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Department of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands
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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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Two separate mechanisms have been proposed for the induction of tumor-specific T cell immunity. In the first model, tumor-specific T cell immunity is induced as a result of direct priming of naive CD8+ T cells by tumor cells that have migrated from the tumor site to the lymphoid tissue. In line with this suggestion, it was recently shown that tumor-specific T cell responses can be detected in experimental settings where viable tumor cells are present in secondary lymphoid organs (5). Furthermore, large numbers of melanoma-specific T cells are generally only observed in patients with detectable tumor infiltration to the lymphoid organs.
Alternatively, tumor-specific T cell responses may result from cross-presentation of tumor cell-derived Ags by professional APCs, such as bone marrow-derived dendritic cells (6, 7, 8). Professional APCs do not exclusively present Ags derived from endogenously produced Ags in the context of MHC class I molecules; in addition, they have the capacity to present Ags derived from exogenous sources (9, 10). This alternative pathway for priming of CD8+ T cells (also referred to as cross-priming) could play a role in the induction of tumor-specific T cell responses, either by capture of tumor Ags in the periphery or by representation of Ags from tumor cells that have migrated to the lymph node.
To determine which of these two possible pathways is sufficient for or required to induce a vigorous tumor-specific T cell response, we developed a murine tumor model that enabled us to follow the induction of Ag-specific T cell responses directly ex vivo. We introduced two virally derived Ags, the influenza A nucleoprotein epitope 366–374 (NP366)4 and the human papilloma virus (HPV) E749 epitope, into the tumor cell lines used in this model to track the T cell immunity induced upon tumor challenge. Interestingly, the resulting tumor cell lines induced massive T cell responses in naive mice, even in the absence of any further antigenic challenge. Using this model, we determined the relative contribution of direct priming and cross-priming for the induction of cytotoxic T cell responses. These results indicate that tumor cells present in the draining lymph nodes (DLNs) can contribute to T cell immunity by direct priming. In addition, when tumor cells devoid of MHC class I molecules are used, equally potent tumor-specific T cell responses are induced. Collectively, these experiments show that both direct priming and cross-priming are remarkably efficient for the induction of tumor-specific T cell responses and can act as redundant mechanisms for T cell activation. Furthermore, CD28-mediated costimulation is crucial for T cell activation through cross-priming, but the induction of Ag-specific CD8+ T cell immunity is only partially affected by CD28 deficiency when direct priming also can take place.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/10, C57BL/6, and MHC class II-deficient mice (11) backcrossed to the C57BL/6 background were obtained from the experimental animal department of the Netherlands Cancer Institute (Amsterdam, The Netherlands). CD28-deficient mice (12) were a gift from Dr. M. A. Oosterwegel (University Medical Center, Utrecht, The Netherlands). TAP1-deficient mice (13) were crossed with recombinase-activating gene (RAG)1-deficient mice to obtain mice that completely lack CD8+ T cells (14). The absence of B/T cells and the absence of MHC class I complexes expressed on the cell surface in the double knockout was confirmed by FACS analysis. NP366-specific, TCR (F5)-transgenic mice (15) backcrossed on a RAG1-deficient background (referred to as F5 mice) were a gift from Dr. D. Kioussis (National Institute of Medical Research, London, U.K.). Mice were handled in accordance with institutional guidelines.
Generation of cell lines expressing influenza A NP366 and HPV16 E749
The H-2Db-restricted epitope NP366 (ASNENMDAM) derived from influenza A/NT/60/68 was fused to the carboxyl terminus of the green fluorescence protein (GFP) gene using the following primers: top, 5'-GGGGGATCCTAAGCCACCATGGTGAGCAAGGGCGAG-3'; and bottom,5'-CCCTTTGCGGCCGCTTACATAGCGTCCATGTTTTCGTTGGAAGCGATCTGAACACCCTTGTACAGCTCGTCCATG-3'. To ensure that the epitope could still be generated from this fusion protein, the four naturally flanking amino acid residues (GVQI) were included in the fusion protein. The H-2Db-restricted HPV16-derived epitope E749 (RAHYNIVTF) was fused to the carboxyl terminus of GFP using the bottom primer (5'-CCCTTTGCGGCCGCTTAGAAGGTCACGATGTTGTAGTGGGCCCTGATCTGAACACCCTTGTACAGCTCGTCCATG-3'). The resulting PCR products were cloned into the BamHI and NotI sites of the retroviral vector pMX (16). As a control, the unmodified GFP gene was cloned into pMX. Sequences were confirmed by sequence analysis. Plasmids containing GFP-NP366, GFP-E749, or GFP were introduced into the cell lines EL4, RMA, and RMA-S by retroviral transduction as previously described (17, 18) to generate EL4-NP, RMA-NP, RMA-S-NP, EL4-HPV, EL4-GFP, and RMA-GFP tumor cells. Briefly, Phoenix-A cells were transfected with plasmid DNA by pfx-2 lipid transfection (Invitrogen, San Diego, CA). After 2 days of culture retroviral supernatant was harvested containing the retrospective retroviruses. Nontreated Falcon petri dishes (BD Biosciences, Mountain View, CA) were precoated with recombinant human fibronectin fragment CH-296 (RetroNectin, Takara Shuzo, Otsu, Japan) for 2 h at room temperature, and blocked with 2% BSA (Sigma, St. Louis, MO) in PBS for 30 min at room temperature. Cell lines were plated on RetroNectin-coated plates (0.5 x 106 cells/plate) in 1 ml retroviral supernatant. After 24 h of culture at 37°C, cells were transferred to 25-cm2 culture flasks (BD Biosciences). Cell lines with stable and comparable gene expression were obtained after two rounds of selection by flow cytometry and single-cell cloning.
All cell lines were cultured in IMDM (Life Technologies, Rockville, MD) supplemented with 5% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.5 x 10-5 M 2-ME.
Tumor challenge and analysis of tumor-specific CD8+ T cell responses
Tumor cells were washed three times with HBSS (Life
Technologies) and resuspended in HBSS. Tumor cells were injected s.c.
in 200 µl HBSS. Every 2–3 days after tumor inoculation, the tumor
size was measured in two dimensions. In addition, 
50 µl peripheral
blood was drawn for analysis of T cell responses at the same time
points. Erythrocytes were removed by incubation in erylysis buffer (155
mM NH4Cl, 10 mM KHCO3, and
0.1 mM EDTA, pH 7.4) on ice for 15 min. Cells were washed twice with
1x PBS containing 0.5% BSA and 0.02% sodium azide (PBA) and stained
with FITC- or PE-conjugated anti-CD8
(BD PharMingen, San Diego,
CA) together with PE- or allophycocyanin-conjugated
NP366 or E749 tetramers
(1, 19) at room temperature for 15 min in PBA. Mononuclear
cells were washed twice and analyzed by flow cytometry (BD
Biosciences). Live cells were selected based on propidium iodide
exclusion.
In vivo depletion of CD8+ T cells by Ab treatment
On days -7, -5, and -3 before tumor challenge, 200 µg
purified anti-CD8 Ab (hybridoma 2.43) was injected i.p. Depletion
of CD8+ T cells was confirmed by flow cytometric
analysis of peripheral blood samples with anti-CD8 and was shown
to be >98%. To maintain efficient depletion of the
CD8+ T cell population, Ab treatment was
continued three times a week after tumor challenge.
Detection of tumor cells in the DLN
Viable tissue sections were prepared as described previously
(20). Briefly, C57BL/10 mice were inoculated s.c. with
1 x 106 EL4-NP tumor cells. On day 8 after
tumor inoculation, DLN (inguinal) were excised and embedded in 4% low
melting temperature agarose (NuSieve GTC; FMC Bioproducts, Rockland,
MA) in PBS. Two hundred- to 500-µm tissue sections were cut
using a Leica VT 1000s microtome (Leica Instruments, Nussloch,
Germany). Tissue sections were incubated overnight at 4°C
with anti-CD8-allophycocyanin or with anti-CD8-FITC (BD
PharMingen) together with allophycocyanin-conjugated MHC tetramers in
PBS, 0.5% BSA, and 10% normal mouse serum. Sections were washed three
times with ice-cold PBS-0.5% BSA and were analyzed by confocal
microscopy.
For semiquantitative analysis of tumor cells present within the DLN, mice were challenged s.c. with 1 x 106 EL4-NP cells, and inguinal lymph nodes were excised 1, 2, 4, 6, and 8 days after tumor challenge. Lymph nodes were meshed and washed once with PBA, and single-cell suspensions were resuspended at a density of 20 x 106 cells/ml. Green fluorescent tumor cells were monitored and counted by fluorescence microscopy.
Purification and CFSE labeling of NP-specific TCR-transgenic F5 cells
Naive F5 spleen cells were labeled with anti-CD8-PE (BD
PharMingen), and MACS purification of CD8+ T
cells with anti-PE MACS beads was performed according to the
manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany).
CD8+ T cells were shown to be >98.5% pure by
FACS analysis with Db-NP tetramers. Purified
F5 cells were labeled with CFSE as described
previously (21). Briefly, 5 x
107 cells/ml were incubated with 5
µM CFSE in RPMI medium without FCS for 10 minat 37°C. Cells were
washed once with RPMI 1640 supplemented with 10% FCS and twice
with HBBS before adoptive transfer into TAP/RAG-deficient
mice.
Intracellular cytokine staining
Spleen cells (1 x 106) were cultured
for 5 h in complete medium supplemented with 50 U/ml human rIL-2
and 1 µl/ml brefeldin A (GolgiStop, BD PharMingen) in the presence or
the absence of 0.1 µg/ml NP366. Cells were
stained with anti-CD8-PE and washed twice, and subsequently
intracellular cytokine stains were conducted using a CytofixCytoPerm
kit (BD PharMingen) according to the manufacturer’s protocol.
Intracellular staining was performed with FITC-conjugated
anti-IFN-
(clone XMG 1.2).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has previously been shown that upon influenza A infection of
mice, a rapid and prominent CD8+ T cell response
against NP366 (ASNENMDAM) develops (19, 22). In this viral infection model, peak values >10% of
NP366-specific CD8+ T cells
in peripheral blood, spleen, and lung have been observed. To evaluate
whether viral infection is a prerequisite for the development of this
pronounced T cell response, we introduced the
NP366 epitope as a COOH-terminal fusion with
enhanced GFP into the murine tumor cell line EL4 (EL4-NP, Fig. 1
A). Remarkably, when naive
mice are inoculated with EL4-NP cells, very high percentages of
NP366-specific CD8+ T cells
can be detected in both peripheral blood and secondary lymphoid organs
(Fig. 1, B and C; data not shown). Ag-specific
CD8+ T cells are detectable in peripheral blood
and DLN starting from day 6 and in the spleen from day 9 after tumor
challenge (data not shown). NP366-specific
CD8+ T cells infiltrate efficiently into the
EL4-NP tumor, with a percentage of Ag-specific T cells comparable to
that observed in peripheral blood. Tumors that express the
NP366 neo-Ag are rejected 12–14 days after
inoculation, whereas control tumors expressing GFP, but lacking the
NP366 epitope (EL4-GFP) grow progressively (Fig. 1D). Furthermore, when naive mice are inoculated with the
Ag-bearing EL4-NP tumor at one flank and with the control tumor EL4-GFP
at the contralateral flank, EL4-NP tumors are rejected, whereas EL4-GFP
tumors grow out (Fig. 2A). To
directly establish whether this epitope-dependent tumor rejection is
due to CD8+ T cell recognition, the EL4-NP tumor
growth pattern was studied in mice that were depleted of
CD8+ T cells by in vivo Ab treatment. In these
mice the EL4-NP tumors grow out with comparable kinetics as EL4-GFP
control tumors (Fig. 2B), providing direct evidence for the
contribution of CD8+ T cells to the rejection of
NP366-expressing tumors.
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) and the LCMV-derived gp33 Ag
(KAVYNFATC; N. Brouwenstijn and T. N. M. Schumacher,
unpublished observations) induce equally potent Ag-specific T cell
responses when introduced into tumor cells. Thus, viral Ags are capable
of inducing very strong T cell immunity independent of their viral
environment. When the kinetics of tumor growth and tumor-specific T
cell expansion are compared, it is apparent that in all cases tested
(see also below) maximal numbers of tumor-specific T cells are observed
shortly after the tumor has started to regress. This suggests that the
increased liberation of Ag or endogenous adjuvants upon tumor cell
death may provide a further stimulus for the expansion of the
Ag-specific T cell repertoire, as shown previously for TCR-transgenic T
cells (24, 25). Taken together, these results indicate
that murine tumor cell lines can induce a readily detectable
tumor-specific CD8+ T cell response provided that
sufficiently immunogenic epitopes are present. By virtue of this fact,
this murine model system provides the means to dissect some of the
relevant parameters that determine the induction of T cell immunity to
solid tumors or the lack thereof.
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We next investigated the underlying mechanism responsible for the
induction of these readily detectable tumor-specific T cell
responses. The activation and subsequent expansion of naive
NP366-specific T cells may either
result from direct activation of this T cell population by
Ag-presenting tumor cells or alternatively through cross-presentation
of the NP366 epitope by professional APCs. For
the direct priming pathway it is hypothesized that tumor cells need to
be present within the lymph nodes where naive T cells reside. To
determine whether in this model tumor cells are able to migrate to
secondary lymphoid tissues, we analyzed draining lymph nodes for the
presence of EL4-NP tumor cells based on GFP gene expression by confocal
microscopy of viable tissue sections (Fig. 4, A and B). These
experiments directly demonstrate the presence of small but measurable
numbers of tumor cells within the lymph node tissue, supporting recent
observations by Zinkernagel and colleagues (5).
Furthermore, simultaneous staining of these sections with
Db-NP366 tetramers
indicates that MHC tetramer-positive CD8+ T cells
can colocalize with tumor cells present within the draining lymph node.
Semiquantitative analysis of tumor cells present within the draining
lymph node reveals 1–10 tumor cells/lymph node in the first 4 days
after tumor challenge. At later time points (days 6 and 8), when
tumor-specific T cell immunity was already initiated (see above), the
number of tumor cells expanded by 5- to 10-fold. These findings
indicate that the neo-tumor Ag is accessible to the immune system as a
result of migrating tumor cells by early time points following tumor
inoculation.
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C). In contrast, no proliferation of
NP366-specific F5 T cells
was observed in mice that did not receive tumor cells. As a further
control, TAP/RAG-deficient mice were challenged with
NP366-expressing RMA-S tumor cells. RMA-S cells
have a strongly reduced capacity to present endogenously produced Ags
to Ag-specific T cells compared with wild-type RMA cells as a result of
their TAP deficiency (26) (data not shown). Even upon
inoculation of high numbers of TAP-deficient RMA-S-NP cells, only
limited proliferation of NP-specific T cells was induced (Fig. 4C). Collectively, these experiments show that MHC class
I-proficient tumor cells present within the draining lymph nodes can
induce activation of Ag-specific CD8+ T cells in
a setting where TAP-dependent Ag cross-presentation is prevented.
Furthermore, the observation that this T cell activation is largely
abolished when Ag-expressing, but TAP-deficient, RMA-S tumor cells were
used points to direct priming of T cells rather than TAP-independent
cross-presentation as the mechanism involved.
To study a possible contribution of the cross-priming pathway to the
induction of tumor-specific T cell immunity, we again made use of the
Ag-positive, TAP-deficient RMA-S-NP cells. Remarkably, upon tumor
challenge of wild-type mice with RMA-S-NP, a tumor-specific T cell
response is induced that is quantitatively similar to the response
induced by TAP-proficient tumors (Figs. 5 and 6A). Furthermore, the
NP366-specific CD8+ T cells
that are induced by cross-priming produce IFN- in an Ag-dependent
fashion, indicating that these tumor-specific T cells are also
functional (see below). T cell induction by cross-priming was first
demonstrated by Bevan (9) by injection of Ag-containing
cells that do not express the relevant MHC restriction element into
mice that do express this MHC allele. In line with this, significant
NP366-specific T cell responses are induced when
NP366-expressing H-2d tumor
cells are inoculated into H-2d/b mice (M. C.
Wolkers, unpublished observations). These experiments indicate that
tumor cells can directly activate tumor-specific T cells in a setting
where measurable numbers of tumor cells are present within the lymphoid
tissue. In addition, equally dramatic tumor-specific T cell responses
can be elicited through Ag cross-presentation. Based on these findings,
it may be concluded that CD8+ T cell activation
through direct priming and cross-priming can act as redundant
mechanisms.
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The current data demonstrate that MHC class I-deficient tumors are able to induce massive tumor-specific T cell responses by cross-priming, and that these polyclonal T cell responses can be directly monitored ex vivo. This provides a straightforward experimental system in which the contributions of different cellular interactions to Ag cross-priming can be studied in a setting that is not affected by confounding factors such as a requirement for in vitro restimulation or functional assays.
To further dissect the pathway of cross-priming, we analyzed whether Th
cells are required for the induction of tumor-specific
CD8+ T cells. To this purpose we challenged MHC
class II-deficient mice with the Ag-positive, TAP-deficient RMA-S-NP
tumor. In this setting the tumor-specific CD8+ T
cell response is completely intact and may even be slightly enhanced in
MHC class II-deficient mice compared with that in wild-type mice, with
respect to both the kinetics and the magnitude of the response (Fig. 6A). Furthermore, these NP366-specific
CD8+ T cells are fully functional despite the
CD4+ T cell deficiency, as judged from their
ability to produce IFN- upon antigenic stimulation (Fig. 6B). Notably, the total independence of
CD4+ T cell help for the induction of tumor
immunity through cross-priming is not an intrinsic property of the
GFP-NP366 fusion protein, as the induction of
CD8+ T cell immunity with the identical fusion
gene by means of DNA vaccination shows an absolute dependence on
CD4+ T cell help (M. C. Wolkers, M. Toebes,
J. B. A. G. Haanen, and T. N. M. Schumacher,
manuscript in preparation).
Although the induction of APC maturation through cognate
CD4+ T cell help is clearly not required for
efficient cross-priming, costimulation by the APC does play an
essential role. CD28-deficient mice not only show a 10-fold reduction
of the Ag-specific T cell population compared with the response
observed in wild-type mice, but the onset of the
CD8+ T cell response is also significantly
delayed (Fig. 6A). However, the small population of
tumor-specific T cells that is induced is capable of producing IFN-
with a similar efficiency as Ag-specific T cells derived from wild-type
mice (Fig. 6B), indicating that the difference is
quantitative rather than qualitative. Previous studies have revealed
that continuous TCR stimulation by repetitive peptide vaccination can
overcome T cell unresponsiveness in CD28-deficient mice
(27). Increased and continuous liberation of Ag upon tumor
cell death in larger tumors could explain why cytokine-producing
CD8+ T cells are detectable to some extent in
CD28-deficient mice, and why the onset of the
CD8+ T cell response is delayed in the absence of
costimulation via CD28. Importantly, when CD28-deficient mice are
inoculated with MHC class I-proficient RMA-NP cells, vigorous T cell
responses comparable in magnitude to T cell responses in wild-type mice
are observed in three of five mice tested (at the peak of the response
(day 15), 2.6, 2.2, and 23.0% NP366-specific
CD8+ T cells in peripheral blood; representative
FACS plots in Fig. 6C). Consistent with our findings that
direct priming and cross-priming are redundant pathways for the
induction of T cell immunity, these data suggest that direct priming
can partially compensate in the induction of tumor-specific T cell
responses when cross-priming is defective due to the lack of
costimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 2, and 6) (19, 22, 29). This higher variation in
tumor-specific T cell immunity vs virus-specific T cell immunity is
likely to reflect the variability of parameters such as tumor growth or
vascularization. Nevertheless, for three different Ags tested,
detectable tumor-specific T cell responses have been observed in all
mice tested to date, rendering this system highly suitable for the
dissection of tumor and Ag requirements in the induction of
tumor-specific T cell immunity.
A number of studies have provided evidence for a role for either direct
priming or cross-priming in the induction of tumor-specific
CD8+ T cell responses in different model systems
(5, 6, 30, 31). Here, we have assessed the capacity of
these two processes to induce T cell immunity in one well-defined model
system by direct quantification of the Ag-specific
CD8+ T cell response. These studies demonstrate
that a low, but significant, number of tumor cells can be directly
visualized in the draining lymph nodes of mice carrying s.c. growing
tumors. Furthermore, we show that such tumor cells can directly prime
Ag-specific T cells when TAP-dependent cross-presentation is prevented
(Fig. 4C).
In addition to the role of direct priming in the induction of
tumor-specific T cell immunity, we show that tumor-specific T cell
responses can likewise be induced through cross-presentation of tumor
Ags derived from MHC class I-deficient tumors (Fig. 5). Tumor-specific
T cell responses induced by cross-priming are equally pronounced as
tumor-specific T cell responses induced against MHC class I-proficient
tumors, indicating that cross-priming can be a pathway of equal merit
in the induction of T cell responses to tumor Ags. In MHC class
I-deficient TAP/RAG-deficient mice, a small percentage of
NP366-specific T cells divide when stimulated
with tumors devoid of MHC class I (Fig. 4C). Recent studies
have suggested that Ags can be cross-presented independently of TAP,
albeit with reduced efficiency, which may provide an explanation for
this observation (31, 32, 33).
Our data demonstrate that direct priming and cross-priming can act as redundant mechanisms in the induction of tumor-specific T cell responses to solid tumors in a murine model system. However, it remains to be evaluated which of these mechanisms is predominantly involved in the induction of CD8+ T cell responses to different types of human tumors. Some of the factors that are likely to influence the relative contributions of these two processes are the efficiency with which the tumor environment results in APC maturation and the efficiency of tumor cell migration to the draining lymph nodes, possibly through the expression of chemokine receptors (34). In this regard, it is noted that the presence of pronounced CD8+ T cell immunity to human tumor Ags often correlates with the presence of tumor cells in the lymphoid organs, providing at least some support for a contribution of direct priming in the human setting. The relative contribution of Ag cross-presentation in the induction of human tumor-specific T cell responses is challenging to estimate. However, very large tumor-specific CD8+ T cell expansions have been observed in patients with seemingly MHC class I-deficient metastases, suggesting that Ag cross-presentation may at least suffice to maintain tumor-specific T cell responses in cancer patients.
The current data show that T cell help is not a prerequisite for
CD8+ T cell responses induced by cross-priming
(Fig. 6). This may appear contradictory with other studies that have
indicated an essential role for CD4+ T cells in
the maturation and activation of professional APCs before
CD8+ T cell priming (35). Maturation
and activation, however, can also be induced in the process of Ag
cross-presentation (36, 37, 38, 39). Therefore, it may be
speculated that CD4+ T cell help is required for
efficient T cell activation through cross-priming for Ags that are
cross-presented with lower efficiency, e.g., as a consequence of low
MHC affinity or suboptimal Ag processing. However, the current results
directly demonstrate that the requirement for T cell help is
conditional rather than absolute. Importantly, costimulation via CD28
is clearly essential for the induction of high magnitude
CD8+ T cell responses through cross-priming. An
interesting observation is that tumor-specific T cell immunity induced
by MHC class I-proficient RMA-NP tumor cells is only affected to a
limited extent by CD28 deficiency. Thus, whereas T cell activation by
cross-priming does require CD28 costimulation, the T cell response
against the very same Ag can occur in the absence of costimulation when
activation occurs through direct priming. These findings are compatible
with the idea that costimulation does not provide an essential danger
signal, but rather serves as a signal amplifier (27). The
observation that the small residual T cell population induced through
cross-priming in CD28-deficient mice is fully functional is also
consistent with the hypothesis that costimulation functions as a
facilitator of T cell responses rather than a factor that controls T
cell activation vs T cell tolerance or anergy.
The data we have presented in this paper show that direct priming and cross-priming can function as fully redundant mechanisms for the induction of vigorous tumor-specific T cell responses. Several factors, including affinity of the epitope for MHC class I complexes and tumor cell apoptosis, have previously been suggested to significantly affect the efficiency of CD8+ T cell activation through direct priming or cross-priming (40, 41). The tumor model system described here provides us with the tools to directly assess the contributions of such factors in one well-defined setting.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ton N. M. Schumacher, Department of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. E-mail address: tschum{at}nki.nl
3 M. G. C. T. van Ooijen, S. G. Elias, G. C. de Gast, M. P. W. Gallee, T. N. M. Schumacher, and J. B. A. G. Haanen. Melanoma-specific CD8+ T lymphocytes correlate with poor clinical outcome in advance stage melanoma patients. Submitted for publication.
4 Abbreviations used in this paper: NP366, influenza A nucleoprotein epitope 366–374; HPV, human papilloma virus; DLN, draining lymph node; RAG, recombinase-activating gene; GFP, green fluorescent protein.
Received for publication May 16, 2001. Accepted for publication July 18, 2001.
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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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R. Arens, K. Schepers, M. A. Nolte, M. F. van Oosterwijk, R. A.W. van Lier, T. N.M. Schumacher, and M. H.J. van Oers Tumor Rejection Induced by CD70-mediated Quantitative and Qualitative Effects on Effector CD8+ T Cell Formation J. Exp. Med., June 7, 2004; 199(11): 1595 - 1605. [Abstract] [Full Text] [PDF]
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M. C. Wolkers, N. Brouwenstijn, A. H. Bakker, M. Toebes, and T. N. M. Schumacher Antigen Bias in T Cell Cross-Priming Science, May 28, 2004; 304(5675): 1314 - 1317. [Abstract] [Full Text] [PDF]
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M. Zoller Tumor Vaccination after Allogeneic Bone Marrow Cell Reconstitution of the Nonmyeloablatively Conditioned Tumor-Bearing Murine Host J. Immunol., December 15, 2003; 171(12): 6941 - 6953. [Abstract] [Full Text] [PDF]
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T. Walzer, A. Marcais, F. Saltel, C. Bella, P. Jurdic, and J. Marvel Cutting Edge: Immediate RANTES Secretion by Resting Memory CD8 T Cells Following Antigenic Stimulation J. Immunol., February 15, 2003; 170(4): 1615 - 1619. [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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M. C. Wolkers, M. Toebes, M. Okabe, J. B. A. G. Haanen, and T. N. M. Schumacher Optimizing the Efficacy of Epitope-Directed DNA Vaccination J. Immunol., May 15, 2002; 168(10): 4998 - 5004. [Abstract] [Full Text] [PDF]
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 Z. Qin, C. Harders, X. Cao, C. Huber, T. Blankenstein, and B. Seliger Increased Tumorigenicity, but Unchanged Immunogenicity, of Transporter for Antigen Presentation 1-deficient Tumors Cancer Res., May 1, 2002; 62(10): 2856 - 2860. [Abstract] [Full Text] [PDF]
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) tumors
were measured in two dimensions, and the average of these two values is
presented. The mice were sacrificed when tumors became necrotic or
reached a diameter of >15 mm. The results are representative of three
experiments.
) or were not (•) depleted of CD8+ T cells by i.p.
administration of anti-CD8 Ab. Alternatively, naive mice were
injected with EL4-GFP control tumor cells (
) or without (
)
NP366 for 5 h in the presence of brefeldin A, and the
percentage of IFN-