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Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production. Surprisingly, reducing the number of adoptively transferred CD8+ T cells led to a parallel reduction in the proportion of the activated cells that exhibited effector functions, suggesting that CTL differentiation was induced by the large numbers of activated CD8+ T cells and not the tumor environment. Provision of tumor-specific CD4+ helper cells provided the signals required to promote both the development of CTL effector functions and increased clonal expansion, resulting in tumor eradication. Considering that only small numbers of tumor-specific CD8+ T cells would be present in a conventional T cell repertoire, these data suggest that tumor growth alone may not provide the inflammatory signals necessary to support the development of CD8+ T cell effector functions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is of interest to compare these results with the consequence of the interaction of naive CD8+ T cells with self-Ags from normal tissue. In particular, our laboratory has extensively investigated the fate of naive clone 4 CD8+ T cells specific for a model Ag, influenza hemagglutinin (HA),4 ectopically expressed in the pancreatic 
cells of InsHA mice. T cells first become aware of the presence of HA when it is acquired by tissue resident dendritic cells (DCs) that constitutively cross-present tissue Ags in draining lymph nodes (LNs) (7, 8, 9). This process is part of the strategy used by the immune system to inform naive T cells circulating through the lymphoid organs of the Ags present in parenchymal tissues (10, 11). Under noninflammatory conditions, the DCs presenting these tissue Ags are not activated to express the level of costimulatory molecules and cytokines required for the activation of a productive immune response. T cells that recognize Ag on these quiescent DCs go through an abortive form of T cell activation that leads to deletion and peripheral tolerance (7, 8, 12, 13, 14, 15). Furthermore, recent studies suggest that CD8+ T cells undergoing peripheral tolerance do not develop effector functions such as cytokine production and cytolytic activity (8, 9, 15). In contrast, activation of the cross-presenting DCs, either through inflammatory signals delivered through the innate immune system as occurs during microbial or viral infection (16, 17) or through interaction with Ag-specific CD4+ T cells or anti-CD40 Abs (14, 15, 18, 19, 20), results in vigorous proliferation and development of effector function by the activated CD8+ T cells.
The discrepancy observed between the successful stimulation of CTL effector function by cross-presented tumor Ag vs the lack of effector function during the induction of peripheral self-tolerance would suggest that tumor growth may provide the inflammatory environment necessary to promote the development of effector function by the activated CD8+ T cells (21). In this report, we address the question of whether spontaneously arising tumors expressing HA as a tumor-associated Ag (TAA) are capable of promoting the type of inflammatory environment that is required for the development of effector function by clone 4 CD8+ T cells. To this end, we have followed the fate of clone 4 CD8+ T cells transferred into mice that spontaneously develop HA-expressing pancreatic cell tumors due to transgenic expression of both HA and the SV40 large T Ag under the control of the rat insulin promoter.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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InsHA-transgenic mice on the B10.D2 background have been previously described (22). Clone 4 TCR (23), RIP-Tag2 (24), and SFE TCR (25) mice were each backcrossed with B10.D2 mice for at least eight generations. Clone 4 TCR mice express a TCR specific for the HA518–526 epitope (Kd-HA) restricted by MHC class I H-2Kd. SFE TCR mice express a TCR specific for the HA111–119 epitope restricted by MHC class II I-Ed. The RIP-Tag2-HA line was generated by crossing RIP-Tag2 mice with InsHA mice. All RIP-Tag2-HA animals used in these experiments were Tag2+/– and HA+/+, and all InsHA animals were HA+/+. Ly5.1+/+ (also referred to as CD45.1+/+ or Ptprca/a) animals on the C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME) and backcrossed to B10.D2 for at least four generations before mating to clone 4 TCR mice. All animals were housed at The Scripps Research Institute’s animal facility, and all procedures were performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Cell lines
The SV40-transformed H-2d cell line B10.D2 was originally obtained from Dr. B. Knowles, (University of Pennsylvania, Philadelphia, PA). B10.D2 cells were maintained in complete RPMI medium-RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% (v/v) heat-inactivated FCS (Gemini Biological Products, Calabasas, CA), 2 mM glutamine (Life Technologies), 5 x 10–5 M 2-ME (Sigma-Aldrich, St. Louis, MO), and 50 mg/ml gentamicin (Gemini Bio-Products, Woodland, CA). Cells were cultured in a humidified incubator at 37°C with 5% (v/v) CO2 and were used as targets in CTL assays.
In vitro cytotoxicity assays
Mice were infected i.p. with 1200 HA units of influenza virus A/PR/8/34 H1N1 (PR8). After 3 wk, splenocytes from infected mice were restimulated with irradiated syngeneic splenocytes (3000 rad) pulsed with the Kd-restricted influenza nucleoprotein (NP) epitope, (TYQRTRALV), or Kd-HA (IYSTVASSL) peptide for 6 days in complete RPMI. Effector cells were then harvested and counted, and cytolytic activity against peptide-loaded (1 µM or 1 nM) or unloaded target cells was measured in a 5-h 51Cr release assay. Target cells were prepared by incubating B10.D2 cells at 37°C with 200 µCi sodium 51Cr (New England Nuclear, Boston, MA) for 1 h in the presence or the absence of Kd-NP or Kd-HA peptide as indicated. Target cells were washed four times, resuspended in complete RPMI, and seeded into 96-well round-bottom plates at 1 x 104 cells/well in 100 µl. Effector CTL were seeded into duplicate wells containing the target cells at various E:T cell ratios, making a final volume of 200 µl. Plates were incubated at 37°C in a humidified incubator with 5% (v/v) CO2 for 5 h. Plates were centrifuged, and 100 µl of supernatant was removed from each well to assess isotope release using an ICN Isomedic beta radiation counter (VWR Scientific, San Diego, CA). Percent specific lysis was calculated with the following formula: 100 x (sample release – spontaneous release)/(maximum release – spontaneous release).
Preparation and adoptive transfer of naive TCR-transgenic T cells
CD8+ T cells were isolated from the LNs of clone 4 TCR mice (6–8 wk of age) by negative selection using the MACS CD8+ T cell isolation kit (Miltenyi Biotec, Auburn, CA). T cell purity was >85% with no contaminating CD4+ cells. SFE-transgenic CD4+ T cells were similarly isolated with the MACS CD4+ T cell isolation kit (Miltenyi Biotec) with >95% purity and no contaminating CD8+ cells. Where indicated, purified clone 4 CD8+ T cells or SFE CD4+ T cells were labeled with CFSE (Molecular Probes, Eugene, OR). Briefly, cells were incubated in 5 µM CFSE in HBSS for 10 min at 37°C and then washed in cold HBSS before adoptive transfer. For adoptive transfer experiments, the indicated number of cells was injected through the tail vein in a volume of 200 µl of HBSS.
Preparation of target cells for in vivo CTL assays
In vivo CTL activity was measured as previously described (3, 26). Briefly, B10.D2 splenocytes were isolated and divided into two populations, one of which was pulsed with 1 µg/ml Kd-HA peptide (IYSTVASSL) for 60 min at 37°C. After two washes in HBSS, HA peptide-pulsed cells were labeled with 5 µM CFSE (CFSEhigh) for 15 min at 37°C. Unpulsed cells were labeled with 0.5 µM CFSE (CFSElow). Cells were washed and resuspended at a 1:1 ratio in HBSS at a total cell concentration of 3–5 x 107 cells/ml. Two hundred microliters of the target cell suspension was injected through the tail vein. In all experiments, cells were recovered from the spleen and/or LNs 16–18 h after transfer and analyzed by FACS to detect and quantify CFSE-labeled cells.
Intracellular cytokine staining
To assess the ex vivo production of IFN- in response to Ag, pancreatic lymph nodes (pLNs) were dissociated and cells were incubated in complete medium with 1 µg/ml Kd-HA peptide and 1 µl/ml GolgiPlug solution (BD PharMingen, San Diego, CA) for 5 h at 37°C. Cells were then incubated in 100 µl of 2.4G2 hybridoma supernatant (American Type Culture Collection, Manassas, VA) for 10 min at 4°C to block FcRs. PerCP-conjugated anti-CD8 and PE-conjugated anti-Thy1.2 (or anti-Ly5.1) were then added, and cells were incubated for an additional 30 min at 4°C. After two washes, intracellular IFN- staining was performed according to the manufacturer’s instructions using the Cytofix/Cytoperm Plus kit (BD PharMingen) and allophycocyanin-conjugated rat anti-mouse IFN-. Cells were analyzed on a BD Biosciences FACSort flow cytometer, and data were analyzed using CellQuest software. All Abs for flow cytometry were purchased from BD PharMingen.
Assessment of tumor growth/eradication by blood glucose monitoring
Mice were assessed for tumor growth (hypoglycemia) or tumor destruction (hyperglycemia) by weekly monitoring of blood glucose. Mice were considered hypoglycemic when blood glucose levels were below 70 mg/dl and were euthanized after consecutive blood glucose readings below 40 mg/dl.
Ab treatment
For transient depletion of endogenous CD4+ cells, mice were treated once i.p. with 100 µl of ascites fluid from GK1.5 cells (American Type Culture Collection) 5 days before adoptive transfer of clone 4 cells. Depletion of CD4+ cells was determined to be 100% by FACS analysis of peripheral blood at the time of adoptive transfer.
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Results |
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RIP-Tag2-transgenic mice express the SV40 large T Ag (Tag) under the control of the rat insulin promoter (24) and develop progressive hypoglycemia starting at 10–12 wk of age due to uncontrolled insulin production by growing cell tumors. These mice are tolerant to Tag, and do not mount a spontaneous immune response to the growing tumors (27). To generate mice that express HA as a tumor Ag, RIP-Tag2 mice were mated and backcrossed with InsHA mice to obtain mice that were Tag2+/– and HA+/+ (22). Mice homozygous for the HA transgene are profoundly tolerant due to both thymic and peripheral tolerance mechanisms (28, 29). To determine whether the expression of HA as a TAA could break tolerance and impede tumor growth, blood glucose was monitored weekly in RIP-Tag2-HA animals to assess tumor progression. Although control InsHA mice maintained normal glucose levels over time, RIP-Tag2-HA mice consistently became hypoglycemic between 10 and 12 wk of age (Fig. 1A). Also, development of hypoglycemia (tumor growth) occurred with identical kinetics in RIP-Tag2 and RIP-Tag2-HA mice (data not shown). Thus, expression of HA on growing cell tumors did not appear to trigger a spontaneous, protective antitumor response. No infiltration was observed in hyperplastic islets of these mice (data not shown), further supporting a lack of tumor-specific immunity.
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Development of effector function by CD8+ clone 4 T cells correlates with tumor burden
Our previous studies revealed that clone 4 CD8+ T cells undergoing tolerance in the pLNs of InsHA recipients do not develop effector function (8). To determine whether clone 4 cells develop effector function in response to HA expressed by tumor cells, 3 x 106 clone 4 CD8+ T cells were labeled with CFSE and transferred into B10.D2 mice, InsHA mice, or RIP-Tag2-HA mice with small (6 wk of age) or large (10 wk of age) tumor burdens. Four days later, pLN cells were harvested and stimulated in vitro for 5–6 h with the HA peptide, followed by intracellular staining for IFN- (Fig. 2A). As previously observed, clone 4 cells transferred into InsHA mice showed little proliferation and minimal IFN- production (4.8% of dividing clone 4 cells). In contrast, clone 4 T cells from both 6- and 10-wk-old RIP-Tag2-HA recipients showed increased proliferation and a significantly higher proportion of IFN--producing cells. Furthermore, the levels of proliferation and IFN- production appeared to increase with increasing tumor burden (48.3% IFN-+ in 10-wk-old mice vs 23.6% IFN-+ in 6-wk-old mice with smaller tumors). As expected, clone 4 cells transferred into control B10.D2 mice did not proliferate. It should be noted that we consistently observe low levels of IFN- production by undivided clone 4 cells upon ex vivo stimulation (see Figs. 2A and 3). The basis for IFN- secretion by undivided cells is not known and the use of RAG–/– clone 4 CD8+ T cells or CD44low sorted clone 4 cells did not diminish this IFN- secretion (data not shown).
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Clone 4 differentiation into effector cells is not dependent on endogenous CD4 help, but is dependent on the number of adoptively transferred clone 4 cells
Development of effector function by Ag-specific T cells is thought to require Ag presentation by an activated DC that can provide signal 1 (TCR stimulation), signal 2 (costimulation), and a third signal in the form of IL-12 or other inflammatory cytokines (9, 30). Quiescent DCs, on the other hand, provide signal 1 in the absence of sufficient costimulation and other accessory signals and do not efficiently promote T cell effector function (13, 14). The observed differentiation of clone 4 CD8+ cells into effector cells in RIP-Tag2-HA mice suggested that the cross-presenting DCs responsible for the presentation of HA may have been activated by an inflammatory environment, presumably established by an innate immune response to tumor growth. However, another possible explanation for the observed development of effector function by the clone 4 cells in RIP-Tag2-HA animals was that the mice may contain residual endogenous tumor-specific CD4+ T cells. In previous studies we showed that when cotransferred into InsHA recipients with clone 4 CD8+ T cells, activated HA-specific CD4+ helper cells could promote the induction of clone 4 effector function (11). To test this possibility, endogenous CD4+ T cells were depleted from RIP-Tag2-HA recipients before clone 4 transfer and proliferating clone 4 CD8+ cells were analyzed for IFN- production. As shown in Fig. 3A, depletion of CD4+ T cells did not lead to diminished clone 4 effector function. Surprisingly, we observed increased proliferation and IFN- production in the absence of the endogenous CD4+ T cells.
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. Surprisingly, dilution of the number of transferred T cells resulted in a progressive decrease in the overall percentage of dividing cells capable of producing IFN- (Fig. 3B). Also, when cells that had undergone the same number of divisions were compared between the three different groups, the percentage of IFN--producing clone 4 cells progressively increased in RIP-Tag2-HA recipients that received higher numbers of transferred cells (see Fig. 3 legend). Furthermore, in subsequent experiments where even lower numbers of clone 4 cells (
1 x 105) were transferred, IFN- production was even further diminished to 0.8–5.1% of proliferating cells (see Fig. 6A and data not shown). Thus, naive clone 4 CD8+ T cells develop into cytokine-producing effectors in response to cross-presented tumor Ag only when large numbers are transferred. These data suggest that, in the RIP-Tag2 model, tumor growth does not provide the inflammatory environment required to activate resident DCs and promote development of CD8+ T cell effector functions, as we would expect both high and low numbers of CD8+ clone 4 cells to develop effector function in such an inflammatory setting.
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Cross-presentation of self-Ags in a noninflammatory environment results in the tolerance of Ag-specific CD8+ T cells, either via deletion or functional inactivation (10). Since the growing tumors did not appear to induce inflammatory signals in the RIP-Tag2-HA mice, we hypothesized that clone 4 T cells should become tolerized in the tumor-bearing recipients as previously observed in InsHA recipients (7). To test for tolerance, clone 4 CD8+ T cells were adoptively transferred into RIP-Tag2-HA mice, tumor-free InsHA mice, or B10.D2 control mice. Tolerance induction was then assessed over time by determining whether the mice retained clone 4 cells that could respond to secondary Ag challenge in the form of influenza PR8 infection. In the first experiment, RIP-Tag2-HA recipients received 2.5 x 103 clone 4 CD8+ T cells and were infected with PR8 at 1, 3, or 5 wk posttransfer to expand any remaining (nontolerized) clone 4 cells. Five days after virus infection, in vivo CTL activity against the Kd-HA peptide was assessed (Fig. 4A). As expected based on our previous studies, T cells were completely tolerized after 5 wk in InsHA mice, as indicated by a lack of in vivo CTL activity against the injected HA peptide-loaded target cells (CFSEhigh) as compared with equivalent numbers of unloaded cells (CFSElow). Furthermore, clone 4 T cells were also tolerized in RIP-Tag2-HA mice (Fig. 4A). In fact, tolerance occurred more rapidly in these tumor-bearing recipients, since HA-specific CTL activity was eliminated by 3 wk posttransfer. This is consistent with our previous observation that the rate of tolerance is directly proportional to the amount of available cross-presented Ag (7). It should be noted that clone 4 T cells were not tolerized in control B10.D2 mice, since high levels of lytic activity against HA-loaded targets were observed after virus infection at all time points (data not shown).
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Clone 4 CD8+ T cells cannot protect mice from tumor progression (hypoglycemia) without cotransfer of tumor Ag-specific CD4+ helper T cells
Since the transfer of larger numbers of clone 4 cells successfully led to the development of effector CTL, it was of interest to determine whether these CTL could protect RIP-Tag2-HA animals from tumor development. Therefore, 3 x 106 clone 4 cells were transferred into RIP-Tag2-HA recipients at 8–9 wk of age and mice were monitored weekly for tumor growth by assessing blood glucose levels (Fig. 5B). Although some mice became transiently hyperglycemic, indicating antitumor activity by activated clone 4 cells, all animals eventually developed severe hypoglycemia with kinetics similar to those of untreated RIP-Tag2-HA mice (Fig. 5, A and B). Transfer of up to 1 x 107 clone 4 CD8+ cells was also unable to provide lasting protection against cell tumor growth (data not shown). Thus, despite development of effector function, clone 4 CD8+ T cells alone were unable to eradicate tumors.
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CD4+ T cell help leads to a significant increase in tumor-specific clone 4 effector function and accumulation
Considering that the provision of CD4 help in the form of naive SFE cells had such a dramatic impact on the ability of clone 4 CD8+ T cells to eliminate growing tumors, we thought it likely that SFE cells were either enhancing the development of effector function by clone 4 cells and/or promoting the accumulation of activated clone 4 cells. To address this issue, clone 4 cells were transferred into RIP-Tag2-HA recipients in the presence or absence of SFE helper cells and assessed for their ability to produce IFN- ex vivo 4 days later (Fig. 6A). As described earlier, transfer of a low number (1 x 105) of clone 4 CD8+ cells alone resulted in proliferation, yet only 
5% of the proliferating cells produced IFN-. In contrast, cotransfer of 3 x 106 SFE helper cells led to a dramatic increase in effector function development, as nearly 60% of proliferating cells produced IFN- upon short-term in vitro stimulation. In addition, roughly 3-fold more Ly5.1+ clone 4 cells were recovered from the draining LN of mice receiving SFE CD4+ T cell help (Fig. 6B). Thus, provision of help increased both the accumulation and the effector function of clone 4 CD8+ T cells in response to the cross-presented HA tumor Ag. As expected, development of clone 4 effector function correlated with the number of SFE helper cells cotransferred (Fig. 6A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by the activated T cells. In the current study, we wished to determine whether the tumor environment provided the type of inflammatory milieu that was necessary to activate the Ag-presenting DCs and thereby promote development of TAA-specific CD8+ T cell effector function. Initially, our experiments suggested that the tumor environment in the RIP-Tag2-HA mice was sufficient to promote an immune response, as adoptively transferred clone 4 CD8+ T cells demonstrated considerable production of IFN- and developed into CTL (Fig. 2) capable of significant levels of islet cell destruction (Fig. 5). Clone 4 effector function was not dependent on endogenous CD4+ T cell help, as elimination of all CD4+ cells actually enhanced the activation and IFN- production by clone 4 CD8+ T cells in RIP-Tag2-HA mice. This suggests that the mice may harbor a population of CD4+ cells that can inhibit the development of CD8+ T cell effector functions. Several candidate suppressor cell populations have already been described in the literature, including CD4+ NKT cells (36) and CD4+CD25+ regulatory T cells (37, 38, 39), both of which have been shown to inhibit tumor-specific T cell activity. Alternatively, depletion of CD4+ T cells in the RIP-Tag2-HA recipient mice could allow for the enhanced clone 4 activation due to increased expansion under lymphopenic conditions.
Having ruled out the possibility that tumor-specific CD4+ T cells were responsible for the development of effector function by the clone 4 CD8+ T cells, we next considered the possibility that the CD8+ T cells themselves may be responsible for promoting the development of their effector function. Several reports in the literature have shown that large numbers of activated CD8+ T cells can provide their own help by directly activating DCs (31, 34). Such activation was dependent on the amount of Ag and the numbers of cognate CD8+ T cells (34). To determine whether this was responsible for the development of effector function in RIP-Tag2-HA mice, we tested whether the ability to produce IFN- was dependent on the number of injected CD8+ T cells. Surprisingly, we observed a direct correlation between the number of adoptively transferred clone 4 cells and the proportion of cells that could produce IFN-. This implies that it was not the tumor environment, but rather the activated CD8+ T cells that were responsible for the development of CTL effector function. Based on these data, we conclude that in the RIP-Tag2 model the growing tumor does not induce the type of inflammatory environment that is necessary to activate DCs and promote a productive immune response.
We next addressed the question of whether the adoptively transferred clone 4 CD8+ T cells are eliminated in RIP-Tag2-HA mice. We previously devised an assay that monitors tolerance of clone 4 CD8+ T cells in InsHA recipients by following the rate of elimination of HA-responsive cells in InsHA recipients. Small numbers of clone 4 CD8+ cells were transferred into groups of InsHA mice that were subsequently infected with influenza at progressive time points to determine how long it takes for elimination of responsive clone 4 cells. The absence of cells that can respond to influenza virus is taken as an indication of their tolerance and deletion. However, we do not rule out the possibility that small numbers of tolerant CD8+ T cells may persist that are unable to clonally respond to influenza and therefore may be below our level of detection. Two different methods were used to detect residual, influenza HA-responsive clone 4 CD8+ T cells, an in vivo CTL assay and direct detection by flow cytometry. Both assays indicated that elimination of virus-responsive cells occurred in both InsHA and RIP-Tag2-HA mice. In fact, it appeared that elimination of responsive cells occurred somewhat more rapidly in tumor-bearing RIP-Tag2-HA recipient mice than in InsHA recipients. This was not unexpected since there is more Ag available for cross-tolerance in tumor-bearing mice, and we showed previously that the rate at which cells were eliminated correlated directly with the expression level of the cross-presented Ag (7).
Our findings that CD8+ T cells do not develop effector function and are eliminated by tolerance in tumor-bearing mice contrasts with the results of Ohashi and coworkers (5, 40) who also used RIP-Tag2 mice to study the fate of CD8+ T cells in tumor-bearing mice. They used the lymphocytic choriomeningitis virus glycoprotein (GP) as a TAA and GP-specific P14 TCR-transgenic cells as the tumor-specific CD8+ T cell population. They concluded from their studies that P14 T cells develop effector function and are not tolerized in RIP(GP x Tag2) recipients. The different conclusions drawn from their study is likely due to several differences between their experiments and ours. First, they used very large numbers (5 x 106) of adoptively transferred T cells, a number that we too find leads to development of CTL and tumor destruction. The basis for their observed lack of tolerance and development of effector function could also be due to a fundamental difference between the GP and HA models. In both the RIP-GP and the RIP(GP x Tag2) mice, there remain substantial numbers of endogenous T cells that are responsive to GP (40, 41). This contrasts with InsHA and RIP-Tag2-HA mice, both of which are profoundly tolerant to HA. The reasons for the differences between the two models are not known, but the remaining endogenous GP-specific CD4+ and/or CD8+ T cells could prevent the tolerance of P14 T cells in their tumor model.
The fact that low numbers of transferred clone 4 CD8+ T cells undergo activation but do not differentiate into effector CTL (Fig. 6A) suggests that, at least in the RIP-Tag2-HA model, tumor growth does not promote the activation of the DCs that cross-present tumor Ags. However, we show that this activation signal can be exogenously provided by SFE CD4+ helper T cells or by large numbers of clone 4 CD8+ T cells. Although DC activation by CD4+ T cells is thought to occur through CD40-CD40 ligand interactions, the mechanism for CD8+ T cell-mediated activation of DCs is not known (31, 34). In both situations though, T-DC interaction leads to the up-regulation of costimulatory molecules and cytokines by the cross-presenting DC (34), which is likely critical for CTL differentiation in the absence of other inflammatory signals. Indeed, we have observed that when CD28-deficient clone 4 CD8+ T cells are transferred into RIP-Tag2-HA recipients, even in large numbers (3 x 106), they do not exhibit effector function (M. Lyman, unpublished observation).
The transfer of large numbers of clone 4 CD8+ T cells into RIP-Tag2-HA mice led to the development of CTL effector function and detectable in vivo antitumor activity (transient hyperglycemia), but was ultimately unable to prevent tumor outgrowth. In contrast, cotransfer of HA-specific CD4+ T cells promoted rapid tumor eradication. It is often assumed that CD8+ T cells are efficiently primed by growing tumors, but are unable to survive due to Ag-induced cell death or "exhaustion" (42). Previous studies have demonstrated an important role for CD4+ T cells in preventing such CTL exhaustion (43, 44). In addition, recent studies have revealed a requirement for CD4 help in the establishment of CD8+ memory T cells (45, 46, 47, 48). Thus, it has been suggested that a critical role of CD4 help in antitumor responses is enhancement of CTL survival and maintenance of CTL effector function in the face of chronic exposure to tumor Ags (3, 49). We show here that in addition to their proposed role in CTL maintenance, activated tumor-specific CD4+ T cells are critical for the early accumulation and development of effector function by responding CTL, especially when the pool of responding CD8+ T cells is limiting and there are no other inflammatory signals available to activate the DCs. These helper effects may be exerted by the cross-presenting DC and through CD4+-CD8+ T cell interactions (50). It is also possible that the adoptively transferred SFE helper cells contribute directly to tumor eradication through their production of effector cytokines (51).
In conclusion, our data present evidence for a model in which tumor growth leads to efficient tolerance of tumor-specific CD8+ T cells without the development of CD8+ T cell effector function. The simplest explanation for these results is that the tumor environment does not promote the activation of the cross-presenting DCs. In support of this, we find evidence for the development of effector function when large numbers of CD8+ T cells simultaneously undergo activation, a situation in which it has been shown that CD8+ T cells can promote DC activation and, accordingly, provide their own help. However, we hesitate to conclude that our results imply the absence of immune surveillance until we learn more about the early events that occur during tumor establishment. A successful tumor may be responsible for the induction of anti-inflammatory molecules that inhibit DC activation (21). Indeed, the observation that removal of CD4+ T cells from tumor-bearing mice results in enhanced activation and effector function by the CD8+ T cells hints at the layers of complexity that are involved.
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Acknowledgments |
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This is manuscript number 16108 from the Department of Immunology at TSRI.
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Footnotes |
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2 Current address: Cell Genesys, Inc., South San Francisco, CA 94080.
3 Address correspondence and reprint requests to Dr. Linda A. Sherman, Department of Immunology, The Scripps Research Institute, IMM-15, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: lsherman{at}scripps.edu
4 Abbreviations used in this paper: HA, influenza hemagglutinin; RIP, rat insulin promoter; Tag, SV40 large T Ag; TAA, tumor-associated Ag; LN, lymph node; pLN, pancreatic lymph node; DC, dendritic cell; NP, nucleoprotein.
Received for publication October 8, 2003. Accepted for publication March 22, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 315:115.[Medline]
cells. Nature 325:223.[Medline]
) monoclonal antibody. Cancer Res. 59:3128.This article has been cited by other articles:
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  G. Verdeil, K. Marquardt, C. D. Surh, and L. A. Sherman Adjuvants targeting innate and adaptive immunity synergize to enhance tumor immunotherapy PNAS, October 28, 2008; 105(43): 16683 - 16688. [Abstract] [Full Text] [PDF]
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 P. Schuler, E. Contassot, M. Irla, S. Hugues, O. Preynat-Seauve, F. Beermann, A. Donda, L.E. French, and B. Huard Direct Presentation of a Melanocyte-Associated Antigen in Peripheral Lymph Nodes Induces Cytotoxic CD8+ T Cells Cancer Res., October 15, 2008; 68(20): 8410 - 8418. [Abstract] [Full Text] [PDF]
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A. Bai, E. Higham, H. N. Eisen, K. D. Wittrup, and J. Chen From the Cover: Rapid tolerization of virus-activated tumor-specific CD8+ T cells in prostate tumors of TRAMP mice PNAS, September 2, 2008; 105(35): 13003 - 13008. [Abstract] [Full Text] [PDF]
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 G. Willimsky, M. Czeh, C. Loddenkemper, J. Gellermann, K. Schmidt, P. Wust, H. Stein, and T. Blankenstein Immunogenicity of premalignant lesions is the primary cause of general cytotoxic T lymphocyte unresponsiveness J. Exp. Med., July 7, 2008; 205(7): 1687 - 1700. [Abstract] [Full Text] [PDF]
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 M. Y. Gerner, K. A. Casey, and M. F. Mescher Defective MHC Class II Presentation by Dendritic Cells Limits CD4 T Cell Help for Antitumor CD8 T Cell Responses J. Immunol., July 1, 2008; 181(1): 155 - 164. [Abstract] [Full Text] [PDF]
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G. Plesa, A. E. Snook, S. A. Waldman, and L. C. Eisenlohr Derivation and Fluidity of Acutely Induced Dysfunctional CD8+ T Cells J. Immunol., April 15, 2008; 180(8): 5300 - 5308. [Abstract] [Full Text] [PDF]
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S. B. J. Wong, R. Bos, and L. A. Sherman Tumor-Specific CD4+ T Cells Render the Tumor Environment Permissive for Infiltration by Low-Avidity CD8+ T Cells J. Immunol., March 1, 2008; 180(5): 3122 - 3131. [Abstract] [Full Text] [PDF]
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M. Long, A. M. Slaiby, S. Wu, A. T. Hagymasi, M. A. Mihalyo, S. Bandyopadhyay, A. T. Vella, and A. J. Adler Histone Acetylation at the Ifng Promoter in Tolerized CD4 Cells Is Associated with Increased IFN-{gamma} Expression during Subsequent Immunization to the Same Antigen J. Immunol., November 1, 2007; 179(9): 5669 - 5677. [Abstract] [Full Text] [PDF]
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M. J. Anderson, K. Shafer-Weaver, N. M. Greenberg, and A. A. Hurwitz Tolerization of Tumor-Specific T Cells Despite Efficient Initial Priming in a Primary Murine Model of Prostate Cancer J. Immunol., February 1, 2007; 178(3): 1268 - 1276. [Abstract] [Full Text] [PDF]
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L. A. Johnson, B. Heemskerk, D. J. Powell Jr., C. J. Cohen, R. A. Morgan, M. E. Dudley, P. F. Robbins, and S. A. Rosenberg Gene Transfer of Tumor-Reactive TCR Confers Both High Avidity and Tumor Reactivity to Nonreactive Peripheral Blood Mononuclear Cells and Tumor-Infiltrating Lymphocytes J. Immunol., November 1, 2006; 177(9): 6548 - 6559. [Abstract] [Full Text] [PDF]
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P. Otahal, T. D. Schell, S. C. Hutchinson, B. B. Knowles, and S. S. Tevethia Early Immunization Induces Persistent Tumor-Infiltrating CD8+ T Cells against an Immunodominant Epitope and Promotes Lifelong Control of Pancreatic Tumor Progression in SV40 Tumor Antigen Transgenic Mice. J. Immunol., September 1, 2006; 177(5): 3089 - 3099. [Abstract] [Full Text] [PDF]
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E. Ambrosino, M. Spadaro, M. Iezzi, C. Curcio, G. Forni, P. Musiani, W.-Z. Wei, and F. Cavallo Immunosurveillance of erbb2 carcinogenesis in transgenic mice is concealed by a dominant regulatory T-cell self-tolerance. Cancer Res., August 1, 2006; 66(15): 7734 - 7740. [Abstract] [Full Text] [PDF]
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R. G. van der Most, A. Currie, B. W.S. Robinson, and R. A. Lake Cranking the Immunologic Engine with Chemotherapy: Using Context to Drive Tumor Antigen Cross-Presentation towards Useful Antitumor Immunity Cancer Res., January 15, 2006; 66(2): 601 - 604. [Abstract] [Full Text] [PDF]
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A. I. Garbe, B. Vermeer, J. Gamrekelashvili, R. v. Wasielewski, F. R. Greten, A. M. Westendorf, J. Buer, R. M. Schmid, M. P. Manns, F. Korangy, et al. Genetically Induced Pancreatic Adenocarcinoma Is Highly Immunogenic and Causes Spontaneous Tumor-Specific Immune Responses Cancer Res., January 1, 2006; 66(1): 508 - 516. [Abstract] [Full Text] [PDF]
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Z. F. H. M. Boonman, G. J. D. van Mierlo, M. F. Fransen, R. J. W. de Keizer, M. J. Jager, C. J. M. Melief, and R. E. M. Toes Maintenance of Immune Tolerance Depends on Normal Tissue Homeostasis J. Immunol., October 1, 2005; 175(7): 4247 - 4254. [Abstract] [Full Text] [PDF]
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X. Martinez, H. T. C. Kreuwel, W. L. Redmond, R. Trenney, K. Hunter, H. Rosen, N. Sarvetnick, L. S. Wicker, and L. A. Sherman CD8+ T Cell Tolerance in Nonobese Diabetic Mice Is Restored by Insulin-Dependent Diabetes Resistance Alleles J. Immunol., August 1, 2005; 175(3): 1677 - 1685. [Abstract] [Full Text] [PDF]
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L. A. O'Mara, L. A. Norian, D. Kreamalmeyer, J. M. White, and P. M. Allen T Cell-Mediated Delay of Spontaneous Mammary Tumor Onset: Increased Efficacy with In Vivo versus In Vitro Activation J. Immunol., April 15, 2005; 174(8): 4662 - 4669. [Abstract] [Full Text] [PDF]
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M. A. Lyman, C. T. Nugent, K. L. Marquardt, J. A. Biggs, E. G. Pamer, and L. A. Sherman The Fate of Low Affinity Tumor-Specific CD8+ T Cells in Tumor-Bearing Mice J. Immunol., March 1, 2005; 174(5): 2563 - 2572. [Abstract] [Full Text] [PDF]
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M.-L. Chen, M. J. Pittet, L. Gorelik, R. A. Flavell, R. Weissleder, H. von Boehmer, and K. Khazaie Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-{beta} signals in vivo PNAS, January 11, 2005; 102(2): 419 - 424. [Abstract] [Full Text] [PDF]
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G. Zhou, Z. Lu, J. D. McCadden, H. I. Levitsky, and A. L. Marson Reciprocal Changes in Tumor Antigenicity and Antigen-specific T Cell Function during Tumor Progression J. Exp. Med., December 20, 2004; 200(12): 1581 - 1592. [Abstract] [Full Text] [PDF]
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