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The Journal of Immunology, 2007, 179, 2860 -2869
Copyright © 2007 by The American Association of Immunologists, Inc.
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Targeting Molecular and Cellular Inhibitory Mechanisms for Improvement of Antitumor Memory Responses Reactivated by Tumor Cell Vaccine1

W. Scott Webster*, R. Houston Thompson*, Kimberley J. Harris{dagger}, Xavier Frigola*, Susan Kuntz{dagger}, Brant A. Inman* and Haidong Dong2,*,{dagger}

* Department of Urology and {dagger} Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Development of effective vaccination approaches to treat established tumors represents a focus of intensive research because such approaches offer the promise of enhancing immune system priming against tumor Ags via restimulation of pre-existing (memory) antitumoral helper and effector immune cells. However, inhibitory mechanisms, which function to limit the recall responses of tumor-specific immunity, remain poorly understood and interfere with therapies anticipated to induce protective immunity. The mouse renal cell carcinoma (RENCA) tumor model was used to investigate variables affecting vaccination outcomes. We demonstrate that although a whole cell irradiated tumor cell vaccine can trigger a functional antitumor memory response in the bone marrows of mice with established tumors, these responses do not culminate in the regression of established tumors. In addition, a CD103+ regulatory T (Treg) cell subset accumulates within the draining lymph nodes of tumor-bearing mice. We also show that B7-H1 (CD274, PD-L1), a negative costimulatory ligand, and CD4+ Treg cells collaborate to impair the recall responses of tumor-specific memory T cells. Specifically, mice bearing large established RENCA tumors were treated with tumor cell vaccination in combination with B7-H1 blockade and CD4+ T cell depletion (triple therapy treatment) and monitored for tumor growth and survival. Triple treatment therapy induced complete regression of large established RENCA tumors and raised long-lasting protective immunity. These results have implications for developing clinical antitumoral vaccination regimens in the setting in which tumors express elevated levels of B7-H1 in the presence of abundant Treg cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The ability of memory T cells to respond briskly to antigenic challenge suggests that such memory cells are a significant component of antitumor immunity. When compared with naive T cells, memory T cells require less costimulation to be activated, release a broader spectrum of cytokines, and are multifunctional after reactivation (1). In certain tumors, such as colorectal carcinoma, tumor infiltration by effector memory CD8+ T cells is associated with a better prognosis (2). Although the presence of memory T cells within tumors may be a favorable prognostic variable, these T cells are usually not sufficient to control cancer progression.

Few functional tumor-specific memory CD8+ T cells are found in the peripheral blood of cancer patients (3, 4). In contrast, functionally active and tumor-specific memory T cells have been identified in the bone marrow of patients with breast cancer, myeloma, or pancreatic cancer (5, 6, 7). The apparent preferential distribution of antitumor memory T cells within bone marrow rather than peripheral blood and effector sites suggests inhibitory regulatory mechanisms may be adversely affecting the function and/or survival of antitumor T cells in tumor and peripheral lymphoid tissues. Therefore, it would be of significant relevance to characterize the nature and extent of regulatory mechanisms that negatively impact peripheral memory responses to cancer.

Several groups have reported that the presence of infiltrating mononuclear cells, including intratumor CD4+ T cells, paradoxically, identifies patients with a poor prognosis (8, 9). This observation suggests that a subset of tumor-infiltrating CD4+ T cells may impede antitumor T and NK cell function (10). This inhibitory T cell subset may include CD4+CD25+Foxp3+ T cells (referred to as regulatory T (Treg)3 cells) capable of significantly inhibiting T cell immunity (11, 12). In support of this scenario are studies reporting that in vivo depletion of CD4+ T cells is moderately effective in raising immunity against established tumors. It may be possible to remove inhibitory CD4+ T cells while simultaneously preserving antitumor CD8+ effector T cells (13, 14). The impact of CD4+ T cell depletion on the recall response of antitumor memory T cells has not been systematically studied, particularly when combined with antitumor immunotherapy.

It has been observed that a systemic or persistent presence of tumor Ag can impair memory CD8+ T cell function (15). Continuous triggering of the TCR may lead to an up-regulation of negative regulatory molecules and an accumulation of inhibitory Treg cells. B7-H1 (also referred to as CD274 or PD-L1) is a coinhibitory molecule that binds to the programmed death-1 (PD-1) receptor on T cells (16, 17). B7-H1/PD-1 engagement results in T cell apoptosis, altered T cell cytokine production, diminished proliferation, and reduced cytotoxicity of effector T cells (18, 19, 20). CD8+ T cells are more sensitive to the regulation of B7-H1 signals, as demonstrated by the delayed hepatic deletion of activated CD8+ T cells and increased expansion and survival of CD8+ T cells in B7-H1-deficient mice (21, 22). Blocking B7-H1 signaling has been shown to improve antitumor immunity and immunotherapy (23, 24). However, the role of B7-H1 in regulating memory T cell responses is largely unknown.

PD-1 has been reported to be detected on exhausted T cells from hosts with chronic viral infections such as lymphocytic choriomeningitis virus in mice and HIV in humans (25, 26, 27, 28). Blocking the binding of PD-1 to B7-H1 significantly improved the recall responses of viral-specific T cells and resulted in the clearing of virus (25). Stimulation of PD-1 receptors on HIV-specific memory T cells induces apoptosis (27). Collectively, these studies suggest that in the setting of viral infection, signaling via the B7-H1/PD-1 pathway is an important mechanism for controlling and down-regulating the response of memory T cells to Ag.

Extrapolating from B7-H1/PD-1 viral data, we hypothesize that negative T cell costimulation via the B7-H1/PD-1 pathway coupled with a presence of inhibitory Treg cells may severely impair antitumor memory T cell responses. Moreover, blocking the inhibitory mechanisms might improve the function of memory T cells and result in efficient clearance of established tumors. To test this hypothesis, we used a murine renal cell carcinoma (RENCA) tumor model to examine the distribution of tumor-specific memory T cells and its relationship with B7-H1 expression. We evaluated the impact of B7-H1 signaling and the presence of Treg cells on the potency of the antitumor immune response generated by memory CD8+ T cells. We also explored a novel, synergistic approach to treating large established tumors by combining a tumor vaccine with B7-H1 blockade and CD4+ T cell depletion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines and mice

Murine RENCA cells were cultured in RPMI 1640 medium (Mediatech) with 10% FBS (Invitrogen Life Technologies), 1 U/ml penicillin, 1 µg/ml streptomycin, and 20 mM HEPES buffer (all from Mediatech). BALB/c wild-type mice and BALB/c Rag–/– female mice were purchased from Taconic Farms. Mice were maintained under specific pathogen-free conditions and used for study at 8–12 wk of age. Studies were conducted in accordance with the National Institutes of Health guidelines for the proper use of animals in research and with local Institutional Animal Care and Use Committee approval.

Flow cytometric analysis and tissue staining

Anti-mouse B7-H1 Ab (clone 10B5, hamster) and anti-mouse CD4 Ab (GK1.5, rat) were purified using a protein G agarose column (Amersham) from hybridoma culture supernatants containing Ultra-Low Ig serum (Invitrogen Life Technologies) (24). Rat IgG and hamster IgG were obtained from Sigma-Aldrich. Fluorescence-conjugated Abs against memory CD3 (mCD3; 145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD122 (5H4), CD25 (7D4), Ly6C (AL-21), CD103 (M290), CD107a (1D4B), and IFN-{gamma} (XMG1.2) were purchased from BD Pharmingen. Rat anti-mouse Foxp3 (FJK-16s) was purchased from eBioscience. Flow cytometry was performed using FACScan and FACSCalibur (BD Biosciences) instrumentation and CellQuest (BD Biosciences) and FlowJo software (Tree Star).

Treatment of RENCA tumors

Mice were inoculated s.c. in the right flank with 1 x 106 RENCA tumor cells in 100 µl of Dulbecco’s PBS (Mediatech). After tumors were established (40–70 mm2), mice were vaccinated i.p. with 1 x 106 irradiated (16,000 rad) RENCA cells (day 0). This procedure was repeated on days 3, 6, and 9. Simultaneously, mice were injected i.p. with 200 µg of anti-B7-H1 Ab (clone 10B5) diluted in 200 µl of PBS. Tumor growth was monitored by measuring the longest bisecting diameters of flank tumors. Mice were sacrificed when the tumors displayed ulceration or reached 250 mm2. Depletion of CD4+ T cells was achieved by i.p. injection of 250 µg of anti-CD4 Ab (GK1.5) performed on days –3, 1, 7, and 14. Depletion of CD25+ T cells was performed using i.p. injection of 400 µg of anti-CD25 Ab (PC61) on day –3. Both CD4 and CD25 depletion were confirmed using flow cytometry in combination with noncross-reactive Abs against CD4 (RM4-5) and CD25 (7D4). Control groups for these studies included cohorts of mice receiving no treatment or irrelevant isotype-matched hamster or rat IgG.

Detection of CTL activity using the CD107a degranulation assay

To monitor functional activity of putative tumor-specific CD8+ CTLs, the CD107a mobilization assay was performed, as previously described, to detect the CTL degranulation (29). Briefly, splenocytes were cultured overnight in CTL medium (IMDM, with 10% FBS, penicillin, streptomycin, and L-glutamine) (Invitrogen Life Technologies) supplemented with 100 U/ml IL-2 (Chiron). Following removal of dead cells using a lymphocyte-M gradient (Cedarlane Laboratories), splenocytes were incubated with target RENCA or EMT-6 (control) cells at a ratio of 1:2 (E:T) for 5 h at 37°C in 5% CO2 in the presence of GolgiStop, GolgiPlug, and FITC-conjugated CD107a Ab (BD Pharmingen). After incubation, samples were then stained with Abs specific for CD8 and CD122, followed by intracellular staining for IFN-{gamma}.

Adoptive transfer of memory CD8+ T cells

Memory CD8+ T cells were purified from long-term (>50 days after treatment) immunotherapy-treated tumor-free mice or tumor cell vaccine-immunized mice, using a mCD8+ T cell enrichment kit (StemCell Technologies), and in some experiments they were further expanded in culture with IL-15 and IL-21 (100 ng/ml each) for 5 days. Female Rag–/– mice were inoculated s.c. on the right flank with 1 x 106 RENCA tumor cells in 100 µl of Dulbecco’s PBS. After the tumors were established, treatment was initiated by injecting 1 x 106 memory CD8+ T cells. Along with memory CD8+ T cell transfer, Rag–/– mice received 200 µg of i.p. anti-B7-H1 Ab (10B5) in 200 µl of PBS twice weekly for 4 wk. In some experiments, purified CD4+CD25+ T cells (1 x 105 cells) or CD4+CD25– T cells (1 x 106 cells) were coinjected with memory CD8+ T cells. Control groups for these studies included mice receiving no treatment or irrelevant hamster IgG.

Functional assay of Treg cells

Lymph nodes from RENCA tumor-bearing mice or naive mice were collected, and single-cell suspensions were used. Magnetic cell sorting was performed to obtain purified CD4+CD25+ Treg cells (Miltenyi Biotec). The CD4+CD25– fraction was labeled with 1 µM CFSE (Molecular Probes), as described in the manufacturer’s manual, and used as responder cells, whereas the CD4+CD25+ cells were used as suppressor cells. CFSE-labeled CD4+CD25– T cells (5 x 104/well) were incubated with an equal number of dendritic cells and titrated numbers of CD4+CD25+ Treg cells in the presence of anti-CD3 (0.25 µg/ml) for 3 days. After incubation, cells were stained with anti-CD4 and analyzed by flow cytometry. Percentage of inhibition was calculated as follows: (1 – % of CFSElow cells with Treg cells/% of CFSElow cells without Treg cells) x 100%.

Statistical analysis

Unless otherwise indicated, all experiments were performed independently at least three times. A two-sided, unpaired Student’s t test was used to assess statistical differences in tumor growth between groups of mice. A p value ≤0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Temporal compartmentalization of the immune response to a tumor vaccine

RENCA in mice is a valuable tumor model because RENCA cells are immunogenic, and when administered as irradiated cells (tumor cell vaccine) protective immunity is induced in naive mice, as measured by their ability to reject subsequent challenge using live RENCA cells (30). Naive and tumor-bearing BALB/c mice were challenged with 1 x 106 irradiated RENCA cells. The memory T cells were distinguished from effector cells by their unique high expression of CD122 (IL-2Rβ/IL-15Rβ). The cytolytic activity of CD8+ T cells was evaluated by CD107a (lysosomal membrane glycoprotein 1) expression (29). CD107a+ functional T cell subsets, CD122high (memory) and CD122low (effector), CD8+ T cells were identified in spleens and bone marrows by flow cytometry at day 7 (Fig. 1, A, C, and E) and day 22 (Fig. 1, B, D, and F) post tumor cell challenge. Functional memory CD8+ T cells (CD107a+CD122high) accumulated in the bone marrow (5.9%, 5.3 ± 0.8 x 104 and 12.5%, 4.9 ± 1.9 x 104 at days 7 and 22) and in the spleen (0.4%, 3.8 ± 0.1 x 104 at day 22) postchallenge in tumor-bearing mice, but were relatively infrequent in the spleen (<0.3% at days 7) and bone marrow (0.3 and 1.7%) of naive mice challenged with tumor cells (Fig. 1, A–D). In contrast, CD107a+CD122low effector CD8+ T cells (Fig. 1E) were more prevalent in the spleen (17.4%, 1.6 ± 1.1 x 105) than bone marrow (2.9%, 0.03 ± 0.04 x 105) at day 7 in tumor mice. At day 22, effector cells in the spleen were reduced to a level (1.2%, 0.1 ± 0.01 x 105) comparable to that seen in the bone marrow from naive and tumor-bearing mice (2.4 and 2.7%, 0.03 ± 0.01 x 105, and 0.032 ± 0.02 x 105) (Fig. 1, B and F). These results suggest that tumor vaccination in tumor-bearing mice elicits a two-phase immune response. In the first phase, occurring during the first week following vaccination, there is an accumulation of memory T cells in the bone marrow and effector CTLs in the spleen. In the second phase, occurring over a 2-wk interval, there is a significant decay in the splenic effector T cell population, whereas the memory T cells persist in the bone marrow.


Figure 1
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  		FIGURE 1. Presence of functional memory T cells in the bone marrow of tumor-bearing mice. Naive BALB/c mice or mice with established RENCA tumors (day 10–14, 40–50 mm2, right flank) were challenged with irradiated tumor vaccine (1 x 106 RENCA cells s.c. left flank). Seven and 22 days later, lymphocytes were isolated from spleen (SP) and bone marrow (BM). Cells were stained with anti-CD8 and anti-CD122 for the detection of memory phenotype. The CTL activity (degranulation) of memory CD8+ T cells was analyzed by the CD107a mobilization, as mentioned in Materials and Methods. Representative data of flow cytometry assays (A, day 7; B, day 22). Circle gates indicate the percentage of functional memory T cells (CD122highCD107a+, TM), and the square gates indicate the percentage of effector T cells (CD122lowCD107a+, TE) in total CD8+ T cells. Summaries of absolute numbers (total cell numbers x gated events/total events collected) of TM cells and TE cells on day 7 (C and E) and day 22 (D and F) are based on the flow cytometry assays (A and B). Values are means ± SD for three mice. *, Denotes a significant difference from naive mice, p = 0.016 (in the bone marrow of C), p < 0.001 (in the spleen of D). Data shown are representative of three independent experiments.

 
Bone marrow dendritic cells demonstrate lower expression of coinhibitory molecules compared with those found in the spleen

To better understand the contrasting distribution of memory and effector T cells, we investigated the variable expression of coinhibitory molecules B7-H1 and B7-2 on resident spleen and bone marrow dendritic cells. It was determined that both spleen CD8{alpha}+CD11c+ and CD8 {alpha}–CD11c+ dendritic cells expressed B7-2, whereas spleen B7-H1 expression was skewed to the CD8{alpha}–CD11c+ dendritic cell population (Fig. 2A). Class II (I-Ad) expression was comparable regardless of CD8 level. B7-H1 and B7-2 expression was lower (p < 0.01) in the CD8{alpha}–CD11c+ bone marrow dendritic cells as compared with spleen dendritic cells (Fig. 2, B and C). Given the preferential accumulation of memory T cells in the bone marrow (where B7-H1 dendritic cell levels are low) and the rapid decay of effector T cells in the spleen (where B7-H1 dendritic cell levels are high), we hypothesized that B7-H1 signaling may be negatively regulating, possibly via interaction with PD-1 receptors, the accumulation and functioning of memory T cells following tumor vaccine treatment.


Figure 2
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  		FIGURE 2. B7-H1 levels in the dendritic cells of spleen and bone marrows. Freshly isolated CD11c+ dendritic cells from spleen and bone marrows of BALB/c mice were stained with anti-CD8{alpha} and anti- B7-H1, anti-B7-2, or anti-I-Ad. A, B7-H1 was mainly expressed by the CD8{alpha}–CD11c+ dendritic cells in the spleen. Numbers indicate the frequencies in each quadrant. B and C, Levels of B7-H1, B7-2, and I-Ad in the CD8{alpha}–CD11c+ dendritic cells of spleen (SP) and bone marrows (BM). Numbers indicate the percentages of positive staining in CD8{alpha}–CD11c+ dendritic cells in B. Values are means ± SD for three mice in C. *, Denotes a significant difference from that of spleen, p < 0.01. Data shown are representative of two experiments.

 
B7-H1 blockade improves the efficacy of functional antitumor memory T cells

To investigate whether B7-H1 signals suppress the reactivation of tumor-specific memory CD8+ T cells, we adoptively transferred tumor-specific memory CD8+ T cells into RENCA tumor-bearing Rag–/– mice alone or with B7-H1-blocking mAb. RENCA tumor-specific memory CD8+ T cells isolated from tumor cell vaccine-immunized mice had a phenotype of CD44highCD62Lhigh, and showed RENCA tumor-specific cytolytic activity (degranulation and IFN-{gamma} production) (Fig. 3A). In addition, low levels of PD-1 expression were found in these memory CD8+ T cells after 5 days of in vitro culture; a process that mimics the in vivo proliferation. Mice receiving no adoptive therapy had tumors of ~250 mm2 at day 41. Mice receiving memory CD8+ T cells and control mAb had slightly smaller tumors (175 mm2). In contrast, the combination therapy of memory CD8+ cell transfer and B7-H1 blockade resulted in the elimination of tumor growth (Fig. 3B). In addition, B7-H1 blockade increased the frequency of CD107a+IFN-{gamma}+ as well as CD107a+IFN-{gamma}– memory CD8+ T cells by 2-fold (4.7 vs 2.3%; 4.5 vs 2.7%) in their ex vivo responses to RENCA tumor targets (Fig. 3C). The tumor specificity of these was confirmed by using control EMT-6 tumor cells as a negative target. These results demonstrate that B7-H1 engagement impairs the quality (degranulation and cytokine production) and quantity of functional memory CD8+ T cells and results in compromised tumor elimination.


Figure 3
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  		FIGURE 3. Blocking B7-H1 improved antitumor activity of memory CD8+ T cells. A, Profile of memory CD8+ T cells. Memory CD8+ T cells were analyzed for their expression of CD44, CD62L, and PD-1 (bold line). Tumor-specific function of memory CD8+ T cells was detected by their degranulation (CD107a assay) and intracellular IFN-{gamma} production in response to RENCA tumor cells and EG7 tumor cells (negative control target). B, Tumor growth in Rag–/– mice. Hamster IgG (control Ab, cAb) or anti-B7-H1 Ab (10B5) injected i.p. (200 µg/mouse) on the same day of memory CD8+ T cell transfer (1 x 106/mouse, i.v.), and twice weekly for 4 wk. Mice without treatment were used as a control group. Values are means ± SD for three to four mice. *, **, Significant difference from control: *, p < 0.05; **, p < 0.01 by Student’s t test. Data shown are representative of two independent studies. C, CTL activity of recovered memory T cells from treated Rag–/– mice (B). Pooled spleen cells were isolated from three mice on day 44 after treatment with anti-B7-H1 or control Ab. Mobilization of CD107a and IFN-{gamma} production in memory T cells was analyzed after incubation with target RENCA or EMT-6 tumor cells for 5 h in vitro. Numbers indicate the percentage of each quadrant gated on CD8+ T cell population. D, Tumor growth in normal mice. A total of 1 x 106 memory CD8 T cells transferred into lightly irradiated (250 rad) tumor-bearing normal mice. Hamster IgG (control Ab, cAb) or anti-B7-H1 Ab (10B5) injected i.p. (200 µg/mouse) on the same day of memory CD8+ T cell transfer (1 x 106/mouse, i.v.), and twice weekly for 4 wk. To deplete Treg cells, anti-CD4 Ab (GK1.5, 250 µg/mouse, i.p.) was injected on days 0 and 4 after memory T cell transfer. Mice without treatment were used as a control group. Values are means ± SD for four to five mice. *, **, Denote significant differences from control: *, p < 0.05; **, p < 0.001 by Student’s t test.

 
Because in Rag–/– mice the only T cells are transferred CD8 T cells, it should be clarified whether the antitumor effect is due to homeostatic Ag-independent expansion of memory/effector CD8 T cells or due to the absence of other suppressive T cells. Therefore, we used normal mice with established RENCA tumors as a model. As shown in Fig. 3D, the transfer of 1 x 106 memory CD8 T cells into lightly irradiated (250 rad) tumor-bearing normal mice was not sufficient to eradicate the tumor in combination with anti-B7-H1 (153 mm2) compared with mice without treatment (192 mm2) or with control Ab (162 mm2). However, the combination of anti-B7-H1 and depletion of CD4+ T cells induced dramatic regressions in tumors (9.6 mm2; p < 0.001 vs control on day 20–27 after treatment). These results suggest that in addition to B7-H1 coinhibitory signals, the endogenous CD4+ T cells also negatively affect the antitumor function of memory CD8 T cells.

CD103+ Treg cells accumulate within tumor-draining lymph nodes

T cells expressing a CD4+CD25+Foxp3+ phenotype have been associated with suppression of immunity and are designated as Treg cells. The proportion of Treg cells detected in regional lymph nodes of tumor-bearing mice was only slightly elevated (11.5%) compared with naive mice (10.1%; data not shown). However, the percentage of CD103+ Treg cells, thought to be a subset of Treg cells (31, 32, 33, 34), was significantly greater in regional lymph nodes of tumor-bearing (17.9 ± 2.1%) compared with naive mice (12.8 ± 1.5%; p = 0.008) (Fig. 4, A and B). In contrast to tumor-draining lymph nodes, the spleens of tumor-bearing mice and naive mice harbored similar levels of CD103+ Treg cells; however, the bone marrow in tumor-bearing mice contained a significantly lower proportion of CD103+ Treg cells (16.9 ± 0.3%) than in naive mice (20.2 ± 0.8%; p = 0.034). Low levels of PD-1 expression were found on these freshly isolated Treg cells (Fig. 4C), indicating they were not fully activated. Treg cells from tumor-bearing and naive mice showed comparable regulatory function in suppressing CD25– T cell proliferation in vitro (Fig. 4D). These data suggest RENCA tumor cells may initiate a migration of functional Treg cells from other reservoir organs (such as bone marrow) to accumulate in tumor regional lymph nodes based on their up-regulation of CD103, a homing ligand to epithelial tissues.


Figure 4
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  		FIGURE 4. Tumor-associated functional Treg cell accumulation. Lymphocytes were isolated from the spleen, bone marrow, and tumor-draining lymph nodes of mice with established RENCA tumors or naive mice and were stained with Abs for CD4, CD25, and CD103, followed with intracellular staining for Foxp3. A, Numbers indicate percentages in each quadrant gated on CD4+CD25+ populations. B, Histograms show the expression of PD-1 on Foxp3+CD4+CD25+ Treg cells. C, A summary of flow cytometry assays of six mice. Values are means ± SD. *, Denotes a significant difference from naive mice, p = 0.008 in tumor-draining lymph nodes, p = 0.035 in tumor bone marrow. D, Functional assay of CD4+CD25+ Treg cells. Purified Treg cells from naive or tumor-bearing mice were incubated at indicated ratios with CFSE-labeled CD4+CD25– responder cells for 3 days in the presence of anti-CD3 and accessory cells. Histograms show the CFSE dilution of responder cells. A summary of suppressive function of Treg cells at different ratios.

 
Treg cells inhibit the effector phase of memory CD8+ T cell responses

To assess whether endogenous CD4+CD25+ Treg cells have functional immunosuppressive properties in antitumor memory responses, adoptive transfer experiments involving CD4+CD25+ Treg cells and antitumor memory CD8+ T cells were undertaken. As shown in Fig. 5, on day 23 control mice had tumors of ~250 mm2, and the adoptive transfer of memory T cells had reduced the average tumor burden to 175 mm2. However, when CD4+CD25+ Treg cells (21.7% of these purified Treg cells are CD103+) were included in the transfer, there was a significant impairment of tumor elimination in that tumor sizes in these mice were the same as control mice (mCD8 vs mCD8 plus Treg; p = 0.039). Incorporation of CD4+CD25– Th cells in the transfer augmented tumor regression (50-mm2 tumors; mCD8 plus Th vs control; p = 0.045). When viewed in terms of survival following adoptive transfer, results are more impressive (Fig. 5B). At day 35 posttreatment, 80 and 100% of mice receiving either memory CD8+ T cells alone or with CD4+CD25– Th cells, respectively, were alive. In contrast, all controls as well as mice receiving CD4+CD25+ Treg cells with memory CD8+ T cells were dead. These results are consistent with the hypothesis that CD4+CD25+ Treg cells are immunosuppressive in the context of antitumor memory response.


Figure 5
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  		FIGURE 5. Antitumor activity of memory CD8+ T cells was inhibited by Treg cells. Memory CD8+ T cells (1 x 106) were adoptively transferred (i.v.) into Rag–/– mice with established RENCA tumors. CD4+CD25+ Treg cells (1 x 105 cells) or CD4+CD25– Th cells (Th, 1 x 106 cells) were cotransferred into RENCA-bearing Rag–/– mice along with memory CD8+ T cells. A, Cotransfer with CD4+CD25+ Treg cells inhibits RENCA tumor rejection, whereas cotransfer of CD4+CD25– Th cells appears to accelerate tumor rejection. Values are means ± SD of three to four mice. *, Significant difference in mCD8 plus Th cells vs control, p = 0.031 (day 23); and in mCD8 vs control or mCD8 plus Treg, p = 0.039 (day 20). There was no significant difference in mCD8 vs mCD8 plus Th. Data are representative of two independent experiments. B, The survival of tumor-bearing mice after treatments.

 
B7-H1 blockade and CD4+ T cell depletion improve the effectiveness of tumor cell vaccination

Having established that administration of irradiated RENCA cells is an effective tumor vaccine (30), that endogenous CD4+CD25+ Treg cells are immunosuppressive, and coupled with the observation that B7-H1 expression varied among lymphoid tissues, we investigated whether the effectiveness of tumor vaccine therapy would be augmented by incorporating either B7-H1 blockade and/or CD4+ or CD25+ T cell depletion (Fig. 6). Mice receiving tumor vaccine and control Ab treatment developed tumors of >250 mm2 by day 21 of treatment (Fig. 6A), whereas tumor vaccine combined with B7-H1 blockade resulted in smaller tumors (175 mm2). Incorporating either B7-H1 blockade or CD4+ T cell depletion as part of the vaccine treatment resulted in a modest reduction in tumor burden (175 and 100 mm2, respectively). Most noteworthy, a triple therapy of tumor vaccine plus B7-H1 blockade and CD4+ T cell depletion resulted in a complete and long-lasting regression of tumors. All mice receiving the triple therapy treatment had long-term survival of greater than 35 days (Fig. 6B). Thus, a triple combination of tumor vaccine, B7-H1 blockade, and CD4+ T cell depletion was most effective in rejecting established tumors.


Figure 6
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  		FIGURE 6. B7-H1 blockade and CD4+ T cell depletion improve the efficacy of tumor vaccine therapy. Mice with established RENCA tumors were treated with tumor vaccine (day 0; VAC) and anti-B7-H1 Ab (10B5), or control hamster IgG Ab (control Ab), on days 0, 3, 6, and 9. A, Anti-CD4 (GK1.5) was injected on days –3, 1, 7, and 14 to deplete CD4+ T cells, or C, anti-CD25 Ab was given on day –3. Tumor size changes following treatments (A and C) are shown in the left panels. Values are means of tumor size (mm2 ± SD) of five mice. *, p < 0.05; **, p < 0.01 vs no-treatment group. Long-term survival (B and D) of mice treated with triple therapy (i.p. vaccine combined with B7-H1 blockade and CD4 T cell depletion) is shown in the right panels, p = 0.009 vs no-treatment group.

 
We then determined whether depletion of CD25+ T cells would be similarly efficient. Under conditions in which untreated mice developed >250-mm2 tumors at day 19 (Fig. 6C), the combination therapy of CD4+ T cell depletion coupled with B7-H1 blockade again resulted in complete tumor elimination and 100% survival at day 35. However, CD25+ T cell depletion did not result in a significant reduction of tumor burden (275 mm2), and all mice receiving CD25+ T cell depletion were dead by day 35 (Fig. 6D).

Memory tumor immunity is preserved after depletion of CD4+ T cells

Because the triple therapy of tumor vaccine, B7-H1 blockade, and CD4+ T cell depletion were curative 35 days posttreatment, it was deemed important to measure some parameters of immunity existing in treated mice. On day 20 after treatment, there was a slight increase in the percentage and absolute numbers of CD122highLy6C+ memory CD8+ T cells comparing vaccine alone (22.2%; 3.5 x 106/spleen)- and triple therapy (33.9; 5.3 x 106/spleen)-treated mice (Fig. 7A). However, there was a nearly 3-fold increase in the percentage and 2-fold increase in absolute numbers of CD122lowCD107a+ recalled effector CD8+ T cells in triple therapy-treated mice (12.1%, 1.8 x 104/spleen) compared with mice treated with tumor vaccine alone (4.7%, 0.8 x 104/spleen) (Fig. 7A). To test the established immunity (memory) to secondary tumor challenge after triple therapy, we rechallenged these tumor-free mice with a lethal dose of RENCA cells. As shown in Fig. 7B, no palpable RENCA tumors were detected at either day 50 postchallenge or as late as 100 days after challenge (data not shown) in these mice after triple therapy. However, the growth of control EMT6 tumor cells injected into the contralateral right flank was not affected (Fig. 7B), indicating this protection was tumor specific.


Figure 7
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  		FIGURE 7. Establishment of antitumoral memory following therapy. A, Presence of functional antitumor memory CD8+ T cells in treated mice. Twenty days after triple therapy (Triple) or vaccine-alone treatment (VAC), splenocytes were isolated and stained with anti-CD8, anti-Ly6C, and anti-CD122 (top panels). The percentages shown are gated on CD8+ T cells. The CTL activity (degranulation) of memory CD8+ T cells was analyzed by ex vivo CD107a mobilization assay after reactivation of spleen cells for 1 wk (bottom panels). The numbers represent the percentages of CD107a+CD122low recalled effector cells in total CD8+ T cells. Absolute numbers (total cell numbers x gated events/total events collected) of gated cells are shown in the parentheses. B, Long-term tumor immunity in treated mice. Fifty days after triple therapy, RENCA tumor-free mice were challenged s.c. with lethal doses of both 1 x 106 RENCA tumor cells (left flank) and 1 x 106 unrelated EMT-6 tumor cells (right flank). Values are mean tumor sizes ± SD of six mice. *, Significant difference from the size of EMT-6 tumors, p < 0.001. C, Recalled responses of anti-RENCA memory CD8+ T cell in tumor-free mice. Naive or treated (tumor free for >40 days after treatment) mice were rechallenged with 1 x 106 RENCA tumor cells s.c. Seven days later, the spleen cells were analyzed for memory phenotype (Ly6C+CD122high) and recalled effector function (CD122lowCD107a+) of these CD8+ T cells. Numbers indicate the percentage of gated area in CD8+ T cell populations. Absolute numbers of gated cells are shown in the parentheses.

 
Again, 40 days after triple therapy, tumor-free mice had increased percentage as well as absolute numbers of both CD122highLy6C+ memory (37.8%, 17.1 x 106/spleen vs 11.9%, 4.2 x 106/spleen) and CD122lowCD107a+ recalled effector (13.9%, 6.3 x 104/spleen vs 5.4%, 1.7 x 104/spleen) CD8+ T cells compared with naive mice when they were rechallenged with fresh tumor cells (Fig. 7C). Thus, the triple therapy protocol mobilized functional populations of memory and effector cells and resulted in the establishment of long-lasting, tumor-specific immunity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We report B7-H1 molecules and CD4+CD25+ Treg cells are involved in suppressing protective T cell memory in the RENCA tumor system. Interfering in this suppression, either with B7-H1 blockade or depletion of populations containing Treg cells, results in optimal generation of antitumor immunity. These results are consistent with the emerging concept of the existence of multiple regulatory mechanisms that thwart the generation of immune responses to tumor cells.

In tumor-bearing mice, we detected an accumulation and persistence of functional CD122high memory CD8+ T cells in bone marrow, followed by emergence of functional CD122low effector CD8+ T cells in the spleen after tumor vaccine therapy. However, with time, the level of effector cells decreased. Interestingly, the distribution of B7-H1 expression on dendritic cells was lower in the bone marrow and higher in the spleen. Thus, it is possible that memory T cells can persist in the bone marrow due to low levels of the inhibitory B7-H1 molecule, whereas long-term survival of effector T cells is adversely affected by increased expression of B7-H1 within the spleen. Supporting this possibility is our observation that B7-H1 blockade as part of tumor vaccine therapy inhibited tumor growth and increased host survivability. Our data are consistent with reports (23, 24) that B7-H1 blockade will be a useful approach in improving antitumor responses, especially in increasing the quality and quantity of tumor-specific memory CD8+ T cells (Fig. 3). It would be of interest to ascertain whether differences in the level of B7-H1 among anatomically distinct (bone marrow vs spleen) dendritic cells contribute to suboptimal immune responses. B7-H1 expression by dendritic cells has been implicated in the induction and maintenance of T cell anergy and tolerance (35, 36). Tumor myeloid dendritic cells express high levels of B7-H1, and the blockade of B7-H1 engagement enhances infiltration of effector CD8+ T cells, thereby conferring improved antitumor immunity (37). However, B7-H1 blockade alone was not sufficient to induce protective immunity. Therefore, additional immune system pathways must be considered when designing antitumor therapies.

Of topical interest is the role of Treg cells in adversely affecting antitumor responses. T cells expressing CD4+CD25+ phenotype have been reported to suppress the proliferation and survival of memory CD8+ T cells in response to either bacterial or alloantigen challenge as well as inhibiting general CD8+ T cell homeostasis (38, 39, 40). Endogenous CD4+CD25+ Treg cells suppress antitumor memory responses, and when included in a transfer of memory CD8+ T cells, host mice become impaired in their ability to eliminate tumor cells. Recently, CD4+CD25+ Treg cells have been subdivided based upon expression of CD103 (41). CD103+ Treg cells are thought to be adaptive, peripherally generated suppressors of specific Ag responses (34). Furthermore, CD103+ Treg cells are capable of migrating to sites of inflammation where, following secretion of IL-10 and TGF-β, they inhibit the cytolytic activities of effector cells within the microenvironment (31, 33). We report the novel observation that CD103+ Treg cells increased within tumor-draining lymph nodes of RENCA tumor-bearing mice. The differential temporal and tissue accumulation of CD103+ Treg cells potentially opens new avenues for identifying novel Treg subsets and delineating their roles in determining immune outcomes.

One of the issues confounding antitumor therapies is the rather controversial topic of the role of CD4+ T cells affecting immune outcomes. Although it is generally accepted that CD4+ T cells play a fundamental role in the immune system activation and memory CD8+ T cell generation (42), it is also realized that CD4+ T cell populations can adversely impact tumor immunity (13, 14, 43). Both Th and Treg cells express the CD4 molecule, and, depending upon the model system, assay conditions, cell preparation, and isolation strategy used, it might be expected that different conclusions could be reached as to the role of CD4+ T cells in affecting immunity. It has been reported that depletion of CD4+ T cells is an effective means of immunotherapy when transferring tumor-specific CD8+ T cells (43). Our results demonstrate that when combined with tumor vaccine and B7-H1 blockade therapy, elimination of CD4+ T cells results in the generation of protective immunity. However, as others have reported (44), adoptive transfer of CD8+ T cells is more effective if CD4+ Th cells are included. Our data in Fig. 5 showed that in the absence of Treg cells in Rag–/– mice, cotransfer of CD4+CD25– Th cells improved the therapeutic effects of transferred memory CD8+ T cells. Thus, the complete depletion of CD4+ T cells may diminish the antitumor immune responses in normal mice. However, we found that blockade of B7-H1 signals improved the antitumor function of memory CD8+ T cells in the absence of CD4+ T cells in both Rag–/– mice and normal mice (Fig. 3, B and D). A recent study made a similar observation in a chronic viral infection model in which blockade of B7-H1 (PD-L1) dramatically restored the function of viral-specific CD8+ T cells in the absence of CD4+ T cells (25). Based on these results, it is speculated that CD4+ Th cells may provide signals to overcome B7-H1/PD-1-mediated negative effects on CD8+ T cells. Thus, in the absence of CD4+ Th cells, blockade of B7-H1 will help to restore the function of CD8+ T cells in vivo.

In conclusion, we report the presence of the coinhibitory molecule B7-H1 and Treg cells suppresses the reactivation of pre-existing tumor-specific memory T cells. Interfering with these immune-suppressing pathways dramatically improves the efficacy of tumor vaccine therapy. Memory T cells tend to accumulate and persist in the bone marrow (site of low B7-H1 expression), whereas cytolytic effector T cell levels elevate rapidly in the spleen. However, because of high levels of B7-H1 expression in the spleen, the proportion of effector cells quickly diminishes. B7-H1 levels in tissues may directly impact the antitumor immune response. A subset of Treg cells, CD103+ Treg cells, accumulates in the regional draining lymph nodes and inhibits the reactivation of tumor-specific memory T cells. The incorporation of B7-H1 blockade and CD4+ T cell depletion into tumor cell vaccine therapy resulted in complete tumor regression and long-lasting, tumor-specific immunity. Given that aggressive forms of many human cancers express high levels of B7-H1 (45, 46, 47), and that many patients harbor elevated levels of CD4+ Treg cells within their tumors (48), our studies may provide useful insights for improving immunotherapeutic approaches to treat advanced cancers in the clinical setting.


   Acknowledgments
 
We thank Dr. Eugene Kwon for critical reading and helpful suggestions, and Dr. Christopher Krco for editing this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 the Fraternal Order of Eagles Cancer Research Fund and Faculty Transition Research Award from Mayo Foundation. Back

2 Address correspondence and reprint requests to Dr. Haidong Dong, Departments of Urology and Immunology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail address: dong.haidong{at}mayo.edu Back

3 Abbreviations used in this paper: Treg, regulatory T; mCD, memory CD; PD-1, programmed death-1; RENCA, renal cell carcinoma. Back

Received for publication January 9, 2007. Accepted for publication June 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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