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Center for Immunology, University of Minnesota, Minneapolis, MN 55455
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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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A major difficulty in attempting to understand why an effective CD8+ CTL response to syngeneic tumor does not normally occur and in developing novel therapeutic approaches that target the CTL response is that the Ag-specific CD8+ T cells are present in very low numbers in the animal and cannot be specifically identified. Thus, their trafficking and response to Ag in vivo cannot be directly tracked. Instead, the location and status of the relevant tumor-specific CD8+ cells can only be assessed by indirect methods that rely on in vitro restimulation and assay of cytolytic activity. These are cumbersome at best and rely upon the ability of the cells to respond in vitro to the stimulus used.
Adoptive transfer of T cells from TCR transgenic mice into normal recipients provides a means of monitoring Ag-specific T cells during a response 11 . The transferred cells can be identified by unique surface markers and serve as an indicator of the endogenous host response. Provided that the cells are transferred in small numbers, to constitute 0.2–0.5% of the spleen and lymph node (LN)3 cells in the recipient, the presence of the transferred cells does not aberrantly skew the normal response, as is the case when attempting to study responses in intact TCR transgenic mice. Adoptive transfer of CD8+ TCR transgenic T cells has been used to study both virus-specific 12, 13 and allogeneic CTL responses 14 . Recent studies of virus-specific responses by adoptive transfer 13 or direct visualization of endogenous Ag-specific cells using tetrameric Ag 15 have shown that the number of specific cells is dramatically underestimated by approaches that rely on in vitro restimulation of the cells, emphasizing the limited information that can be gained by such indirect approaches.
To begin to develop a more detailed understanding of CD8+ T cell responses to syngeneic tumor, we have applied the TCR transgenic adoptive transfer approach to study the response to E.G7, the EL-4 thymoma transfected with the gene for OVA 16 . E.G7 cells grow rapidly in C57BL/6 mice and the host dies within 25–35 days. This cell line, expressing OVA as a pseudo-tumor Ag, has been extensively used in studies of immunotherapy procedures 17, 18, 19, 20, 21, 22 . CD8+ T cells from OT-I mice with the C57BL/6 background express a transgenic TCR that recognizes H-2Kb and OVA257–264 peptide 23 . By adoptively transferring these cells into Thy-1 congenic C57BL/PL mice and challenging them with E.G7, it is possible to identify the Ag-specific cells and determine whether they can respond to the tumor, and if so where and when the response occurs and if it is effective in reducing tumor growth. As described in this report, the OT-I cells do respond at the site of the tumor by clonally expanding and developing cytolytic function, and as a result they transiently control tumor growth. However, at a point where tumor is still present, the OT-I cells migrate from the tumor site into the spleen and LN where they still retain lytic effector function but appear to be anergic to restimulation; the tumor progresses and the mice die. These results clearly define the in vivo induction and progression of a CD8+ T cell immune response to a syngeneic tumor. Furthermore, they identify novel mechanisms involved in the circumvention of the immune response by the tumor and have important implications for devising strategies for effective immunotherapy of cancer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OT-I TCR transgenic mice 23 , a gift from Dr. Francis Carbone (Monash Medical School, Victoria, Australia), were bred to wild-type C57BL/6 mice to generate mice heterozygous for the OT-I TCR transgene, and these mice were used as the source of transgenic T cells in all experiments. C57BL/PL mice congenic for the Thy-1 marker were purchased from The Jackson Laboratory (Bar Harbor, ME) and were used as recipients in all the experiments described. Animals were housed under specific pathogen-free conditions at all times.
Cell lines
EL-4, a thymoma derived from the C57BL/6 mouse (H-2b), was maintained in vitro in complete RPMI medium; RPMI 1640 (Cellgro, Herndon, VA), 10% FCS (Tissue Culture Biologicals, Tulare, CA), 0.2% L-glutamine, 0.1% penicillin/streptomycin, 0.1% HEPES (BioWhittaker, Walkersville, MD), 0.1% nonessential amino acids, 0.01% sodium pyruvate (Cellgro), and 0.05% 2-ME. E.G7 (OVA-transfected EL-4) 16 was maintained in complete RPMI medium containing 400 µg/ml of G418 (Cellgro). Both cell lines were periodically passaged in vivo. B3, a Kb/OVA257–264-specific CTL cell line was a gift from Drs. S. Jameson and K. Hogquist (University of Minnesota, Minneapolis, MN).
Abs and reagents
Directly conjugated mAbs including anti-CD8
-cychrome,
anti-Thy-1.2-PE, anti-CD62L-FITC, anti-CD44-FITC,
anti-CD49d-FITC, anti-CD25-FITC, and anti-CD69-FITC were
purchased from PharMingen (San Diego, CA). Purified anti-CD16/CD32
(Fc Block) was also purchased from PharMingen.
Adoptive transfer of transgenic cells and tumor challenge
LN cells (axillary, brachial, mesenteric, inguinal, cervical, periaortic, and mediastinal) from heterozygous OT-I transgenic mice were removed, homogenized, and washed three or four times in PBS. CD8+/Thy-1.2+ cells (3–4 x 106) were transferred into sex-matched naive C57BL/PL mice by tail vein injection. Recipient mice were rested for a day and were then challenged by i.p. injection of 4 x 106 EL-4 or E.G7 cells in 0.5 ml of PBS or PBS alone as a control.
Analysis by flow cytometry
Mice were sacrificed at varying times, and the spleen and LN were collected (periaortic, mesenteric, axillary, and brachial), homogenized, and treated with ammonium chloride to remove RBCs. The peritoneal cavity was washed twice with 25 ml of PBS each time, and the resulting peritoneal exudate lymphocytes were adherence-depleted for 90 min in complete medium at 37°C. The total number of cells obtained from each site was determined by counting using a hemocytometer.
Cells (1 x 106) isolated from each site were treated
with Fc Block for 30 min on ice and were then stained with
anti-CD8-cychrome, anti-Thy-1.2-PE and a third Ab-FITC
specific for phenotypic marker (see below). After a 1-h incubation on
ice, the cells were washed twice, resuspended in 0.2 ml of 1%
formaldehyde, and analyzed by three-color flow cytometry using the
CellQuest software package (Becton Dickinson, San Jose, CA).
Transferred OT-I cells were identified as
CD8+/Thy-1.2+ cells. Cells from C57BL/PL mice
stained in the same way showed no events in the
CD8+/Thy-1.2+ gate, and cells from mice that
had received OT-I cells by adoptive transfer had <0.05% of events in
this gate when the Thy-1.2-PE mAb was replaced with an isotype-matched
control Ab labeled with PE. These controls were included in every
experiment.
Activation status-associated phenotypic characterization of OT-I cells was performed by staining with a third Ab; these included anti-CD44-FITC, anti-CD25 (IL-2R)-FITC, anti-CD62L (L-selectin)-FITC, anti-CD49d (VLA-4)-FITC, and anti-CD69-FITC. The CD8+/Thy-1.2+ cells were gated, and their numbers and phenotype were determined as a percentage of the total population. In figures examining phenotype, the markers used to determine the percentage of cells expressing high levels of the protein are indicated. The positions of these markers were determined for each Ab by staining with the appropriate FITC-labeled isotype-matched control Ab. The numbers of tumor cells were determined by gating on large granular cells that were Thy-1.2+/CD8-. The total numbers of CD8+/Thy-1.2+ (OT-I) and tumor cells at each location were determined by multiplying the percentage of cells in the population by the total number of cells recovered from each site.
Chromium release assay
The lytic activity of spleen or LN cells obtained from tumor-primed mice was determined using a standard 4-h 51Cr release assay with EL-4 or E.G7 target cells. Briefly, serial dilutions of adherence-depleted spleen and LN cells were plated in 96-well V-bottom plates with 5 x 103 of 51Cr-labeled EL-4 or E.G7 cells in a final volume of 0.2 ml. After 4-h incubation at 37°C, 0.1 ml of supernatant was removed from each well, and radioactivity was determined. The percent specific 51Cr release was determined by subtracting spontaneous release from experimental release and dividing by maximum release minus spontaneous release. Maximum release was determined by treatment of cells with 3% SDS.
Cell proliferation assay
To determine the proliferative ability of spleen or LN cells from tumor-challenged mice, 1 x 105 adherence-depleted spleen or LN cells in complete RPMI medium were cultured in 96-well U-bottom plates with irradiated spleen cells from naive C57BL/6 mice pulsed with OVA257–264 peptide in the presence or the absence of mouse rIL-2 (R&D Systems, Minneapolis, MN). On the indicated days, cultures were pulsed with 1 µCi of [3H]thymidine for 6–8 h and lysed with distilled water, and the amount of [3H]thymidine incorporated into DNA was determined by liquid scintillation counting. The total numbers of CD8+/Thy-1.2+ cells in the cultures was determined before and after the culture period to determine the fold increase in CD8+/Thy-1.2+ cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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E.G7 cells are EL-4 thymoma transfected with the OVA gene 16 and
express about 100 Kb/OVA257–264 complexes/cell
24 . Thus, the OVA peptide is present at a low density, as is likely
to be the case for physiologically relevant tumor Ags. To insure that T
cells from OT-I mice with a TCR-specific for
Kb/OVA257–264 23 could respond to Ag on
E.G7, proliferation in response to irradiated E.G7 cells was examined
(Fig. 1
A). OT-I cells made a
strong proliferative response to OVA257–264-pulsed spleen
cells, and a weaker but still substantial, response to E.G7, while no
response occurred to the parental EL-4 cells. E.G7 cells could also be
killed by OT-I effector CTL, while lysis of EL-4 was minimal (Fig. 1B).
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A). Adoptive
transfer was performed by i.v. (tail vein) injection of cells from OT-I
mice into Thy-1 congenic C57BL/PL recipients to allow detection of the
transferred cells by staining with anti-Thy-1.2 mAb. In C57BL/PL
mice that had not received OT-I, virtually no
Thy-1.2+CD8+ events could be detected in LN
(Fig. 2B) or spleen cells. In mice that received OT-I by
i.v. injection (tail vein), the Thy-1.2 and CD8+ OT-I cells
could be readily detected in spleen and LN (Fig. 2C). The
naive phenotype of the OT-I cells (Fig. 2A) was maintained
in the recipients following adoptive transfer (Fig. 2C).
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EL-4 tumor expresses Ags that can be recognized by C57BL/6
CD8+ T cells 25, 26 , and the E.G7 line expresses in
addition the OVA257–264 CD8+ T cell
determinant. Nevertheless, i.p. injection of live E.G7 results in
progressive tumor growth and death of the host in about 25–35 days.
This could be due to failure to generate a CD8+ CTL
response or failure of the response to control tumor growth. To examine
this, OT-I cells were adoptively transferred by i.v. (tail vein)
injection in C57BL/PL recipients and allowed to equilibrate for 24
h so that their distribution in lymphoid organs reflected that of the
endogenous CD8+ T cells. One day later the recipients were
challenged by i.p. injection of live E.G7, and the number of OT-I cells
(CD8+/Thy-1.2+ cells) in the LN, spleen, and
peritoneal cavity was determined at subsequent times. By day 4 after
challenge, the number of OT-I cells had declined modestly in the spleen
and LN (Fig. 3, A and
B) and increased substantially in the peritoneal cavity
(Fig. 3C), suggesting that they were migrating to the latter
site and undergoing clonal expansion. By day 6, OT-I cell numbers had
begun to decline in the peritoneal cavity and were increasing in the
spleen. Few detectable OT-I cells remained in the peritoneal cavity by
day 10, while increased numbers were present in the LN and spleen and
remained present at these sites through days 18 (Fig. 3, A
and B) and 22 (data not shown). In mice that were not
challenged with E.G7 (Fig. 3, OT-I), the number of OT-I cells declined
for the first 4 days and then remained constant, with no expansion at
any sites. Similarly, no expansion of OT-I cells occurred in adoptively
transferred mice that were challenged with EL-4 tumor (data not shown).
Thus, OT-I cells exhibit an Ag-specific increases in number at the site
of tumor growth in mice challenged with tumor.
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A), the
majority of OT-I in the peritoneal cavities of E.G7-challenged mice had
an activated phenotype (Fig. 4C), with increased CD44, CD25,
and VLA-4 and decreased L-selectin surface levels, and about half were
blasts as judged by FSC. However, the OT-I cells in the spleen and LN
on day 4 maintained a naive phenotype, with the exception of increased
VLA-4 expression on OT-I cells in the LN (data not shown). When
challenge was with EL-4, very few OT-I cells appeared in the
peritoneal cavity on day 4 or later, and they did not increase as a
percentage of the total CD8+ T cells. The small
numbers made precise estimates of phenotypic distribution difficult,
but it is clear that the majority of OT-I cells did not become
activated in response to the EL-4 tumor (Fig. 4B).
Thus, OT-I cells undergo Ag-dependent proliferation in response to the
E.G7 tumor, and clonal expansion occurs preferentially at the site of
tumor growth rather than in draining LN.
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While they were present in expanded numbers in the peritoneal
cavity (days 4–6), OT-I cells limited the rate of growth of the tumor
compared with tumor growth in normal C57BL/PL mice (Fig. 5A). After day 6, when OT-I
cells were declining in the peritoneal cavity, tumor growth rate
increased (Fig. 5A) and paralleled the growth rate in
normal, untransferred C57BL/PL mice. Control of tumor growth by the
OT-I cells is Ag specific; transferred OT-I cells have no effect on the
growth of EL-4 tumor (Fig. 5B). In five of six experiments
the time course of the response, OT-I expansion in the peritoneal
cavity, phenotypic changes, and transient control of E.G7 tumor growth
were comparable to the representative results shown in
Figs. 3–5
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tumor failed to grow in the control mice in the sixth experiment.
Numerous additional experiments examining more limited time points have
further confirmed these findings.
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2, one of the transgenic TCR
chains. This allows detection of OT-I cells with reasonable accuracy
over the low background of CD8+V2+ host
cells, particularly in the peritoneal cavity during the response. The
numbers and kinetics of clonal expansion followed by decline in
OT-I cells and the numbers and kinetics of tumor cells in the
peritoneal cavity were essentially the same as shown in Figs. 3 and 5.
Thus, the Thy-1 difference between the OT-I cells and the C57BL/PL
recipients did not alter the response, consistent with the low
responder status of B6 mice to Thy-1 Ags 27 . OT-I effector CTL migrate out of the peritoneal cavity
The increasing numbers of OT-I cells in the LN and spleen after
day 4 concomitant with the decline in the peritoneal cavity suggested
that the cells might be migrating out of the peritoneal cavity.
Alternatively, a second wave of clonal expansion might occur in the LN
and spleen. Examination of the phenotype of the OT-I cells in spleen
and LN cells on day 10 showed the majority to have a phenotype
consistent with having been previously activated. The cells had high
CD44 and low L-selectin levels, but few were blasts as judged by FSC
(Fig. 6), and they did not have increased
CD25 levels (data not shown), indicating that they were not actively
proliferating. Similar results were obtained when OT-I cells from the
LN and spleen were examined on day 8 (data not shown).
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, E.G7). In contrast, OT-I cells placed
in the peritoneal cavities of mice that received PBS remained in the
peritoneal cavities (Fig. 7, PBS). These results are consistent with
the suggestion that OT-I cells respond to E.G7 in the peritoneal
cavity, control tumor growth transiently, but then migrate out of the
site of tumor growth and into the spleen and LN. Furthermore, the
retransferred CD8+ T cells did not proliferate in response
to tumor, as evidenced by their forward scatter (data not shown).
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Exit of activated OT-I cells from the site of tumor and resumed
tumor growth could occur if cytolysis by the OT-I CTL resulted in
selection of an Ag loss variant, E.G7 cells that no longer expressed
OVA. However, a Kb/OVA257–264-specific cloned
CTL line could kill E.G7 cells isolated on day 18 from the peritoneal
cavities of OT-I-transferred, E.G7-challenged mice (Fig. 8A), demonstrating that the
majority of E.G7 cells still express sufficient OVA peptide to act as
targets.
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B). When fresh OT-I cells were then injected i.v., a second
wave of OT-I expansion occurred in the peritoneal cavity that peaked
3–4 days after the cells were injected and then declined. Thus, the
cells freshly injected on day 8 responded identically to cells present
on day 0 at the time of the initial tumor challenge. Tumor growth was
transiently controlled during both the first and the second response by
OT-I cells (Fig. 8C).
A response also occurred when tumor was allowed to grow uncontrolled in
normal C57BL/PL mice (no adoptive transfer) for 8 days before i.v.
injection of the OT-I cells (Fig. 8B). Again, clonal
expansion of OT-I cells occurred in the peritoneal cavity 4 days after
injection, and tumor growth was controlled while OT-I cells were
present at the site of the tumor (Fig. 8C). Thus, even when
tumor load has reached a high level before introduction of the OT-I
cells, they are still able to respond by clonal expansion and to
transiently control further tumor growth.
OT-I cells from day 10 spleen have lytic effector function but are anergic
When cells were recovered from the spleens and LN of mice on day
10 following the response to tumor, and the CD8+ cells
(which included OT-I cells) were purified, they demonstrated potent
cytolytic activity for E.G7 tumor (Fig. 9A, OT-I/E.G7). In contrast,
killing of E.G7 was low by cells from mice that had received OT-I by
adoptive transfer but had not been challenged with tumor (Fig. 9A, OT-I) or from mice challenged with EL-4 tumor (Fig. 9A, OT-I/EL-4). For all populations there was essentially no
lysis of EL-4 tumor cells (Fig. 9B). Thus, the OT-I cells
that have migrated out of the peritoneal cavity retain Ag-specific
cytolytic activity and could presumably continue to control tumor
growth, but they are no longer in the right location to perform this
function.
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). This suggested that although
the OT-I cells on day 10 can still lyse target cells (Fig. 9), they may
no longer be able to proliferate in response to Ag. We therefore
examined whether cells recovered from LN and spleen on day 10 could
respond in an in vitro proliferation assay. Cells were taken from mice
that had received OT-I cells by adoptive transfer but not been
challenged with E.G7 (OT-I), mice that received OT-I cells and were
challenged with EL-4 (OT-I/EL-4), or mice that received OT-I cells and
were challenged with E.G7 (OT-I/E.G7).
Cells from OT-I or OT-I/EL-4 mice made a vigorous proliferative
response to C57BL/6 spleen cells pulsed with OVA257–264
peptide, as measured by [3H]TdR incorporation at 48
h (Fig. 10A). In contrast,
the OT-I cells from E.G7-challenged mice did not respond (Fig. 10A, OT-I/E.G7). Addition of exogenous IL-2 to the cultures
restored the response of the cells from the OT-I/E.G7 mice (Fig. 10A). IL-2 in the absence of peptide Ag stimulated some
response by the cells from the OT-I/E.G7 mice, but the response was
much stronger when Ag was also present. To confirm that the
[3H]TdR incorporation measured in these experiments
reflected the proliferative capacity of the OT-I cells, we also
determined the changes in absolute numbers of OT-I cells in the
cultures after 3 days (Fig. 10B). This analysis yielded the
same conclusion; cells from OT-I/E.G7 mice can respond in the presence
of exogenous IL-2, but not in its absence. Thus, the cells from the
OT-I/E.G7 mice display an apparent split anergy, able to lyse targets
but unable to undergo proliferation unless exogenous IL-2 is provided.
Induction of this state depends upon Ag recognition in vivo, since OT-I
cells from mice challenged with EL-4 tumor respond without IL-2
addition.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When adoptively transferred mice are challenged by i.p. injection of
EG.7, OT-I CD8+ T cells migrate to the peritoneal cavity
within 3 days, undergo substantial clonal expansion, develop lytic
activity, and control tumor growth over the next 2 days (Figs. 3 and 5). Thus, the OT-I cells do not remain ignorant and are not tolerant to
the Ag; in fact, they make a vigorous response. By day 6, however, the
CTL are leaving the peritoneal cavity and migrating to the LN and
spleen. They continue to have lytic activity through at least day 10
(Fig. 9), but are ineffective because they are not present in the
peritoneal cavity where the tumor has resumed rapid expansion. Failure
to remain at the site and control tumor growth is not due to the
appearance of Ag loss variants, since tumor cells isolated following
the transient CTL response still express Ag and can be killed by the
CTL (Fig. 8A).
Failure to sustain a CTL response at the site of tumor also does not
appear to be due to development of blocking factors or cells with
suppressor activity, since these would be expected to prevent a
response when fresh OT-I cells are transferred into the animal, but
instead a vigorous response occurs that is essentially identical with
the initial response (Fig. 8, B and C). Thus, the
environment present in the tumor-bearing host by day 8 and beyond is
not inhibitory to the generation of an Ag-specific CD8+ T
cell response and does not prevent lytic effector cells from being
active in the peritoneal cavity, nor is the tumor resistant to control
by the CTL that are generated.
The first, somewhat surprising, observation in this system was that
full activation of the OT-I cells, as assessed by CD25 up-regulation
and blast transformation, was first detected in the peritoneal cavity
at the site of tumor rather than in draining LN (Fig. 4 and data not
shown). In fact, most of the clonal expansion that was observed
occurred in the peritoneal cavity, with the cells subsequently
migrating to the LN and spleen (Fig. 7) where they expressed a high
level of CD44, indicating that they had already responded to Ag, but
where few were blasts. Failure to detect full activation and expansion
in the draining LN was unexpected. However, although blast
transformation was not detected in LN at early times, the level of
VLA-4 expression on the OT-I cells did increase in an Ag-dependent
manner (data not show). This occurred at the same time that OT-I cells
were decreasing in number in the spleen and LN and appearing in the
peritoneal cavity. Similar changes were seen when examining 2C TCR
transgenic CD8+ T cells responding to allogeneic tumor
challenge in the peritoneal cavity 14 . These observations suggest the
hypothesis that recognition of Ag by naive CD8+ T cells
results initially in altered expression of receptors involved in
trafficking, including VLA-4, and that this promotes selective
migration of the Ag-specific cells to the peripheral site of tumor
growth where they become fully activated. This could potentially
explain the somewhat paradoxical observation that expression of B7-1 on
a tumor cell enhances the CD8+ T cell response to the
tumor, but that the responding T cells are restricted only by class I
expressed by host APC; T cells specific for class I expressed just by
the tumor make only a small contribution 28 . Ag-specific
CD8+ T cells may need to recognize Ag presented by host APC
in the draining LN, i.e., by cross-priming, so that they then
migrate to the tumor site and undergo potent activation and expansion
in response to the tumor expressing the B7-1 costimulatory ligand. Most
T cells specific for class I on the tumor that is not expressed by the
host APC may never gain access to the tumor.
Although the EG.7 tumor used in these experiments does not express B7 ligands, vigorous clonal expansion of OT-I cells occurs. This may involve cross-priming 28, 29 . Alternatively, ICAM expressed by the tumor may provide sufficient LFA-1-dependent costimulation to support proliferation 30, 31 . Host CD4+ Th cells do not appear to be involved, since the OT-I response through at least day 10 is essentially identical in recipients that have been depleted of CD4+ T cells by in vivo anti-CD4 mAb administration (data not shown).
Following the peak of clonal expansion on day 4, the number of OT-I
cells declines in the peritoneal cavity (Fig. 3). This is probably due
in part to cell death; by using mAb staining for annexin V 32 , about
30% of OT-I cells in the peritoneal cavity were found to be positive
on days 4–6 (data not shown). It is also clear, however, that much of
the decline in number of effector cells at the tumor site is due to
migration out of the peritoneal cavity into LN and spleen (Fig. 7).
Additional experiments have shown that migration out of the peritoneal
cavity is not Ag specific; activated OT-I cells placed in the
peritoneal cavities of mice that have been injected i.p. with either
EL-4 tumor or LPS also leave and appear in the LN and spleen (P.
Shrikant and M. F. Mescher, manuscript in preparation). Thus, it
appears that exit from the peritoneal cavity requires inflammation, but
not recognition of specific Ag by the OT-I cells. OT-I cells placed in
the peritoneal cavities of E.G7-bearing mice can be found in LN and
spleen as early as 2 days after injection (data not shown).
Why do the active lytic effector OT-I cells leave the peritoneal cavity
while tumor cells are still present in large numbers? This is not
because the tumor no longer expresses Ag (Fig. 8). It also does not
appear to be due to the tumor creating an environment that either
prevents T cells from entering the peritoneal cavity or causes them to
leave rapidly, since naive OT-I transferred late can migrate there,
respond, and again transiently control tumor growth (Fig. 8, B and C). The response of 2C CD8+ T
cells to allogeneic tumor in the peritoneal cavity shows almost
identical kinetics of clonal expansion followed by exit from the
peritoneal cavity 14 as does the response of OT-I cells to EG.7, the
difference being that allogeneic tumor has been eliminated by the time
the CTL leave, while the syngeneic tumor is still present in large
numbers.
Taken together, these observations raise the possibility that the CTL developmental program may dictate this behavior and that it is largely Ag independent once the initial activation has occurred. What regulates the location of the CTL is unclear. One possibility is that naive or recently activated CD8+ T cells home very effectively to an inflammatory site, resulting in their accumulation in the tumor-bearing peritoneal cavity, but that within a short time of gaining effector function the cells lose this ability. Thus, once they exit the peritoneal cavity they would be unable to efficiently reenter the site. Another possibility is that Ag recognition during the initial activation results in the cells being held at the site of the tumor, perhaps through constitutive up-regulation of adhesion receptors. Subsequent down-regulation would then allow the cells to release and exit the site; the effector cells might continue to migrate through the peritoneal cavity, but fail to accumulate there after the initial activation period. Consistent with this suggestion, we have found that CD8+ T cells display constitutively active binding to ICAM-1 and fibronectin within about 24 h of in vitro stimulation with Ag, and this disappears by about 72 h when the cells develop effector function (J. M. Curtsinger, D. Lins, and M. F. Mescher, manuscript in preparation).
Whatever the mechanism responsible, it is clear that failure of the cytolytic effector cells to remain at the site of the tumor is the major limitation in the ability of the CD8+ T cell response to control tumor growth. A similar phenomenon may occur for CD8+ T cell responses to human tumors. In recently reported trials 33 , administration of melanoma-specific peptides resulted in increased Ag-specific reactivity of cells from the peripheral blood of the majority of patients, but few tumor responses were seen. In contrast, administering peptide along with IL-2 had little effect on the reactivity of peripheral blood cells, but induced tumor responses in a significant fraction of the patients. The investigators suggested that IL-2 administration might cause the Ag-specific effector CTL to traffic to the tumor site, while in its absence increased numbers of CTL are still generated but are ineffective because they leave the tumor site. We have, in fact, found that IL-2 administration at the appropriate time can cause OT-I cells to remain in the peritoneal cavity in large numbers and control tumor growth for longer times than in the absence of IL-2 (P. Shrikant and M. F. Mescher, unpublished observations).
The OT-I cells that have responded to tumor and then exited the
peritoneal cavity are anergic, in that they cannot respond to Ag by
proliferating unless exogenous IL-2 is provided (Fig. 10). This
somewhat resembles the split anergy described for cloned
CD8+ T cell lines 34 , where a signal 1 stimulus is
sufficient to mediate cytolysis but renders the cells unresponsive to
subsequent costimulation. It also resembles the anergy described for
CD4+ T cells 35, 36, 37 . In both of those cases, however, the
cell are rendered anergic when they recognize Ag in the absence of
costimulation and fail to make an initial response. In contrast, the
results described here demonstrate that OT-I cells become nonresponsive
following a vigorous initial response to the EG.7 tumor. This induction
of anergy may not be unique to tumor Ag, since CD8+ T cell
nonresponsiveness has also been observed following in vivo stimulation
with peptides and superantigen 38, 39, 40, 41, 42 . Whether activation-induced
nonresponsiveness limits the ability of CD8+ T cells to
control tumor growth is unclear, given that the cells are not at the
site of Ag in any case.
Being able to visualize the response of Ag-specific CD8+ T cells to a tumor has provided some novel insights into the factors that lead to the immune system failing to control tumor growth. In the system studied here, this is clearly not a result of a failure of the CD8+ T cells to become activated. Rather, it is a failure of the activated cells to remain at the site of tumor growth, where they could continue to control or eliminate it. Thus, successful immunotherapy in this case would not require manipulations to initiate a response as this occurs anyway, but, rather, manipulations to sustain the response and, critically, to sustain it at the appropriate location.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for Immunology, Box 334 Mayo, 420 Delaware St. S.E., Minneapolis, MN 55455. E-mail address:
3 Abbreviations used in this paper: LN, lymph nodes; PE, phycoerythrin; VLA-4, very late antigen-4; FSC, forward scatter.
Received for publication September 1, 1998. Accepted for publication November 12, 1998.
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References |
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), E.G7 (
), or B6 spleen cells pulsed with OVA (x)).
After 48 h each well was pulsed with 1 µCi of
[3H]thymidine for 12 h, and the incorporation was
determined. Results are shown as the mean of triplicate
determinations. B, Cytolysis of E.G7 targets by OT-I
CTL. Purified CD8+ T cells from LN of OT-I mice were
stimulated for 3 days in vitro with B6 spleen cells pulsed with OVA and
then assayed for lytic activity in a 4-h 51Cr release assay
using 1 x 105 labeled EL-4 or E.G7 as targets. The
percent specific lysis was calculated as described in Materials
and Methods.
). Two animals from each group were sacrificed on
the indicated days after tumor challenge, and cells harvested from
draining and peripheral LN (A), spleen
(B), and peritoneal cavity (C) were
analyzed by flow cytometry to determine the total number of OT-I cells
(CD8+/Thy-1.2+) at each site. The results shown
are the mean ± SD of three separate experiments examining two
mice per group at each time.
