD8+ are missing from the
repertoire of TAP1-/- mice. In the same
experiment, TAP1-/- mice failed to respond to
rVVs encoding full length VSV N, cytosolic minigene versions of VSV
N52–59, SEN NP324–332
(not shown), or the same peptides appended to the COOH terminus of
ER-targeted NP. Taken together, the present (Fig. 1) and previous
results (11, 14) indicate that peptide class I complexes
are generated most efficiently in TAP deficient cells by expressing
ER-targeted peptides. These results indicate that
TAP-/- mice were able to respond only to
rVV-infected cells that express peptide-class I complexes at the
highest densities on the cell surface of TAP-deficient cells.
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We bypassed difficulties with limitations in the TCR repertoire of TAP1-/- mice by reconstituting the mice with purified TCD8+ from TCR Tg mice. To generalize the results, we used two strains of TCR Tg mice; F5, which generate TCD8+ specific for Db-NT60 NP366–374 complexes, and OT-1, which generate TCD8+ specific for Kb-OVA257–264 complexes. TAP1-/- mice generate strong and rapid cytolytic responses against adoptively transferred TCD8+ expressing normal levels of cell surface class I molecules (reviewed in Ref. 33).To avoid rejection of adoptively transferred Tg TCD8+, recipients were irradiated with 800 rad 2 days before transfer. TCD8+ were labeled with CFSE just before injection to enable flow cytometric determination of cell division, and mice were infected with rVVs 2–3 h later. Similar results were obtained by measuring ex vivo cytotoxicity, but this required greater numbers of animals and was much more variable than CFSE staining, which became the method of choice.
Reconstitution of TAP1-/- mice with F5 TCR Tg
TCD8+ followed by infection with VV-S-NT60
NP366–374 caused significant proliferation of
the F5 TCD8+ 2 days postinfection (Fig. 3). Proliferation was undetected 1 day
postinfection under these conditions (not shown), or on day 2 if the
mice were uninfected. The specificity of activation of the F5 cells was
best illustrated by their failure to proliferate after infection with
an rVV expressing an ER-targeted version of an homologous peptide from
the PR8 NP that possesses two amino acid substitutions. Despite binding
to Db molecules with high affinity, this peptide
is not recognized by F5 cells in vitro, even at very high
concentrations (21). In the same experiment, we found that
F5 cells proliferated after infection with VV-S-NT60
NP328–498, but not rVVs expressing the cytosolic
peptide NP366–374 (VV-NT60
NP366–374), normal NP (VV-NT60 NP), or
ER-targeted NP (VV-S-NT60 NP) (Fig. 3). Therefore, in this system, in
vivo priming closely parallels in vitro presentation.
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TAP dependence of CTL priming after reconstitution of TAP1-/- mice with OVA257–264-specific TCR Tg TCD8+
We expanded these findings using the same experimental protocol to
examine the presentation of Ags to OT-1 TCD8+.
TAP1-/- mice reconstituted with CFSE-labeled
OT-1 TCD8+ displayed a strong proliferative
response 2 days postinfection with
VV-S-OVA257–264, whereas no proliferation was
observed if mice were uninfected (data not shown) or infected with a
control rVV encoding S-NP147–155 (Fig. 4). OT-1 TCD8+ also
proliferated after infection with
VV-S-NP-OVA257–264, providing evidence for the
in vivo relevance of the C-end rule (17). The degree of
proliferation was lower than that after
VV-S-OVA257–264 infection, however,
demonstrating that OT-1 proliferation is not all or none but rather is
related to the number of
Kb-OVA257–264 complexes
generated by APCs. In contrast to the findings with the F5 system,
infection with rVV expressing the cytosolic minimal determinant
triggered OT-1 proliferation in TAP1-/- mice,
in this case to a degree similar to that of
VV-S-NP-OVA257–264. The in vivo presentation of
OVA257–264 and NT60
NP366–374 minimal determinant products in
TAP1-/- mice parallels their in vitro
presentation in TAP1-/- cells (Table I).
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Transferred TAP1+/+ cells are not responsible for priming of a response to VV-OVA
As with the F5 system, our interpretation of the OT-1 results are
entirely dependent on the conclusion that presentation is mediated by
TAP1-/- APCs, and not cells transferred from
OT-1 mice. To rigorously support this conclusion, we transferred
CFSE-labeled OT-1 cells into Kbm1 mutant mice.
Due to 3 amino acid substitutions in the helix of the
Kb binding groove, these mice are incapable of
presenting OVA257–264 to OT-1
TCD8+ (34). Thus, if priming were due to
infection of the adoptively transferred OT-1 cells, then the cells
should proliferate in Kbm1 mice after infection
with the appropriate rVVs. Although Kbm1
molecules present many of the same self peptides as wild-type
Kb, an allogeneic response is still mounted
against transferred OT-1 cells; therefore, the same protocol as for
TAP1-/- recipients was followed, with the
Kbm1 mutant mice being irradiated 2 days before
reconstitution with T cells.
Infection of Kbm1 mice with even the strongest
stimulating virus in the OT-1 system,
VV-S-OVA257–264, failed to stimulate
proliferation of transferred OT-1 TCD8+. VV-OVA
was also nonimmunogenic in Kbm1 mice. As a
positive control, mice were infected with two rVVs, one expressing
wild-type Kb and mouse
2m (VV-Kb +
2m), the other expressing
S-OVA257–264 and mouse
2m (VV S-OVA257–264 +
2m). Under these conditions, where cells
doubly infected could express OVA257–264 in
complex with wild-type H-2Kb, a strong
proliferative response was observed (Fig. 5). Each of the rVVs alone was
nonimmunogenic under these conditions. These findings provide strong
evidence that first priming in the OT-1 transfer system is based on
presentation of Ag by TAP1-/- APCs and second
that virus-infected APCs present endogenous ER-targeted peptides to
TCD8+.
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The discrepancy in the presentation of OVA by TAP1-/- cells in vivo and in vitro is consistent with the occurrence of a cross-priming mechanism in which an OVA257–264-containing protein produced by rVV-infected cells is presented by TAP1-/- cells to OT-1 TCD8+. To explore this possibility, we immunized TAP1-/- mice with splenocytes from B6 or Kbm1 mice infected with rVVs encoding OVA, the cytosolic minigene product, or a control rVV.
Immunization of TAP-/- mice with either B6 or
Kbm1 splenocytes expressing virus-encoded OVA or
OVA257–264, but not an irrelevant gene product,
activated transferred OT-1 TCD8+ (Fig. 6). Splenocytes are poorly infected by
VV, and it is unlikely that they are capable of producing sufficient
virus for priming, particularly because relatively large amounts of
VV-OVA (106 PFU) are needed to activate OT-1
cells in vivo (data not shown). It is possible, however, that virus is
transferred by contact of infected cells with host APCs. To demonstrate
that priming was not due to virus transfer, we took advantage of the
fact that minigenes offer an exceedingly small target for
psoralen-enhanced inactivation by UV light (35). By
titrating the time of UV irradiation, we could completely inactivate
the ability of VV to replicate while maintaining its capacity to
synthesize cytosolic minigene (although at reduced levels relative to
untreated virus). Immunization of mice with cells expressing either
Kb or Kbm1 and infected
with UV-irradiated VV-OVA257–264 resulted in
activation of OT-1 cells, demonstrating that true cross-priming occurs
with the cells expressing a cytosolic minigene. The activation of OT-1
cells was clearly less than when using noninactivated virus. Whether
due to decreased expression of the minimal determinant, decreased
VV-induced alterations in infected cells, or spread of VV from cells
infected with noninactivated virus is uncertain.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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What special properties of OVA might account for its cross-priming properties? An important clue comes from the inability of VV-NP-OVA257–264 to activate OVA257–264-specific TCD8+ in TAP1-/- mice. In TAP-expressing cells, OVA257–264 is presented at similar efficiencies from NP-OVA257–264 vs OVA, suggesting that the enhanced TAP-independent immunogenicity of OVA is unlikely to reflect increased levels of cytosolic chaperones bearing OVA257–264-containing peptides. This shifts suspicion to the secretory nature of OVA, which unlike NP (or SEN-NP or VSV N, for that matter) is targeted to the secretory pathway, where it obtains an N-linked oligosaccharide and is efficiently secreted by cells. Although purified OVA is poorly immunogenic, perhaps the immunogenicity of biosynthesized OVA is enhanced by the local environment of a VV infection, or by molecular chaperones that remain associated with a minor fraction of newly secreted OVA. The immunogenicity of OVA257–264 may be further enhanced in the context of OVA by the special properties of the OVA257–264 peptide and its immediate flanking residues (36). Perhaps it is representative of a subset of Ags that are able to withstand proteolysis in the endosomal compartment of pAPCs and associate with class I molecules that recycle to the cell surface for activating TCD8+.
We also provide the initial evidence that expression of a minimal Ag expressed in the cytosol is sufficient for cross-priming. Unlike the case with OVA, this probably occurs through binding of the peptide to either a cytosolic or ER molecular chaperone (37, 38, 39), because free peptides are degraded rapidly by cells (40). The recent finding that heat shock protein 70-mediated presentation of OVA257–264 by pAPCs can be TAP independent (41) is consistent with this possibility.
In contrast to the present findings, it was previously reported that the presentation of VV-OVA to OVA257–264-specific TCD8+ was completely TAP-dependent, inasmuch as the virus did not activate naive TCD8+ in irradiated B6 mice receiving bone marrow from TAP1-/- mice (42). In the same study, the presentation of VV-S-OVA257–264 occurred in a TAP-independent manner, as we observed. We believe the discrepancy in the presentation of OVA is due to the lower sensitivity of the bone marrow chimeras relative to the transferred OT- 1 TCD8+. Indeed, we demonstrate that VV-S-OVA257–264 provides a stronger in vivo stimulus than OVA, and no doubt had we transferred less OT-1 cells we could have set the system to observe a positive response to VV-S-OVA257–264 and negative response to VV-OVA. Similarly, our failure to demonstrate responses to various rVVs in TAP1-/- mice cannot be interpreted as a total inability to present the corresponding Ags, but only as a failure to present Ag at the sensitivity of the method used for detection.
The second significant aspect of the present study is that it provides the initial in vivo demonstration of class I peptide processing in the secretory pathway. We detected TAP-independent presentation of a C-end rule determinant (S-NP-OVA257–264) as well as the endoproteolytic liberation of NP366–374 from a secreted fragment of NP to adoptively transferred TCD8+. The parallels between in vivo and in vitro presentation of these Ags strongly suggest that their presentation occurs exclusively by direct presentation in TAP1-/- mice (and not cross-priming), because it is unlikely that the mechanisms governing the efficiency of TAP-independent cross-priming of various forms of Ag should directly parallel the mechanisms involved in entering the ER in a TAP-independent manner. Although the presentation of peptide can occur in the absence of TAP in vivo, it is possible that TAP may enhance the efficiency of this pathway, perhaps via stabilization of the MHC class I complex in the ER, allowing more peptide receptive MHC class I molecules to reach the secretory pathway.
Although this conclusion is limited to TAP-independent cross-priming, previous findings regarding the immunogenicity of VV suggest that TAP-dependent cross-priming of VV-encoded Ags may be the exception rather than the rule. It was shown a number of years ago that both influenza HA and NP inserted into VV elicit better mouse TCD8+ response when controlled by an early promoter than by a late promoter, even when the late promoter constructs generate far greater quantities of the Ag (43). A study by Bronte et al. (44) provided a likely explanation for these findings; VV infection of mouse DC does not progress past the early phase of viral replication. This is consistent with the hypothesis that direct presentation accounts for most of the priming of rVV-encoded Ags, and, given that late viral proteins will still be produced in large quantities by other infected cells in the immunized animals, cross-priming does not detectably occur for many proteins.
In summary, the present findings support the idea that there are determinant-specific routes of presenting Ags in vivo to naive TCD8+. The routes used in any given circumstance will depend on multiple factors including the species immunized, route of immunization, dose of Ag, vector used or form of Ag administered, context of the immunogenic peptide, and the peptide itself. In combination, these factors will determine the extent to which direct priming vs cross-priming contributes to TCD8+ activation. Cross-priming itself will no doubt also use a number of distinct mechanisms. We have provided the initial evidence that expression of a minimal Ag is sufficient for cross-priming. The most likely mechanism for this activity is the binding of the peptide to either a cytosolic or an ER molecular chaperone (37, 38, 39). We also note that cells expressing the cytosolic OVA257–264 express far greater amounts of the peptide than cells expressing OVA. Therefore, it remains an open question to what extent cross-priming with OVA and other Ags is based on transfer of molecular chaperones bearing peptides as opposed to transfer of free forms of Ags, either full-length or truncated (45). Other crucial questions await answers including the nature of the pAPCs involved in direct and cross-priming and to what extent cross-priming Ags are acquired from live, necrotic, or apoptotic cells (46, 47).
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, Indiana University School of Medicine, Walther Oncology Center, Indianapolis, IN 46202-5254.
3 Address correspondence and reprint requests to Dr. Jack R. Bennink and Dr. Jonathan W. Yewdell, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0440.
4 Abbreviations used in this paper: ER, endoplasmic reticulum; pAPCs, professional APCs; rVVs, recombinant vaccinia viruses; VSV, vesicular stomatitis virus; N, nucleocapsid; NP, nucleoprotein; HA, hemagglutinin; 2m, 2-microglobulin; VVs, vaccinia viruses; 2m, 2-microglobulin; Tg, transgenic; DC, dendritic cell; VV-OVA, rVVs expressing full-length OVA; SEN, Sendai virus; S, ER targeting signal/leader sequence.
Received for publication October 24, 2000. Accepted for publication January 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactosidase provides visual screening of recombinant virus plaques. Mol. Cell. Biol. 5:3403.This article has been cited by other articles:
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M. A. de Witte, A. Jorritsma, A. Kaiser, M. D. van den Boom, M. Dokter, G. M. Bendle, J. B. A. G. Haanen, and T. N. M. Schumacher Requirements for Effective Antitumor Responses of TCR Transduced T Cells J. Immunol., October 1, 2008; 181(7): 5128 - 5136. [Abstract] [Full Text] [PDF]
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M. Coccoris, E. Swart, M. A. de Witte, J. W. J. van Heijst, J. B. A. G. Haanen, K. Schepers, and T. N. M. Schumacher Long-Term Functionality of TCR-Transduced T Cells In Vivo J. Immunol., May 15, 2008; 180(10): 6536 - 6543. [Abstract] [Full Text] [PDF]
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M. A. de Witte, A. Jorritsma, E. Swart, K. C. Straathof, K. de Punder, J. B. A. G. Haanen, C. M. Rooney, and T. N. M. Schumacher An Inducible Caspase 9 Safety Switch Can Halt Cell Therapy-Induced Autoimmune Disease J. Immunol., May 1, 2008; 180(9): 6365 - 6373. [Abstract] [Full Text] [PDF]
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 O. Demirel, Z. Waibler, U. Kalinke, F. Grunebach, S. Appel, P. Brossart, A. Hasilik, R. Tampe, and R. Abele Identification of a Lysosomal Peptide Transport System Induced during Dendritic Cell Development J. Biol. Chem., December 28, 2007; 282(52): 37836 - 37843. [Abstract] [Full Text] [PDF]
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S. Bertholet, R. Goldszmid, A. Morrot, A. Debrabant, F. Afrin, C. Collazo-Custodio, M. Houde, M. Desjardins, A. Sher, and D. Sacks Leishmania Antigens Are Presented to CD8+ T Cells by a Transporter Associated with Antigen Processing-Independent Pathway In Vitro and In Vivo J. Immunol., September 15, 2006; 177(6): 3525 - 3533. [Abstract] [Full Text] [PDF]
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 R. Chavan, K. A. Marfatia, I. C. An, D. A. Garber, and M. B. Feinberg Expression of CCL20 and Granulocyte-Macrophage Colony-Stimulating Factor, but Not Flt3-L, from Modified Vaccinia Virus Ankara Enhances Antiviral Cellular and Humoral Immune Responses. J. Virol., August 1, 2006; 80(15): 7676 - 7687. [Abstract] [Full Text] [PDF]
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 M. A. de Witte, M. Coccoris, M. C. Wolkers, M. D. van den Boom, E. M. Mesman, J.-Y. Song, M. van der Valk, J. B. A. G. Haanen, and T. N. M. Schumacher Targeting self-antigens through allogeneic TCR gene transfer Blood, August 1, 2006; 108(3): 870 - 877. [Abstract] [Full Text] [PDF]
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L.-A. M. Pozzi, J. W. Maciaszek, and K. L. Rock Both Dendritic Cells and Macrophages Can Stimulate Naive CD8 T Cells In Vivo to Proliferate, Develop Effector Function, and Differentiate into Memory Cells J. Immunol., August 15, 2005; 175(4): 2071 - 2081. [Abstract] [Full Text] [PDF]
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S. M. M. Haeryfar, R. J. DiPaolo, D. C. Tscharke, J. R. Bennink, and J. W. Yewdell Regulatory T Cells Suppress CD8+ T Cell Responses Induced by Direct Priming and Cross-Priming and Moderate Immunodominance Disparities J. Immunol., March 15, 2005; 174(6): 3344 - 3351. [Abstract] [Full Text] [PDF]
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 A. M. Thomas, L. M. Santarsiero, E. R. Lutz, T. D. Armstrong, Y.-C. Chen, L.-Q. Huang, D. A. Laheru, M. Goggins, R. H. Hruban, and E. M. Jaffee Mesothelin-specific CD8+ T Cell Responses Provide Evidence of In Vivo Cross-Priming by Antigen-Presenting Cells in Vaccinated Pancreatic Cancer Patients J. Exp. Med., August 2, 2004; 200(3): 297 - 306. [Abstract] [Full Text] [PDF]
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J. Stebbing, B. Gazzard, S. Patterson, M. Bower, D. Perumal, M. Nelson, A. McMichael, G. Ogg, A. Epenetos, F. Gotch, et al. Antibody-targeted MHC complex-directed expansion of HIV-1- and KSHV-specific CD8+ lymphocytes: a new approach to therapeutic vaccination Blood, March 1, 2004; 103(5): 1791 - 1795. [Abstract] [Full Text] [PDF]
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 A. L. Ackerman, C. Kyritsis, R. Tampe, and P. Cresswell Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens PNAS, October 28, 2003; 100(22): 12889 - 12894. [Abstract] [Full Text] [PDF]
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A. Y. Savinov, F. S. Wong, A. C. Stonebraker, and A. V. Chervonsky Presentation of Antigen by Endothelial Cells and Chemoattraction Are Required for Homing of Insulin-specific CD8+ T Cells J. Exp. Med., March 3, 2003; 197(5): 643 - 656. [Abstract] [Full Text] [PDF]
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M. Andrieu, J.-F. Desoutter, E. Loing, J. Gaston, D. Hanau, J.-G. Guillet, and A. Hosmalin Two Human Immunodeficiency Virus Vaccinal Lipopeptides Follow Different Cross-Presentation Pathways in Human Dendritic Cells J. Virol., December 20, 2002; 77(2): 1564 - 1570. [Abstract] [Full Text] [PDF]
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W. M. Cohen, A. Bianco, F. Connan, L. Camoin, M. Dalod, G. Lauvau, E. Ferries, B. Culmann-Penciolelli, P. M. van Endert, J. P. Briand, et al. Study of Antigen-Processing Steps Reveals Preferences Explaining Differential Biological Outcomes of Two HLA-A2-Restricted Immunodominant Epitopes from Human Immunodeficiency Virus Type 1 J. Virol., September 11, 2002; 76(20): 10219 - 10225. [Abstract] [Full Text] [PDF]
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S. Basta, W. Chen, J. R. Bennink, and J. W. Yewdell Inhibitory Effects of Cytomegalovirus Proteins US2 and US11 Point to Contributions from Direct Priming and Cross-Priming in Induction of Vaccinia Virus-Specific CD8+ T Cells J. Immunol., June 1, 2002; 168(11): 5403 - 5408. [Abstract] [Full Text] [PDF]
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G. Delogu, A. Li, C. Repique, F. Collins, and S. L. Morris DNA Vaccine Combinations Expressing Either Tissue Plasminogen Activator Signal Sequence Fusion Proteins or Ubiquitin-Conjugated Antigens Induce Sustained Protective Immunity in a Mouse Model of Pulmonary Tuberculosis Infect. Immun., January 1, 2002; 70(1): 292 - 302. [Abstract] [Full Text] [PDF]
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G. Lautscham, S. Mayrhofer, G. Taylor, T. Haigh, A. Leese, A. Rickinson, and N. Blake Processing of a Multiple Membrane Spanning Epstein-Barr Virus Protein for Cd8+T Cell Recognition Reveals a Proteasome-Dependent, Transporter Associated with Antigen Processing-Independent Pathway J. Exp. Med., October 15, 2001; 194(8): 1053 - 1068. [Abstract] [Full Text] [PDF]
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