Signal 3 Availability Limits the CD8 T Cell Response to a Solid Tumor

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1 This information is current as of July 8, Signal 3 Availability Limits the CD8 T Cell Response to a Solid Tumor Julie M. Curtsinger, Michael Y. Gerner, Debra C. Lins and Matthew F. Mescher J Immunol 2007; 178: ; ; doi: /jimmunol References Subscription Permissions Alerts This article cites 45 articles, 32 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on July 8, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2007 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 The Journal of Immunology Signal 3 Availability Limits the CD8 T Cell Response to a Solid Tumor 1 Julie M. Curtsinger, 2 Michael Y. Gerner, Debra C. Lins, and Matthew F. Mescher CD8 T cells need a third signal, along with Ag and costimulation, for effective survival and development of effector functions, and this can be provided by IL-12 or type I IFN. Adoptively transferred OT-I T cells, specific for H-2K b and OVA, encounter Ag in the draining lymph nodes of mice with the OVA-expressing E.G7 tumor growing at a s.c. site. The OT-I cells respond by undergoing limited clonal expansion and development of effector functions (granzyme B expression and IFN- production), and they migrate to the tumor where they persist but fail to control tumor growth. In contrast, OT-I T cells deficient for both the IL-12 and type I IFN receptors expand only transiently and rapidly disappear. These results suggested that some signal 3 cytokine is available, but that it is insufficient to support a CTL response that can control tumor growth. Consistent with this, administration of IL-12 at day 10 of tumor growth resulted in a large and sustained expansion of wild-type OT-I cells with enhanced effector functions, and tumor growth was controlled. This did not occur when the OT-I cells lacked the IL-12 and type I IFN receptors, demonstrating that the therapeutic effect of IL-12 results from direct delivery of signal 3 to the CD8 T cells responding to tumor Ag in the signal 3-deficient environment of the tumor. The Journal of Immunology, 2007, 178: The immunotherapeutic approach to treatment of cancer is based on observations that immune responses to cancer cells can occur and that activated, tumor-specific T cells can cause tumor regression. Tumor-specific CD8 T cells have been isolated from the tumor, draining lymph nodes (DLN), 3 and blood of patients with a variety of types of cancer, yet in these cases the tumor is not being controlled by the immune response (1 7). Adoptive immunotherapy, using tumorinfiltrating lymphocytes that are expanded in vitro, has been the most successful immunotherapeutic approach thus far and gives reason for optimism that if immune responses can be activated in situ, tumor control could be achieved (8 13). However, although numerous means of activating immune responses in patients have been examined, they have had modest success rates, and none of these methods have approached the success rate achieved by adoptive immunotherapy (14). To determine the best way to activate a tumor-specific response in situ, it is essential to understand how tumors escape control by the immune system even when they express Ags that are known to stimulate an immune response. It is possible that tumors are ignored by the immune system either because T cells that can respond to tumor-associated Ag, which are often self-ag, have been deleted during development, or because tumor Ag is not processed Department of Laboratory Medicine and Pathology, Center for Immunology, University of Minnesota, Minneapolis, MN Received for publication January 8, Accepted for publication March 16, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant R01 CA (to M.F.M.) and National Cancer Institute Training Grant 2 T32 CA (to M.Y.G.). 2 Address correspondence to Dr. Julie M. Curtsinger, Center for Immunology, Box 334 Mayo, 420 Delaware Street SE, Minneapolis, MN address: curts001@umn.edu 3 Abbreviations used in this paper: DLN, draining lymph node; GzmB, granzyme B; wt, wild type; RKO, receptor knockout; DC, dendritic cell; Treg, regulatory T cell; AINR, Ag-induced nonresponsiveness; LN, lymph node. Copyright 2007 by The American Association of Immunologists, Inc /07/$2.00 and presented by the appropriate APCs. However, when CD8 T cells specific for tumor-associated Ag are identified in patients, they often have a phenotype that indicates prior Ag exposure, suggesting that an initial response to the tumor occurred but was not sufficient to control its growth (1, 2, 4 6, 15). Recently, it has been determined that for CD8 T cells to be fully activated, they must respond not only to Ag and costimulatory ligands, but also to an additional signal that can be provided by IL-12 or type I IFN. The requirement for a third signal was initially identified in vitro, where CD8 T cells that are activated in the absence of a third signal proliferate, but do not become lytic, and are deficient in the ability to produce IFN- in response to Ag (16, 17). We and others have now shown that CD8 T cells require a third signal in vivo to undergo optimal clonal expansion, develop effector functions, and establish a memory population, and third signal cytokines can substitute for adjuvant. In these in vivo studies, CD8 T cells evidently detect the presence of Ag, but in the absence of a third signal they fail to respond effectively to Ag in a variety of forms, including soluble peptide or protein Ag, Ag expressed on ectopic heart grafts, self-ag expressed in the pancreas, or infectious Ag (18 24). Because tumors do not have adjuvant properties, we hypothesized that the response to tumor might be another situation in which CD8 T cells recognize Ag, but due to inadequate third signal(s) they do not expand to high numbers, or develop potent effector functions, and therefore are unable to control tumor growth. To test this, OVA-specific OT-I-transgenic T cells were transferred to normal mice, and the mice were then challenged with E.G7, the OVA-transfected EL-4 thymoma. The OT-I cells in tumor-bearing mice proliferated in the DLN and trafficked to the tumor. However, the presence of activated T cells in the tumor did not affect tumor growth. Thus, although there was a response by the transgenic CD8 T cells, it was insufficient to control tumor growth and had the hallmarks of a third signal-deficient response: limited clonal expansion and inadequate effector function. Transgenic OT-I T cells that lack receptors for IL-12 and type I IFN responded even less well to the tumor, suggesting that low levels of one or both of these cytokines were present to provide a weak

3 The Journal of Immunology 6753 third signal to the wild-type (wt) OT-I cells. Treating tumor-bearing animals with a single dose of IL-12 resulted in enhanced clonal expansion of OT-I T cells and significant control of tumor growth in mice that had received wt transgenic cells, while mice that received receptor-deficient cells did not respond to IL-12 therapy, demonstrating that the effect is directly on the CD8 T cells. These results also suggest that insufficient third signal cytokine production in the tumor-bearing animal contributes to the deficient CD8 T cell response. Materials and Methods Mice, cell lines, and reagents OT-I mice having a transgenic TCR specific for H-2K b and OVA were a gift from Dr. F. Carbone (University of Melbourne, Melbourne, Australia). OT-I mice were crossed with Thy1-congenic B6.PL-Thy1a/Cy (Thy1.1) mice (The Jackson Laboratory) and bred to homozygosity to generate the OT-I/PL line. IFN-IR-deficient mice on a C57BL/6 background were a gift from Dr. J. Sprent (The Scripps Institute, La Jolla, CA). The IFN-IR-deficient mice were crossed to the OT-I/IL-12R 1-deficient mice and bred to homozygosity. OT-I-transgenic mice deficient in both the IL- 12R 1 and the IFN-IR are referred to as OT-I receptor knockout (OT-I/ RKO) mice. The OT-I/PL and OT-I/RKO TCR-transgenic mice were maintained under specific pathogen-free conditions at the University of Minnesota. C57BL/6NCr and CD45.1-congenic B6 (B6.Ly5.2) mice were purchased from the National Cancer Institute. Experiments were performed in compliance with relevant laws and institutional guidelines and with the approval of the Institutional Animal Care and Use Committee at the University of Minnesota (Minneapolis, MN). E.G7 tumor cells (EL-4 thymoma transfected with OVA) were maintained by in vitro culture in RPMI 1640 medium supplemented with 10% FBS (RP-10) and 250 g/ml G418 (Mediatech). Anti-human granzyme B (GzmB) PE- or allophycocyaninconjugated Ab (which cross-reacts with mouse GzmB) was purchased from Caltag Laboratories. All other directly conjugated fluorescent Abs were purchased from BD Pharmingen, ebioscience, or BioLegend). Adoptive transfer of transgenic cells Pooled LN from OT-I/PL or OT-I/RKO mice were disrupted to yield single-cell suspensions and washed with PBS. Before transfer, the cells were analyzed by flow cytometry to determine the percentage of CD8 cells. Their CD25, CD69, and CD44 phenotypes were determined to confirm that the cells that were transferred were not activated. In some experiments, transgenic LN cells were labeled with CFSE before transfer to recipient mice as described previously (21). A total of CD8 cells in 0.3 ml of PBS was transferred via tail vein injection into age- and sex-matched naive 4- to 8-wk-old recipients. Recipient mice were rested for at least 24 h before challenge with E.G7 tumor. Tumor challenge and IL-12 therapy E.G7 cells were thawed from liquid nitrogen and grown for no more than 2 wk in culture before being injected into recipient mice and were always in logarithmic growth phase when harvested for injection. E.G7 cells were counted, washed two times with Dulbecco s PBS, and a total of cells were injected s.c. in the groin of recipient mice, in a volume of 0.2 ml of Dulbecco s PBS. Mice were monitored daily until tumor was detected, then biweekly tumor measurements were made. Calipers were used to measure the tumor in its longest dimension and the perpendicular dimension. The product of the two measurements is the tumor area, reported in square millimeters. Tumor-bearing mice were sacrificed before they exhibited signs of pain or distress. Some mice received a single therapeutic dose of 1 g of recombinant murine IL-12 (Wyeth) delivered i.v. via tail vein at the indicated time. Statistical analysis of IL-12 effects on OT-I cell responses and on tumor growth was by a one-tailed t test. Flow cytometry analysis of transferred cells Mice were sacrificed at the indicated times after adoptive transfer and tumor challenge. Spleens were removed from all animals. The LN analyzed were the inguinal node on the same side as the tumor (DLN) and the opposite side from the tumor (nondraining LN, or NDLN), or, in some experiments, all axillary, brachial, and inguinal nodes were collected and pooled. Spleens, LN, and tumors were disrupted with a Dounce homogenizer to obtain a single-cell suspension, and cells were stained with Abs to CD8 and either Thy 1.1 or CD45.2 to detect the transferred OT-I cells. Stained cells were collected on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star). Cell counts were FIGURE 1. Adoptively transferred OT-I cells do not alter the growth kinetics of E.G7 growing as a solid tumor (TUM). OT-I lymph node (LN) cells were adoptively transferred into C57BL/6 mice ( CD8 cells/mouse). The next day, mice without ( ) and with (F) adoptively transferred OT-I cells were challenged with E.G7 cells s.c. in the groin in a volume of 0.2 ml. Tumors were measured biweekly and the mean and SD of tumor area for the three mice in each group is shown. obtained by adding a known number of PKH26 reference microbeads (Sigma-Aldrich) to an aliquot of each cell suspension, and then the cell/ bead mixtures were run on the flow cytometer. The ratio of lymphocytes to beads collected for each sample was multiplied by the number of beads per milliliter in the sample tube and the dilution factor for the cells to obtain the number of lymphocytes per milliliter in the sample. Intracellular staining For detection of IFN- production, cells harvested from tumor-bearing mice were incubated in RP-10 with 1.0 M OVA After 1hof incubation at 37 C, the monensin-containing solution GolgiStop (BD Pharmingen) was added and cultures were incubated for an additional 3 4 h at 37 C. Cells were washed, stained with Abs to mark the OT-I cells, fixed in Cytofix buffer (BD Pharmingen) for 15 min at 4 C, and permeabilized in saponin-containing Perm/Wash buffer (BD Pharmingen) for 15 min at 4 C before staining with Ab to IFN- for 30 min at 4 C. Cells were washed once with Perm/Wash buffer and once with PBS containing 2% FBS. Intracellular GzmB staining was the same, except that the cells were not restimulated in vitro before surface stain, fixation, permeabilization, and staining with Ab to GzmB. All samples were collected on a FACSCalibur flow cytometer using CellQuest software and analyzed using FlowJo software. Results Tumor-specific CD8 T cells proliferate and migrate to tumor early in growth of tumor OVA-specific OT-I-transgenic T cells were adoptively transferred into C57BL/6 mice that were then challenged s.c. with the OVAexpressing E.G7 tumor. Earlier studies showed that transferred OT-I cells had minimal effects on the E.G7 tumor growing in the peritoneal cavity (25). To establish that OT-I cells did not alter the growth kinetics of a solid tumor, we compared tumor growth in animals with or without adoptively transferred OT-I cells. As seen in Fig. 1, tumor growth was comparable in both groups of animals, with only a slight delay in growth of the tumor in animals that received OT-I cells. Thus, the adoptively transferred OT-I cells did not appear to mount an effective immune response to the tumor. To visualize early events in CD8 T cell responses to E.G7 growing as a solid tumor, OT-I cells were labeled with CFSE before adoptive transfer. We could detect dividing OT-I cells in the DLN as early as 6 days after tumor injection (Fig. 2A), before there was measurable tumor growth. At this early time, dividing OT-I cells were found only in the DLN; cells in the contralateral NDLN were uniformly CFSE high. Although there was not measurable tumor at this time, when one of the animals was dissected, a small tumor was found under the skin. There were OT-I cells in this tumor, and they were uniformly CFSE low. After six to seven cell divisions, the intensity of CFSE fluorescence is equivalent to that of unlabeled

4 6754 INSUFFICIENT SIGNAL 3 LIMITS CD8 T CELL RESPONSE TO TUMOR Ag FIGURE 2. Adoptively transferred OT-I cells proliferate in the DLN of E.G7 tumor-bearing mice and traffic to the tumor, but their clonal expansion is limited. OT-I/PL LN cells were adoptively transferred into C57BL/6 mice ( CD8 cells/mouse). The next day these mice were challenged with E.G7 cells s.c. in the groin. A, OT-I/PL cells were labeled with CFSE before adoptive transfer. At 6, 9, and 13 days after tumor injection spleens (Spl), DLN, contralateral NDLN, and tumors (when present) were removed and processed for flow cytometry analysis. Staining for CD8 and Thy1.1 identified OT-I/PL cells and their CFSE profile is shown. The profiles shown are representative of two animals assessed at each time point in this experiment and similar results were obtained in six independent experiments. B, At 5, 8, and 15 days after tumor injection, staining for CD8 and Thy1.1 identified OT-I/PL cells in spleens and inguinal LN, and total numbers of OT-I cells at these sites were determined. Averages from two (days 0 and 8, range is indicated) or three (days 5 and 15, SD is indicated) mice are shown. C, AsinB, except that OT-I numbers were assessed on days 11, 20, and 32 after tumor injection. Averages and ranges from two mice at each time point are shown. Results in B and C are representative of six independent experiments. cells. Thus, at this early time, there were OT-I cells that had divided at least six times, presumably in the DLN, and had left the node and trafficked to the tumor site. At 9 and 13 days after tumor challenge, there were increased numbers of dividing OT-I cells in the DLN, spleen, and tumor. Cells that had divided between one and six times were primarily found in the DLN, indicating that this continued to be the primary site of initial Ag presentation and proliferation. Cells that were CFSE low were found in the NDLN and spleen, most likely representing OT-I cells that had left the DLN and entered the circulation. There continued to be undivided cells in the NDLN and, to a lesser extent, in the spleen. In contrast, the tumor contained only CFSE low cells, indicating that OT-I cells traveled to the tumor site only after dividing at least six times. Total numbers of OT-I cells in the spleen and LN were calculated at various times after tumor challenge, revealing that there was an initial expansion of OT-I cells that occurred between 5 and 10 days after tumor challenge (Fig. 2B). In other experiments, OT-I numbers were followed for longer times and, after the initial clonal expansion, the numbers of OT-I cells remained relatively constant FIGURE 3. Adoptively transferred OT-I cells in E.G7 tumor-bearing mice show evidence of recent encounter with Ag in the DLN and tumor. OT-I/PL LN cells were adoptively transferred into C57BL/6 mice ( CD8 cells/mouse). The next day these mice were challenged with E.G7 cells s.c. in the groin. After 14 days, spleens, DLN, NDLN, and tumors were removed and processed for flow cytometry analysis. Staining for CD8- and CD45.2-identified OT-I cells (CD8 CD45.2 gate in the left panels; events shown are gated on lymphocytes as determined by forward scatter and side scatter profile) and their CD69 expression is shown (right panels, open histograms). Shaded histograms in the right panels show the CD69 expression of the endogenous CD8 population (CD8, CD45.2- gate in the left panels); these were used to set the marker for high CD69 expression. The percentage of OT-I cells that are CD69 high is shown in each histogram and the mean fluorescence intensity of CD60 staining of the total OT-I population is shown in parentheses in each histogram panel. Results are representative of two mice assessed in this experiment and similar results were obtained in five independent experiments. SPL, Spleen. despite continuing tumor growth (Fig. 2C). We attempted to determine the number of OT-I cells in the tumors; however, these numbers are probably low estimates because it was difficult to disrupt the tumors and many of the lymphocytes and tumor cells that were obtained were dead. The numbers of OT-I cells in the tumors continued to increase as the tumor grew, but when expressed as cells per unit of tumor area, this number remained fairly constant even after 21 days (data not shown). Together, these data suggest that OT-I cells do respond to OVA produced by the E.G7 tumor cells but do not accumulate in large numbers after an initial period of expansion. Responding CD8 T cells have an effector phenotype Although the OT-I cells responded to OVA Ag early after tumor challenge, these cells did not control the growth of the tumor (Fig. 1). To test the possibility that the cells that entered the tumor were not able to recognize Ag in that environment, CD69 expression was examined on OT-I cells at various sites 10 days after tumor challenge, when measurable tumor was detected. As seen in Fig. 3, a significant proportion of cells in the DLN were CD69 high, indicating that these cells had recently encountered Ag, possibly on APCs. In contrast, 10% of the OT-I cells in the contralateral nodes or spleen were CD69 high. Because Ag presentation does not occur in the spleen (Fig. 2A), we conclude that the phenotype of spleen cells is representative of cells that have left the DLN and entered the circulation. Because the OT-I cells in the spleen were CD69 low, they apparently did not encounter Ag in the periphery. When OT-I cells in the tumor were examined, it was found that a majority of the cells were CD69 high (Fig. 3). This suggested that

5 The Journal of Immunology 6755 FIGURE 4. Adoptively transferred OT-I cells in E.G7 tumor-bearing mice express GzmB and produce the effector cytokine IFN-. OT-I LN cells were labeled with CFSE and adoptively transferred into B6.Ly5.2 (CD45.1-congenic) mice ( CD8 cells/mouse). The next day these mice were challenged with E.G7 cells s.c. in the groin. After 15 days, tumors (A and C) and DLN (B and D) were removed and processed for flow cytometry. A and B, Cells were stained with Abs to CD8 and CD45.2 to identify the transferred OT-I cells. After fixation, the cells were permeabilized and stained to detect GzmB expression in the OT-I population, shown here as a function of number of cell divisions as indicated by CFSE dye dilution. C and D, Cells were restimulated with OVA peptide in the presence of monensin (GolgiStop) for 4 h. Cells were stained with Abs to CD8 and CD45.2 to identify the transferred OT-I cells. After fixation, the cells were permeabilized and stained to detect IFN- expression in the OT-I population, shown here as a function of number of cell divisions. Results are representative of analysis of two mice at this time point and four additional mice analyzed 7 and 10 days after tumor injection in this experiment. Similar results were seen in four independent experiments. the OT-I cells had re-expressed CD69 after entering the tumor and were therefore still capable of recognizing Ag in the tumor environment, either on APCs in the tumor or directly on the tumor cells. CD8 T cells that do not receive the third signal typically proliferate but clonal expansion and effector functions are diminished (16, 17, 21).To test the hypothesis that the OT-I cells failed to control tumor growth because they had not received an adequate third signal from inflammatory cytokines, the functional status of OT-I cells in the DLN and in the tumor was examined 15 days after tumor inoculation, when the tumor is well established. To indirectly assess lytic potential, cells were stained with Ab to GzmB, one of the proteases found in the lytic granules of effector CTL, but not naive or memory CD8 T cells (26 28). GzmB expression correlates with lytic function and is dependent on third signal cytokines, at least in the context of immunization with peptide Ag (29). In Fig. 4, intracellular GzmB staining is shown as a function of CFSE fluorescence intensity. As seen before, all of the OT-I cells in the tumor had divided at least six times. Approximately one-half of these cells were positive for GzmB (Fig. 4A), consistent with the acquisition of lytic effector function. There were also GzmB OT-I cells in the DLN, but only in cells that had divided at least six times, as indicated by CFSE dilution (Fig. 4B). The restriction of GzmB expression to cells that have divided more than six times suggests a developmental program in CD8 T cells in which production of this component of the lytic granules is a relatively late event and is consistent with our earlier observation that IL-12 can drive the acquisition of lytic effector function even if added late in the CD8 T cell response. These results also suggest either that a third signal cytokine is available or that GzmB expression can be induced by another mechanism. Although GzmB cells were only 15% of the total OT-I cells in the DLN, they represented 33% of the cells that had divided more than six times, comparable to the percentage of OT-I cells in the tumor that were GzmB (Fig. 4B). Similar restriction of GzmB to those cells that had divided more than six times was also seen at 7 or 10 days after tumor inoculation, although there were not as many GzmB cells at those earlier times (data not shown). The GzmB staining results suggest that the OT-I cells in the tumor may be capable of lytic function. Another effector function of CD8 T cells that is enhanced by exposure to third-signal cytokines is IFN- production. IFN- can activate macrophages, enhance Ag processing, and increase MHC expression, including up-regulation of class I expression on tumor cells, making them more susceptible to CTL lysis. IFN- produced by CD8 T cells responding to tumor Ag has been shown to be critical for E.G7 tumor rejection in an adoptive immunotherapy model (30). To determine whether the OT-I cells that were responding to tumor-derived Ag were capable of producing IFN-, cells isolated from the tumor and DLN were restimulated with OVA-peptide Ag in vitro and then stained with Abs to IFN-. As seen in Fig. 4C, 22% of the OT-I cells that were isolated from the tumor expressed IFN- when restimulated in vitro with peptide Ag. About the same percentage of OT-I cells from the DLN also expressed IFN- (Fig. 4D). The DLN-derived IFN- cells were present in all generations of cells rather than being restricted to those cells that had divided at least six times, as was the case for GzmB production. The proportion of OT-I cells in the tumor that could make IFN- in response to peptide was low, especially when compared with the 50% of OT-I cells that can make IFN- when they have been stimulated in vivo with Ag and adjuvant. In addition, the intensity of staining with the IFN- Ab was at least 10- fold less than was seen in cells immunized with Ag and adjuvant (21). Overall, the IFN- response by OT-I cells to a tumor-associated Ag was weak relative to a response to peptide Ag given with adjuvant, both in the fraction of cells that could respond and in the amount of cytokine that was made by those cells that did respond. Thus, phenotypic analysis of the OT-I cells revealed GzmB expression, consistent with lytic effector function, but there was no clonal expansion between 7 and 15 days after tumor challenge (Fig. 2C), and expression of the effector cytokine, IFN-, was relatively low. These results suggested that the OT-I cells responding to the tumor were receiving a limited third signal, which could explain why tumor growth was not controlled. CD8 T cells lacking receptors for third-signal cytokines have a defective response to tumor To address this possibility directly, either wt OT-I cells or OT-I cells that lacked the receptors for both IL-12 and type I IFN (OT- I/RKO) were transferred into C57BL/6 mice; IL-12 and type I IFN are two cytokines that can provide a third signal to CD8 T cells. These mice were challenged with E.G7 tumor. One week after challenge with tumor, expanded numbers of both the wt OT-I cells and the OT-I/RKO cells were found in the spleen (Fig. 5A). One week later, however, the numbers of OT-I/RKO cells had dropped dramatically. The numbers of OT-I/RKO cells were significantly less than the numbers of wt OT-I cells which remained approximately the same, as seen in earlier experiments. Similar numbers of OT-I cells were found in LN (data not shown). The phenotype of the OT-I/RKO cells was examined 7 and 15 days after tumor challenge. GzmB expression and IFN- production by wt OT-I and OT-I/RKO cells were similar at 7 days after tumor challenge (data not shown). However, the OT-I/RKO cells still present 15 days after tumor challenge were uniformly low producers of IFN- (Fig.

6 6756 INSUFFICIENT SIGNAL 3 LIMITS CD8 T CELL RESPONSE TO TUMOR Ag FIGURE 5. Tumor-specific CD8 T cells that lack receptors for IL-12 and type I IFN decrease in number after an initial period of expansion and lose effector phenotype. LN cells from OT-I/PL mice or OT-I/RKO mice were adoptively transferred into B6.Ly5.2 (CD45.1-congenic) mice. The next day these mice were challenged with E.G7 tumor (TUM) s.c. in the groin. Mice were sacrificed on days 7 and 15 and spleens (SPL), DLN, NDLN, and tumors were removed and processed for flow cytometry analysis. Staining for CD8-, Thy1.1-, and CD45.2-identified OT-I/PL and OT- I/RKO cells. A, Total numbers of transferred cells in spleens of mice given OT-I/PL cells (F) or OT-I/RKO cells (Œ) were determined on days 7 and 15. Numbers of OT-I/RKO cells were significantly lower than wt OT-I cells (p 0.036). B, Cells collected from each site on day 15 were restimulated with OVA peptide in the presence of monensin (GolgiStop) for 4 h. Cells were stained with Abs to CD8, Thy1.1, and CD45.2 to identify the transferred cells. After fixation, the cells were permeabilized and stained to detect IFN- expression in the OT-I population. Results shown are the percentage of OT-I cells that were IFN- positive. C, Cells collected from each site on day 15 were stained with Abs to CD8, Thy1.1, and CD45.2 to identify the transferred cells. After fixation, the cells were permeabilized and stained to detect GzmB expression in the OT-I population. Results shown are the percentage of OT-I cells that were GzmB positive. Percentages of IFN- and GzmB OT-I/RKO cells were significantly lower than for wt OT-I cells in the spleen, NDLN, and DLN (p 0.05 in each case), but not in the tumor. In each panel, averages and SD from four mice from two independent experiments are shown. 5B) and had significantly lower GzmB expression than wt OT-I cells in the spleen and LN, although not in the tumor (Fig. 5C). These results suggest that the initial response to tumor Ag is independent of IL-12 and type I IFN. However, continued survival of tumor-specific CD8 T cells after this initial response requires that cells can respond to IL-12 or type I IFN, because without receptors for one of these cytokines, the tumor-specific cells rapidly decrease in number; those that do remain make little IFN-, FIGURE 6. A single dose of ril-12 results in significant control of tumor growth. Mice without (A) or with (B) adoptively transferred OT-I cells were challenged with E.G7 cells s.c. in the groin. After 10 days, mice were left untreated (F) or were given a single i.v. dose of ril-12 (E). Tumors were measured biweekly and the mean and SD of tumor area for the three mice in each group are shown. At times marked with an asterisk, tumor sizes were significantly different between treated and untreated mice as determined by Student s t test (p 0.05). and their expression of GzmB is reduced at sites other than the tumor, indicating a loss of effector functions. Survival of receptorsufficient OT-I cells after the first week appears to be dependent on signals through the IL-12 and/or type I IFN receptors. However, the phenotype of these cells suggested that the endogenous levels of these signals are not sufficient to allow further clonal expansion and substantial production of the effector cytokine IFN-, possibly explaining their failure to control tumor. Therefore, we tested whether an exogenous third signal might improve the quantity and quality of the tumor-specific response and allow control of tumor growth. Exogenous IL-12 acts on CD8 T cells causing clonal expansion and control of tumor growth Tumor therapy with IL-12 has been effective in a variety of animal models and has been used for cancer therapy in clinical trials. To examine the effect of IL-12 as an exogenous third signal for the OT-I response in the s.c. tumor model, mice with and without adoptively transferred OT-I cells were challenged s.c. with E.G7 tumor. After 10 days, when measurable tumor was detected in all of the animals and when OT-I cells have begun to proliferate and traffic to the tumor (Fig. 2A), one-half of the animals in each group received a single i.v. dose of ril-12 and tumor growth was monitored. As seen in Fig. 6A, in the animals without OT-I cells, there was a small decrease in mean tumor size in the IL-12-treated group, although this difference was statistically significant only on day 20 ( p 0.011). This modest effect was reproducible, because in six independent experiments, tumor either regressed or grew more slowly in 20 of 24 mice that received IL-12 therapy, but it did not achieve statistical significance in the majority of cases. It is not surprising that a single dose of IL-12 would not be sufficient to control tumor growth indefinitely, but these results confirm earlier studies showing modest efficacy of IL-12 for tumor therapy in unmanipulated animals. In the mice that received OT-I cells, the IL-12 effects were much more dramatic. As seen in Fig. 6B, tumor

7 The Journal of Immunology 6757 FIGURE 7. IL-12 control of tumor growth depends on functional IL-12 receptor on tumor-specific CD8 T cells. B6.Ly5.2 mice, without OT-I cells or with either wt OT-I or OT-I/RKO LN cells transferred the previous day, were challenged with E.G7 cells s.c. in the groin. On day 10, some mice were given 1 g of IL-12. Tumor areas on day 16 are shown. The horizontal bar shows the mean tumor area for each group. For the mice given OT-I/PL cells, the average tumor areas were significantly different between untreated and IL-12-treated mice as determined by Student s t test (p 0.021). growth was significantly delayed in animals that were given a single dose of IL-12 on day 10 on days 16, 20, and 23. In six independent experiments, 36 mice received OT-I cells and were challenged s.c. with E.G7 tumor and then received IL-12 therapy between days 8 and 10. Of these 36, tumor growth was unaffected in 11 mice, while tumor stopped growing in 5 mice and regressed in 20 mice. IL-12 is known to have effects on other cell types in addition to CD8 T cells, including CD4 T cells, NK cells, and dendritic cells (DC) (31). The fact that tumor control was more effective in animals that received transferred OT-I cells suggested that the therapeutic effect of IL-12 was at least partially a result of direct action on the OT-I CD8 T cells. To confirm this, we transferred wt OT-I cells or OT-I/RKO cells into C57BL/6 mice, challenged these with E.G7 tumor, and gave IL-12 on day 10, when measurable tumor was present. In this experiment, a single dose of IL-12 resulted in complete tumor regression in five of six mice that received wt OT-I cells (Fig. 7). In mice that did not receive OT-I cells or were given OT-I/RKO cells, there was a slight decrease in the average tumor size on day 16, but this difference was not statistically significant. Thus, when the transferred OT-I cells cannot respond to IL-12, the effect of IL-12 on tumor growth is no better than in animals without OT-I cells (Fig. 7). These results confirm that the therapeutic effect of IL-12 is largely a result of direct action on CD8 T cells. To determine whether IL-12 therapy changed the numbers or phenotype of the responding OT-I cells, we analyzed the OT-I cells from tumor-bearing animals at day 8, just before giving IL-12 therapy, and at day 15, which was 7 days after IL-12 therapy. There were large numbers of OT-I cells in the spleens and LN of animals that received IL-12 on day 8, 10-fold higher than in the animals that were untreated (Fig. 8A). This effect of IL-12 on clonal expansion was seen in three separate experiments, with approximately the same fold increase in numbers in each case. In one experiment, animals were sacrificed at 3 wk, nearly 2 wk after IL-12 therapy, and the increased number of OT-I cells persisted, even though at this time tumors were nearly gone in two of the three mice analyzed (data not shown). We also examined the effector phenotype of the OT-I cells on day 15 in mice that were given IL-12 therapy or were left untreated. There was a modest increase in the proportion of OT-I cells that expressed GzmB (Fig. 8B) and IFN- (Fig. 8C) in mice that received IL-12 therapy. Although the change in the fraction of OT-I cells that express GzmB FIGURE 8. IL-12 treatment of tumor-bearing mice causes increased clonal expansion of tumor-specific CD8 T cells. OT-I/PL LN cells were transferred to C57BL/6 mice. The next day mice were injected with E.G7 cells s.c. in the groin. On day 8, some mice were given 1 g of IL-12. Mice without tumor (day 0) and mice at days 8 and 15 were sacrificed and spleens (SPL) and DLN were removed and processed for flow cytometry analysis. Staining for CD8- and Thy1.1-identified OT-I/PL cells. A, Total numbers of transferred cells recovered from untreated mice (solid lines) and from mice given IL-12 (dashed lines) are shown. B, Cells from day 15 were fixed, permeabilized, and stained to detect GzmB expression. Results shown are the percentage of OT-I cells that were GzmB positive. C, Cells from day 15 were restimulated with OVA peptide in the presence of monensin (GolgiStop) for 4 h. After fixation, the cells were permeabilized and stained to detect IFN- expression. Results shown are the percentage of OT-I cells that were IFN- positive. In each panel, average and range for two mice at each time point are shown. Results are representative of three independent experiments. tx, Treatment; TUM, tumor. or IFN- was small, the number of cells with an effector phenotype increased significantly due to the 10-fold increase in numbers of OT-I cells following IL-12 therapy. The results reported here are consistent with a model in which CD8 T cells initially respond to tumor Ags and would therefore exhibit a phenotype consistent with previous Ag exposure. However, the availability of third signal cytokines is limited and the CD8 T cells stop expanding and fail to develop effector function, particularly the ability to make IFN-. In our model system, a single dose of IL-12 was sufficient to cause a 10-fold increase in the number of tumor-specific CD8 T cells and therefore a similar increase in the number of effector phenotype cells, resulting in significant control of tumor growth.

8 6758 INSUFFICIENT SIGNAL 3 LIMITS CD8 T CELL RESPONSE TO TUMOR Ag Discussion This study characterizes the earliest stages of a CD8 T cell response to tumor Ag. OT-I cells adoptively transferred to a recipient before challenge with E.G7 tumor initiate a response in the LN draining the tumor site, undergo multiple rounds of division, and migrate to the tumor site. Some of the cells contain GzmB and produce modest amounts of IFN- when restimulated in vitro, suggesting that they have some effector functions. After an initial 10-fold expansion, however, the number of OT-I cells reaches a plateau around 10 days after tumor challenge. Although cells continue to enter the pool of divided cells after this time, there is no additional accumulation of OT-I cells, and the activated OT-I cells do not affect the rate of tumor growth. We demonstrated previously that although Ag and costimulatory ligands are sufficient to stimulate in vitro proliferation of CD8 T cells, a third signal, provided by an inflammatory cytokine, is necessary for full activation (16). The in vivo requirement for a third signal was initially revealed by immunization with peptide, bypassing a requirement for Ag processing (18, 19, 21). In this approach, Ag presentation presumably occurs by passive loading of peptide onto DC or other cells that express costimulatory molecules such as CD80 or CD54 (32, 33). In response to in vivo challenge with peptide alone, CD8 T cells expand, but their expansion is limited, they fail to develop lytic effector function, and the cells are tolerized. A single dose of IL-12, given along with peptide, changes the response from tolerizing to immunizing. We reported that type I IFNs can also provide the third signal in vitro (17), and recently Murali-Krishna s group (23) demonstrated that type I IFNs are critical for an optimal in vivo CD8 T cell response to lymphocytic choriomeningitis virus. In their studies, transgenic, lymphocytic choriomeningitis virus-specific CD8 cells that lacked the type I IFN receptor failed to accumulate in response to viral challenge and did not form a memory population. In an ectopic heart transplant model, Ingulli and coworkers (22) showed that CD8 T-mediated transplant rejection was also dependent on a third signal. The third signal was provided via CD4 helper T cell stimulation of IL-12 production by cross-presenting DC. Alternatively, the requirement for CD4 help could be bypassed by provision of exogenous IL-12. Thus, a third signal cytokine can replace adjuvant in a peptide immunization and is required for an effective response to viral and transplant Ag. Third signal requirements in response to infectious Ag appear to vary, depending at least in part on whether IL-12, type I IFNs, or both are produced in response to the infection (Z. Xiao and M. F. Mescher, manuscript in preparation and Ref. 34). In the s.c. tumor model described here, we show that responses by tumor-specific CD8 T cells require third-signal cytokine(s), because OT-I cells lacking receptors for IL-12 and type I IFNs are profoundly deficient in clonal expansion and effector functions as compared with wt OT-I cells. Endogenous third signals appear to be inadequate, however, because even the response by receptor-sufficient OT-I cells is ineffective, but a single dose of exogenous IL-12 results in a 10-fold expansion of these cells and significant control of tumor growth. Thus, the requirement for third signal cytokines can be extended to generation of an effective CD8 T cell response to tumor-associated Ag. The characteristics of the early response to E.G7 tumor suggest that there might be some third signal available, because the OT-I cells do expand and some acquire an effector phenotype. However, the early response by IL-12 and type I IFN receptor-deficient OT-I cells is very similar to wt cells, implying that the earliest response is independent of a third signal. This is consistent with in vitro and in vivo studies showing that stimulation with only two signals results in substantial OT-I proliferation (16, 17, 21). A third signal is necessary, however, for enhanced survival leading to significant clonal expansion. The increased survival caused by the third signal depends at least in part on up-regulation of Bcl-3, a transcription factor that presumably regulates expression of genes involved in survival (35). The lack of a survival factor would explain why the number of OT-I cells in tumor-bearing mice does not increase after an initial period of expansion, but after IL-12 therapy that number increases 10-fold. OT-I cells stimulated with only two signals also make some IFN- when restimulated with OVA peptide (21), consistent with the phenotype of OT-I cells responding to tumor. There is a correlation between GzmB expression and lytic function in OT-I cells examined at the time of peak lytic activity both in vitro and in vivo, and the GzmB expression is dependent on a third signal. However, OT-I cells stimulated in vitro with only two signals do make GzmB early in the response, before lytic activity develops, but a third signal is required to maintain GzmB expression. Thus, it is possible that OT-I cells in tumor-bearing mice might be only transiently expressing GzmB and that expression may be lost before lytic function is acquired (29). Whether or not the initial response requires a third signal, the rapid decline in numbers of the receptor-deficient OT-I cells indicates that CD8 T cells quickly become dependent on IL-12 or type I IFN for both survival and continued expansion. The fact that receptor-sufficient OT-I cells persist but do not expand or control tumor suggests that there is some third signal available, but not enough for an effective antitumor response. These results raise the question of what limits third-signal cytokines in the immune response to tumor Ag. IL-12 and type I IFNs are produced by DC in response to signals delivered by ligands for TLRs. Alternatively, DC can be stimulated to produce these cytokines via ligation of CD40 by CD154 expressed on activated CD4 T cells. Thus, the limited third signal could be due to the absence of ligands that stimulate DC maturation in the tumor environment. To address this possible defect, numerous clinical trials have taken the approach of direct activation of DCs through ligation of TLRs (14). Alternatively, the limited third signal could be a result of an inadequate CD4 response to the tumor Ags, and in several mouse models activated CD4 helper T cells have been shown to augment the CD8 T cell response to tumors (36, 37). There can also be active inhibition of maturation of DC in a tumor environment resulting, for example, from tumor cell production of vascular endothelial growth factor, a factor that also promotes tumor angiogenesis (38). Recent studies have revealed a significant role for regulatory T cells (Treg) in defective immune responses to tumor Ags. Treg functions are mediated in part through secretion of immunosuppressive cytokines IL-10 and TGF. IL-10 can inhibit DC Ag presentation and cytokine secretion while TGF inhibits proliferation and effector functions of both CD4 and CD8 T cells (39). Interestingly, a recent study found that IFN- could inhibit the generation of Treg, revealing a mechanism by which effector CD8 T cells can counter immune suppression (40). Thus, it is possible that as an immunosuppressive environment develops in the growing tumor, DC that secrete third-signal cytokines become scarce. This might be due to an absence of DC activation signals, active suppression of DC activation, or inhibition of activated CD4 T cells which could themselves activate DC. This could explain why the vigorous early response by OT-I cells is followed by such a lethargic one. Whatever the cause of the inadequate third signal, the defect can be bypassed by providing exogenous cytokine. Another possible explanation for the early OT-I response to tumor is suggested by studies from Sherman and coworkers (36) using another model of adoptive transfer of tumor-specific CD8 T

9 The Journal of Immunology cells into tumor-bearing mice. In this model, the number of transferred T cells affected the phenotype of the responding cells. Large numbers of transferred cells created an inflammatory environment that led to acquisition of effector function. Transfer of smaller numbers of transgenic cells resulted in a decreased percentage of cells having an effector phenotype. We are transferring fewer cells than in those experiments, but it is possible that the relatively large numbers of cells that are responding enhance the early response. This effect would not be due to production of IL-12 or type I IFN because the OT-I and OT-I/RKO cells responded similarly. In a model similar to ours, Malek and coworkers (41) analyzed responses by adoptively transferred OT-I cells to E.G7 tumors growing s.c. In this model system, the OT-I cells did not respond to tumor. It is likely that the differences can be attributed to the use of a different subline of the E.G7 tumor as was noted by the authors. However, it is also possible that this discrepancy could be explained by the difference in experimental approach; in the studies by Malek s group (41), the OT-I cells were transferred when there was an established s.c. tumor. It may be that the tumor already had developed mechanisms to suppress an immune response and therefore the OT-I response was blocked. Another similar adoptive transfer model from Combadiere and coworkers (42) yielded results similar to ours. They saw proliferation of OT-I cells in the DLN of mice with s.c. E.G7 tumors and trafficking of activated cells to the tumor only after four or more divisions. However, in their system, the OT-I cells only responded if there was a preexisting tumor, and when the tumors were small the OT-I response eliminated the tumor. These discrepancies might also be due to differences in the E.G7 subline used. Responses by adoptively transferred OT-I cells have been measured when OVA-expressing tumors are growing at various sites, with notable differences in both T cell responses and in therapeutic outcomes. When the E.G7 tumor is growing in the peritoneal cavity, OT-I cells initially respond, go to the tumor site, and briefly control tumor, but then leave and do not control tumor long term. The OT-I cells were shown to be in a state of Ag-induced nonresponsiveness (AINR) that could be reversed by IL-2, either provided exogenously or by in situ-activated CD4 T cells (25, 43 45). In contrast, in a lung metastasis model, in which B16 melanoma cells transfected with OVA are injected i.v., adoptively transferred OT-I cells remain ignorant until the tumor is too large to control (F. Popescu and M. F. Mescher, manuscript in preparation). In this tumor model, IL-12 alone has minimal efficacy. However, providing both Ag and third-signal cytokine has a therapeutic effect. In the studies described here, OT-I cells clearly recognize Ag on E.G7 tumor growing at a s.c. site, but the lack of sufficient third signal prevents control of tumor growth. Taken together, these results point out the importance of determining the status of tumorspecific CD8 T cells at the time of therapy. If the immune system has not detected the tumor Ag, as in the case of the B16-OVA lung metastasis model, then it is not surprising that providing IL-12 would have no effect on the CD8 T cell response to tumor, while providing both Ag and IL-12 has an effect. If it appears that tumor Ag has been recognized, however, as in the i.p and s.c. tumor models, then it would be important to determine whether the cells are AINR or third-signal deficient, with IL-2 providing effective therapy in the former case, and IL-12 or another third-signal cytokine being effective in the latter case. 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