Published online before print January 3, 2008
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Department of Pediatrics, Steele Children’s Research Center, University of Arizona, Tucson, Arizona, USA
1 Correspondence: University of Arizona, Department of Pediatrics, 1501 N. Campbell Ave., P.O. Box 245073, Tucson, AZ 85724-5073, USA. E-mail: katsanis{at}peds.arizona.edu
ABSTRACT
CD4+CD25+ regulatory T lymphocytes (Tregs) critically contribute to the mechanisms of cancer-induced tolerance. These cells suppress anti-tumoral CD8+ and CD4+ T lymphocytes and can also restrain the function of APCs. We have previously documented the immunostimulatory effects of a chaperone-rich cell lysate (CRCL) anti-cancer vaccine. Tumor-derived CRCL induces tumor immunity in vivo, partly by promoting dendritic cell (DC) and macrophage activation. In the current study, we evaluated the effects of CD4+CD25+forkhead box P3+ Tregs isolated from mice bearing 12B1 bcr-abl+ leukemia on DC and macrophages that had been activated by 12B1-derived CRCL. CRCL-activated DC and macrophages resisted Treg suppression, as the production of proinflammatory cytokines, the activation of transcription factor NF-
B, and their immunostimulatory potential was unaffected by Tregs. Our results thus highlight CRCL as a powerful adjuvant endowed with the capacity to overcome tumor-induced Treg-inhibitory effects on APCs.
Key Words: dendritic cells • monocytes-macrophages • CD4+CD25+ regulatory T lymphocytes • chaperone-rich cell lysate
INTRODUCTION
A small population of CD4+ T cells coexpressing CD25 [CD4+CD25+ regulatory T cell lymphocytes (Tregs)] critically contributes to the complex regulatory processes that govern the maintenance of peripheral self-tolerance [1 2 3 4 ]. More specifically characterized by the expression of forkhead box P3 (FoxP3), a major transcription factor involved in their lineage commitment, development, and function [4 5 6 7 ], naïve CD4+CD25+ Tregs express several cell-surface markers also detected on conventional, nonsuppressive T cells, including CTLA-4, glucocorticoid-induced TNFR, CD62 ligand, or OX-40 [6 ]. It has become increasingly evident that these cells are endowed with the capacity to suppress immune responses to self and foreign antigens, which places them at a crucial checkpoint in modulating autoimmunity, infections, or cancer [8 , 9 ]. However, the molecular basis for the suppressive activity of CD4+CD25+ Tregs and their relationship to other members of the growing family of regulatory cells remains contentious. Although some of the mechanisms by which Tregs exert their suppressive control may involve a cell-to-cell, contact-dependent inhibition of IL-2 production by effector T cells, immunosuppressive cytokines such as TGF-β and IL-10 may also be involved [9 10 11 12 13 ].
CD4+CD25+ Treg induced during tumor progression participate in the establishment and persistence of cancer-mediated immune tolerance [3 , 8 , 14 15 16 ], representing, therefore, an obstacle for successful immunotherapy. An increase in the frequency of these cells has been detected in the blood, lymph nodes, and spleen of tumor-bearing hosts [17 18 19 20 21 ], and their therapeutic depletion improves responses to cancer immunotherapy [17 , 19 , 22 23 24 ]. Expanded Tregs compromise the function of anti-tumor effector CD8+ T cells and can also curtail CD4+ T cell help [14 , 15 , 19 , 25 , 26 ].
Although effector CD4+ and CD8+ T lymphocytes have first been identified as the primary targets of Tregs, recent reports have indicated that CD4+CD25+ Tregs may exert their inhibitory effects on APCs as well. In particular, Tregs may down-regulate monocyte/dendritic cell (DC) costimulatory molecule expression and proinflammatory cytokine production, impair the capability of APCs to present antigens to T cells [27 28 29 30 31 ], or promote DC-suppressive properties [32 ]. These effects may be critical, as tumor-induced Tregs can maintain DC in an immature/tolerogenic state, thus preventing them from initiating a productive immune response. This may constitute an additional mechanism by which cancer cells evade the immune system.
A cancer cell-based vaccine has been developed in our laboratory and consists of tumor cell lysates enriched for chaperone proteins using an isoelectrofocusing technique [33
34
35
36
37
]. We have previously reported that this chaperone-rich cell lysate (CRCL) vaccine elicits specific T cell responses, resulting in tumor regression [33
, 36
, 37
]. We have also documented that CRCL triggers DC activation and that vaccination with tumor CRCL-loaded DC induces long-term, tumor-specific immunity [38
]. In addition, this tumor vaccine can induce the activation of macrophages (unpublished data), a subset of immune cells reported to be involved in the regulation of tumor immunity [39
, 40
]. The current study was designed to evaluate the susceptibility of tumor-derived, CRCL-treated DC and macrophages to inhibition by CD4+CD25+FoxP3+ Tregs harvested from mice bearing a BCR-ABL+ leukemia (12B1). Our data indicate that CRCL-activated bone marrow-derived DC and macrophages resist Treg-mediated suppression. The production of DC or macrophage proinflammatory cytokines and NF-B activation induced by CRCL is not curtailed by Tregs, and Tregs do not induce phosphorylation of the STAT3 transcription factor involved in IL-10 signaling in CRCL-treated cells. A putative, direct effect of CRCL on Tregs is unlikely, as their survival, FoxP3 expression, and immunosuppressive function are not negatively modulated by CRCL exposure.
MATERIALS AND METHODS
Mice
Mice were housed under specific, pathogen-free conditions and cared for according to the guidelines of the University of Arizona Institutional Animal Care and Use Committee (Tucson, AZ, USA). Female BALB/c (H2d) and C57BL6 (H2b) mice were obtained from the National Cancer Institute (Bethesda, MD, USA) and used at the age of 6–8 weeks.
Cell line
The murine leukemia cell line 12B1 was generated by retroviral transformation of BALB/c bone marrow cells with the human bcr-abl (b3a2) fusion gene. These cells express the p210 bcr-abl protein. This is an aggressive leukemia, and the LD100 is 100 cells after tail-vein injection [41
]. The cells were cultured at 37°C and in 5% CO2 in RPMI medium (Gibco/BRL, Gaithersburg, MD, USA), supplemented with 10% heat-inactivated FBS (Gemini Bio-products, Woodland, CA, USA). The 12B1 cell line was obtained from Dr. Wei Chen (Cleveland Clinic, Cleveland, OH, USA) and was tested routinely and found to be free of Mycoplasma contamination.
Generation of 12B1 tumors
For injection, 12B1 cells were first washed three times in PBS (Gibco/BRL) and then counted and adjusted to a concentration of 5 x 104 cells/mL. Female BALB/c mice were injected with 0.1 mL (5x103 cells) s.c. in the right groin and were monitored for tumor development.
Bone marrow-derived DC and peritoneal macrophages
DC were generated from BALB/c bone marrow cells harvested from femurs and tibiae and filtered through a Falcon 100-µm nylon cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ, USA). RBCs were lysed in a hypotonic buffer (150 mM NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA), and the marrow was cultured at a density of 5 x 105 cells/ml in a complete RPMI medium (Gibco/BRL) containing 10% FBS (Gemini Bio-products). Murine GM-CSF (Peprotech, Rocky Hill, NJ, USA) and IL-4 (Peprotech) were added at a concentration of 10 ng/ml each. Complete RPMI medium containing GM-CSF and IL-4 was added on Day 2. On Day 5, nonadherent and loosely adherent cells were collected, washed in complete RPMI, and used in further experiments. Flow cytometry analysis indicated that 
70% of the cells expressed CD11c.
Macrophages were obtained by washing the BALB/c naïve mouse peritoneal cavity with 5 ml cold RPMI medium. Macrophage number was determined by counting adherent cells after a 1-h attachment period on a hemocytometer glass surface at 37°C in 5% CO2. Cells were seeded and used after a 24-h adherent step in a flat-bottom, 96-well plate after the elimination of floating cells by extensive washes.
CRCL preparation
12B1 tumor-derived CRCL was prepared as described previously [33
, 36
, 37
]. The endotoxin activity of CRCL was determined using the limulus amebocyte lysate assay kit (Cambrex Bio Science, Walkersville, MD, USA), according to the manufacturer’s instructions. The level of endotoxin in CRCL was lower than that in media control (<0.01 EU/µg).
Magnetic cell sorting and cultures
Spleens and lymph nodes isolated from mice bearing established (2000–3000 mm3) 12B1 tumors or from naïve animals were dissociated, and cells were pooled together. RBCs were lysed in a hypotonic buffer, and two washes were performed in MACS buffer (PBS supplemented with 10% FBS and 2 mM EDTA). CD4+CD25+ and CD4+CD25– T lymphocytes were purified by magnetic cell sorting using a mouse CD4+CD25+ Treg isolation kit and an autoMACSTM separator, according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA, USA). Both CD4+ T lymphocyte populations (1x105 cells per well) were separately activated for 24 h in round-bottom, 96-well plates coated with anti-CD3 antibody (5 µg/ml, clone 145-2C11, BD Biosciences PharMingen, San Diego, CA, USA) and 5 µg/ml soluble anti-CD28 antibody (clone 37.51, BD Biosciences PharMingen) in the presence of IL-2 (100 U/ml, R&D Systems, Minneapolis, MN, USA).
Day 5 DC or peritoneal macrophages were cultured alone or with CD4+CD25+ Tregs at a DC or macrophages:CD4+CD25+ T lymphocytes ratio of 1:1 and activated for 24 h with 10 ng/ml LPS (Sigma Chemical Co.) or with 12B1-derived CRCL (25 µg/mL). In some experiments, cell culture supernatants were collected, centrifuged to remove cell debris, frozen, and stored at –80°C until further use. In other experiments, DC were separated from CD4+CD25+ Tregs using anti-CD25 microbeads and the autoMACSTM separator (Miltenyi Biotec) for further analysis.
Flow cytometry analysis and antibodies
For FoxP3 detection, CD4+CD25+ or CD4+CD25– T cells, purified by magnetic cell sorting, were fixed, permeabilized, stained using an APC anti-mouse FoxP3 staining set, following the provider’s instructions (clone FJK-16, eBioscience, San Diego, CA, USA), and analyzed using a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). A minimum of 10,000 events was collected for each sample, and data analysis was carried out with CellQwest software (Becton Dickinson Immunocytometry Systems).
DC surface expression of specific antigens was determined as followed. Cells were washed twice in PBS containing 3% heat-inactivated FBS and 0.09% sodium azide (Sigma Chemical Co.). A total of 2 x 105–1 x 106 cells was transferred in each well of 96-well U-bottom microplates. Cells were incubated first with an FcR-blocking antibody (BD Biosciences PharMingen) for 5 min and then with saturating amounts of the appropriate primary antibodies for 30 min. The cells were then washed three times in PBS containing 3% heat-inactivated FBS and 0.09% sodium azide, fixed with PBS containing 1% paraformaldehyde (Polysciences, Warrington, PA, USA), and analyzed using a FACScan (Becton Dickinson Immunocytometry Systems). The primary antibodies used to monitor DC surface marker expression included PE-conjugated anti-CD11c and FITC-conjugated anti-CD-40 and anti-CD86 and were purchased from BD Biosciences PharMingen. Isotype control antibodies were obtained from BD Biosciences PharMingen (PE-conjugated rat IgG1; FITC-conjugated rat IgG2a).
T cell proliferation and suppression assays
CD4+CD25– T cells (1x105) from naïve BALB/c mice (H2d) were cocultured for 60 h in round-bottom, 96-well plates with 1 x 105 APC (CD90-depleted splenocytes from C57BL/6 mice, H2b) and anti-CD3 antibody (2 µg/ml), with or without activated CD4+CD25+ T cells (1x105) from naïve (data not shown) or 12B1 tumor-bearing mice. [3H]-Thymidine (ICN Pharmaceuticals, Costa Mesa, CA, USA) was then added (1 µCi per well) for an additional 12 h. The cells were then harvested using a 96-well Packard cell harvester, and the radioactivity was measured on a Packard β counter (Packard Biosciences, Meriden, CT, USA). In other experiments, the ability of bone marrow-derived DC to stimulate allogeneic T cells in MLR after their incubation with CD4+CD25+ T cells was evaluated. DC were purified from the coculture, as described above, treated with Mitomycine C (20 min, 50 µg/ml), and plated with responder C57BL/6 splenocytes at a DC:C57BL/6 cell ratio of 1:5. [3H]-Thymidine incorporation by the responder splenocytes was assessed as described above. All cultures were set up in triplicate.
Detection of cytokine and chemokine production by ELISA
The concentration of TNF-
or IL-12 in CD4+CD25+ T cells and DC or macrophage culture supernatants was determined using ELISA kits, according to the manufacturer’s procedures (eBiosciences).
Detection of I-B and STAT3 phosphorylation by Western blotting
Following the culture with CD4+CD25+ Tregs, DC were purified as described above and lysed in lysis buffer (1% Nonidet P-40, 50 mM Tris HCl, pH 7.4, 2 mM EDTA, 100 mM NaCl, 0.2 mg/mL aprotinin, 0.2 mg/mL leupeptin, 1 mM PMSF, 10 mM NaF, 30 mM NaPPi, 10 mM Na3VO4). DC treated with LPS (10 ng/mL) were used as a positive control for I-B phosphorylation. Negative controls consisted in DC cultured alone. Western blot analysis was then performed as described [31
], using antiphospho-I-B, anti-I-B, antiphospho-STAT3, or anti-STAT3 antibodies (Cell Signaling Technologies, Beverly, MA, USA).
Detection of NF-B activation by DNA-binding transcription factor ELISA assay
Nuclear extracts were performed from DC (1x106 cells) or macrophages (5x105 cells) using a nuclear extract kit (Active Motif, Carlsbad, CA, USA). Then NF-B P50 DNA-binding activity was measured with 15 µg nuclear extract with a NF-B P50 Trans-AMTM kit, according to the manufacturer’s recommendations (Active Motif). DC treated with LPS (10 ng/mL) were used as positive controls for NF-B activation.
Treg depletion in vivo
Cyclophosphamide (CY)-induced Treg depletion has been successfully associated with immunotherapy to treat established tumors [17
, 42
]. In the current study, we applied this strategy to eliminate Tregs in 12B1 tumor-bearing mice. One single i.p. injection (15 mg/kg body weight) of CY was performed 14 days after tumor cell challenge. This dose has been optimized in our laboratory and results in a significant decrease in the ratio CD4+CD25+:CD4+, which reaches its nadir 4–6 days after the injection.
Statistical analysis
All experiments were reproduced three times and performed in triplicate. Student’s t-tests were used for the significance of data comparison.
RESULTS
CRCL-induced phenotypic maturation of DC is not inhibited by tumor-derived Tregs
Treg are defined by FoxP3 expression, which confers them the ability to inhibit nonregulatory effector T cells. As expected, CD4+CD25+ T cells isolated by magnetic cell sorting from the spleen of mice bearing established 12B1 tumors (2000–3000 mm3) expressed FoxP3 (Fig. 1A
) and were capable of inhibiting the proliferation of CD4+CD25– cells induced by APC and anti-CD3 antibodies (Fig. 1B)
. Similar results were obtained after analysis of CD4+CD25+ T cells from naïve mice (results not shown).
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Figure 1. CD4+CD25+ T cells from mice bearing established 12B1 tumors express FoxP3 and abrogate the proliferation of CD4+CD25– T lymphocytes. (A) CD4+CD25+ but not CD4+CD25– T cells purified from the spleen and draining lymph nodes of mice bearing established (2000–3000 mm3) tumors express FoxP3, as determined by flow cytometric analysis. Representative histograms of seven independent experiments are presented. (B) Inhibition of the proliferation of CD4+CD25– cells by CD4+CD25+ T lymphocytes from tumor-bearing mice. Responder BALB/c CD4+CD25– T cells were stimulated with APC (CD90-depleted C57BL6 splenocytes) and anti-CD3 in the presence or absence of CD4+CD25+ T cells isolated from the spleen of mice bearing 12B1 (2000–3000 mm3) tumors. The data are shown as means ± SD of triplicate wells of 3H-thymidine incorporation. Results are representative of three independent experiments. *, Significant difference when compared with the corresponding control without CD4+CD25+ Tregs (P<0.05).
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Figure 2. CRCL-induced DC costimulatory molecule expression and production of cytokines by DC and macrophages are not down-regulated by Tregs. Tregs (A) do not suppress CRCL-induced DC phenotypic maturation. Day 4 bone marrow-derived DC were cultured in the presence or absence of CD4+CD25+ Tregs isolated from tumor-bearing mice (tumor volume=2000–3000 mm3) and subjected to LPS or CRCL treatment. After 24 h, cells were stained with PE-conjugated anti-CD11c mAb and FITC-conjugated anti-CD40 or anti-CD86 mAb and analyzed by FACS. Representative dot-plots of three independent experiments (left panel) and histogram analysis of the flow cytometry data (right panel) are presented. (B and C) Tregs obtained as described were cultured for 24 h with Day 5 bone marrow-derived DC or peritoneal macrophages (M ), with or without LPS or 12B1-derived CRCL. The culture supernatants were collected and analyzed by ELISA. DC cultured in medium alone were used as controls. Results are representative of three experiments performed in triplicate wells. Determination of IL-12 and TNF- production by (B) DC and (C) macrophages. Significant difference when compared with the corresponding control without Treg; *, P < 0.05; **, P < 0.01; NS, no statistical difference compared with the corresponding group without Tregs.
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by DC and macrophages is not affected by Tregs was determined in Treg-DC cultures using ELISA. As expected, DC maturing in the presence of tumor-derived CRCL or LPS produced significant levels of both cytokines. Consistent with CD40 and CD86 expression, the addition of Tregs in the cultures curtailed the DC response to LPS but not to CRCL stimulation (Fig. 2B)
.
Based on our unpublished observations that tumor-derived CRCL are powerful activators of macrophages, we examined whether Tregs may modulate CRCL-induced production of inflammatory cytokines by naïve peritoneal macrophages. In line with our findings with DC, tumor-derived CRCL-induced production of IL-12 and TNF- by peritoneal macrophages was not affected significantly in the presence of Tregs (Fig. 2C)
. These data therefore indicate that DC and macrophage activation by CRCL can be maintained, despite the presence of immunosuppressive tumor-derived Tregs. Similar results were obtained with Tregs from tumor-free mice (results not shown).
CRCL-induced ability of DC to elicit T cell proliferation is not curtailed by Tregs
We then investigated whether DC activated with CRCL, in the presence of Tregs, retain their ability to stimulate T lymphocyte proliferation. DC cultured with Tregs isolated from 12B1 tumor-bearing BALB/c mice were exposed to LPS or 12B1-derived CRCL. DC were then isolated by depletion of Tregs and were subsequently cultured with allogeneic mouse (C57BL/6) splenocytes. Consistent with our previous report [31
], the capacity of LPS-treated DC to induce proliferation of allogeneic splenocytes was restrained by Tregs (Fig. 3
). However, the presence of Tregs in the DC cultures during CRCL exposure did not negatively influence the allostimulatory activity of DC (Fig. 3)
.
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Figure 3. CRCL-induced DC immunostimulatory function is not compromised by Tregs. Day 5 DC were precultured for 24 h, with or without Tregs isolated from 12B1 tumor-bearing mice in the presence or absence of LPS or 12B1-derived CRCL. At the end of the coculture, DC were separated from Tregs, washed, and used as stimulators in an allogeneic MLR. C57BL6 splenocytes (C57) were the responder cells. The results are representative of two independent experiments. The data are shown as mean ± SD of triplicate wells of 3H-thymidine incorporation. *, Significant difference when compared with the corresponding group without Tregs (P<0.05).
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B and do not modify STAT3 activation status in DC and macrophages exposed to CRCLB is a central transcription factor in DC and macrophages. Activated in response to various external stimuli or cytokines, it regulates the expression of key genes such as IL-12 and TNF-. NF-B activation depends on the phosphorylation of its inhibitor, I-B by I-B kinase, leading to the translocation of the transcription factor in the nucleus, where it binds to specific DNA promoter sequences. I-B phosphorylation was detected by Western blot in cellular extracts from DC (Fig. 4A
) exposed to LPS or 12B1 tumor-derived CRCL for 24 h. To confirm these results, the DNA-binding activity of NF-B to an immobilized oligonuleotide probe containing the consensus NF-B site was assessed. Consistently, LPS and CRCL treatment of DC enhanced NF-B P50 binding to its specific oligonucleotide probe (Fig. 4B)
. In peritoneal macrophages, NF-B was also activated significantly by 12B1 tumor-derived CRCL but to a lesser extent compared with DC (Fig. 4C)
. The presence of Tregs during DC or macrophage treatment with tumor-derived CRCL did not significantly hamper NF-B activation, as I-B phosphorylation and NF-B P50 DNA-binding activity were maintained after the culture with the suppressive T cells (Fig. 4)
.
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Figure 4. CRCL-induced NF-B activation is not hampered by Tregs. (A) Day 5 bone marrow-derived DC were incubated for 24 h with Tregs, with or without LPS or 12B1-derived CRCL. DC were separated from Tregs. Total cell extracts were performed, and phospho-I-B and I-B expression was analyzed by Western blot. Negative controls consisted of DC cultured alone and positive control of DC cultured for 30 min with LPS. A similar analysis was performed using anti-phopho-STAT3 and anti-STAT3 antibodies. (B and C) CD4+CD25+ Tregs do not modulate NF-B DNA-binding activity in DC and macrophages. At the end of the coculture with CD4+CD25+ Tregs, DC (B) or macrophages (C) were separated from tumor-derived Tregs, as mentioned previously. Nuclear extracts were performed, and the DNA-binding activity of NF-B P50 to a consensus DNA probe was assessed, as described in the experimental procedures. Negative control consisted in DC or macrophages cultured alone and positive control in DC or macrophages cultured for 30 min with LPS. The data are representative of three experiments and are shown as means ± SD of duplicate wells of NF-B P50 activation determined as the OD value of 450 nm, as indicated by the manufacturer. *, Significant difference when compared with the corresponding group without Tregs (P<0.05).
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Target cells simultaneously exposed to LPS and CRCL are not subjected to Treg-immunosuppressive effects
We then sought to determine whether CRCL may overcome Treg-suppressive effects on LPS-treated peritoneal macrophages. Macrophages were incubated for 24 h with LPS, 12B1 tumor-derived CRCL, or both LPS and CRCL, in the presence or absence of activated Tregs purified from 12B1-tumor bearing mice. The response of macrophages to LPS stimulation was reduced significantly following coculture with Tregs. However, macrophages incubated with CRCL and LPS retained their ability to secrete TNF- in the presence of Tregs (Fig. 5A
).
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Figure 5. (A) Treg-suppressive effects on LPS-stimulated cells are overcome by CRCL. Macrophages were cultured for 24 h, with or without Tregs, in the presence or absence of LPS, 12B1-derived CRCL, or LPS and CRCL. The culture supernatants were collected, and TNF- production was determined by ELISA. Representative results of three experiments performed in triplicate are shown. **, Significant difference when compared with the corresponding group without Tregs (P<0.01). (B) CRCL activates macrophages preincubated with Tregs. Peritoneal macrophages were first preincubated for 24 h with Tregs isolated from 12B1 tumor-bearing mice and were then exposed to CRCL for an additional 24 h. TNF- concentration was detected in the culture supernatant by ELISA. ***, Significant difference compared with macrophages preincubated with CD4+CD25+ Tregs and not treated with CRCL (P<0.005).
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following CRCL stimulation but to a lower extent compared with their counterparts cultured in the absence of the suppressive cells (Fig. 5B)
. This suggests that CRCL may partly revert the suppressive effects of Tregs on APC.
The refractory status of CRCL-treated target cells to Treg-mediated suppression does not depend on CRCL concentration
We next sought to address whether Treg down-regulation of macrophage TNF- production was dependent on the strength of the signal to which the cells were exposed. Peritoneal macrophages were thus cultured with increasing concentrations of LPS or CRCL. The data depicted in Figure 6A
indicate that Treg-immunosuppressive effects could be overcome with higher concentrations of LPS. However, toxic effects of LPS on macrophages were observed at these higher concentrations (10 µg/ml). To explore whether a similar phenomenon was also responsible for the observed resistance of CRCL-treated macrophages to Treg-suppressive activity, we performed identical experiments with different concentrations of 12B1-derived CRCL. Tregs did not exhibit suppressive effects on macrophages, even at the lowest concentration of CRCL for which a stimulating response in macrophage was detected (15 µg/ml; Fig. 6B
). Therefore, these data indicate that unlike LPS, high concentrations of CRCL are not required for target cells to resist Treg-mediated immunosuppression.
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Figure 6. LPS but not CRCL concentration influences Treg-suppressive effects. Macrophages were cultured for 24 h, alone or with CD4+CD25+ Tregs, and simultaneously exposed to the indicated concentration of (A) LPS or (B) CRCL. The culture supernatants were collected, and TNF- production was determined by ELISA. Results are representative of three independent experiments. Data are the means ± SD of duplicate wells. (A) **, Significant difference when compared with the corresponding group without Tregs (P<0.01). (B) For each CRCL concentration, no statistical difference was observed between macrophages cultured with or without Tregs.
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Figure 7. CRCL-treated Tregs retain their immunosuppressive potential. (A) Activated Tregs were treated for 24 and 48 h with 12B1-derived CRCL, and cell survival was assessed by MTT cytotoxic assays. (B) FoxP3 expression was examined by flow cytometry in Tregs treated or not for 24 h with 12B1-derived CRCL. (C and D) Tregs were pretreated for 24 h with tumor-derived CRCL, washed, and tested for their ability (C) to inhibit the proliferation of CD4+CD25– T cells as described in Materials and Methods or (D) to suppress LPS-induced TNF- secretion by macrophages. The data are representative of three (A and B) or two (C and D) independent experiments performed in triplicate (A, C, and D). (A) No statistical difference was observed between each group. (C and D) *, P < 0.05; **, P < 0.01.
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Finally, to rule out any direct effect of CRCL on Tregs, CD4+CD25+ cells were cultured for 24 h in the presence of 12B1 tumor-derived CRCL, washed, and tested for their ability to inhibit the proliferation of CD4+CD25– cells induced by allogeneic APC and anti-CD3 antibodies (Fig. 7C)
or to suppress LPS-induced TNF- production by macrophages (Fig. 7D)
. Consistent with the previous results, Treg preincubation with CRCL did not demonstrate an altered, suppressive potential (Fig. 7C
and 7D)
. Together, these observations indicate that a putative, direct modulation of Tregs by tumor-derived CRCL is unlikely.
CRCL partly reverts tumor-induced suppression of APC and may be efficiently combined with Treg depletion to treat established 12B1 tumors
To further define whether CRCL may affect the immunosuppressive state of APC induced in vivo by progressing 12B1 tumors, CD11c+ cells were isolated from 1000 to 2000 mm3 12B1 tumors (tumor-infiltrating CD11c+, TiCD11c+) or from the spleen of naïve mice (splenic CD11c+, SpleCD11c+) and were cultured for 24 h, with or without 12B1-derived CRCL. TNF- production by tumor-infiltrating cells was induced by the chaperone-based vaccine but to a lower extent compared with splenic CD11c+ cells (Fig. 8A
).
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Figure 8. (A) Effects of CRCL on 12B1 tumor-infiltrating CD11c+ cells. (B) Combination of CRCL vaccination and Treg depletion to treat established 12B1 tumors. (A) CD11c+ cells were isolated by magnetic cell sorting (Miltenyi Biotec) from 12B1 tumors (TiCD11c+) or from the spleen of naïve mice (SpleCD11c+) after tissue dissociation. Cells were cultured for 24 h in 96-well plates, with or without CRCL (25 µg/ml), and TNF- production was detected by ELISA. The data are representative of two independent experiments performed in duplicate. **, P< 0.01; ***, P < 0.005. (B) Balb/c mice were injected with 5000 12B1 tumor cells (s.c.) at Day 0. Some mice were treated at Days 10, 14, and 18 with CRCL (25 µg s.c. per mouse) prepared from 12B1 tumors (CRCL) or with CY to deplete Tregs, as indicated in Materials and Methods, or with CY and CRCL (CY+CRCL); n = 8 mice per group. Results are representative of three independent experiments.
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DISCUSSION
The exploitation of CD4+CD25+FoxP3+ Treg cells by cancer contributes to the occurrence and persistence of tumor-induced tolerance. In support of this concept, studies in humans and animal models have demonstrated that neutralization or depletion of Tregs promotes anti-tumoral immunity [15 , 17 , 22 23 24 , 43 , 44 ]. The mechanisms by which tumor cells foster Treg expansion remain controversial. However, a recent extensive analysis of Treg induction in several tumor models has highlighted the occurrence of a conversion process of CD4+CD25– T cells into CD4+CD25+ rather than a proliferation of pre-existing Tregs [45 ]. Similarly, the mechanisms underlying the suppressive activity of these cells are not completely defined, and the nature of Treg target cells is versatile. Previous studies have indicated that Tregs compromise the function of effector CD8+ T cells, impede CD4+ T cell help [14 , 15 , 19 , 25 , 26 ], and may down-regulate DC function [31 ]. Inhibition of DC by tumor-induced Tregs is a relevant issue, as immunocompromised/tolerogeneic DC are not only capable of inducing T cell anergy [46 47 48 49 50 ] but may also be involved in the further generation of Tregs [47 , 51 , 52 ]. We have proposed that this positive-feedback loop, by which tolerogenic DC may induce Tregs, which in turn enhance DC-suppressive function, may contribute to the persistence of tumor tolerance [31 ]. Attempts to disrupt this suppressive Treg-DC amplification circle would thus promote anticancer immunity.
CRCL prepared from tumor tissues is a potent vaccine that not only carries a diversity of tumor antigens but also exerts adjuvant activity [38
, 53
], leading to the activation of DC [38
]. These data prompted us to examine whether CRCL may modulate APC sensitivity to Treg-suppressive effects. Consistent with our previous work [31
] and with other reports [27
28
29
, 54
], Tregs were able to dampen LPS-induced DC phenotypic maturation, cytokine production, and their capacity to activate T cells. Our current results indicate that tumor-derived CRCL-induced DC activation resists tumor Treg suppression. CRCL-induced maturation and immunostimulatory activity of DC were not altered by Treg cells. Similarly, DC proinflammatory cytokine production induced by the tumor-derived CRCL vaccine was not affected by Tregs. In line with these results, CRCL-dependent activation of the transcription factor NF-B was maintained in target cells exposed to Tregs, and the activation level of STAT3 was not affected by the presence of the immunosuppressive cells. In addition, DC precultured with Tregs still responded to CRCL stimulation. Tumor-infiltrating DC are often compromised by the tumor environment [48
49
50
, 55
]. CRCL stimulated the production of TNF- by CD11c+ cells isolated from 12B1 tumor tissues, further suggesting that this vaccine may partially overcome the immunosuppressive state of DC induced during tumor growth in vivo. We have previously documented that in vivo, the growth of pre-established tumors can be delayed but not suppressed completely by CRCL vaccination [33
, 38
]. The result depicted in Figure 8B
of the current study confirms these data and further indicates that Treg depletion dramatically fosters the efficiency of the CRCL vaccine. This result emphasizes that in vivo, CRCL by itself, although capable of inducing DC resistance to Tregs, may not be sufficient to overcome the complex mechanisms of tumor-induced immmunosuppression. Direct suppression of anti-tumoral CD4+ and CD8+ T lymphocytes by Tregs has been described previously [14
, 15
, 19
, 25
, 26
]. It is therefore probable that additional inhibition of these effector cells by Tregs may occur in the 12B1 tumor model, explaining the observed, synergistic effects of Treg elimination with CRCL administration and further underlining the need for combination therapies associating CRCL vaccination with Treg depletion/inactivation.
Macrophages represent a population of cells located at the nexus between innate and adaptive immunity. They may act as APCs as well as effector killers or suppressor cells [39
, 40
, 56
]. Tumor-associated macrophages are primarily classified in two groups, M1 and M2 [57
]. M1 cells produce IL-12 and TNF- and can induce protective tumor immunity leading to tumor regression. Conversely, M2 cells that do not secrete proinflammatory cytokines may foster tumor growth and angiogenesis [57
58
59
]. Macrophages may contribute to the immunosuppressive microenvironment of progressive tumors [60
], but their relationship with tumor-induced Tregs is not fully understood. Based on the prominent role of these cells in tumor growth control and on our recent observation that tumor-derived CRCL fosters macrophage activation, we sought to define the modulatory role of Tregs on macrophages exposed to CRCL. Consistent with the DC data, Tregs were capable of suppressing LPS-induced proinflammatory cytokine production and NF-B induction and failed to inhibit macrophage activation triggered by CRCL.
Through their cell-surface receptors, DC and macrophages may perceive a diversity of signals, with the end result being cell activation or inhibition. Activating stimuli, such as the ligation of TLRs or proinflammatory cytokine receptors by their corresponding ligands, trigger intracellular signaling cascades, resulting in the up-regulation of genes involved in cell activation (costimulatory molecule expression, production, and secretion of proinflammatory cytokines or chemokines) [61 ]. Conversely, immunosuppressive factors such as IL-10 or TGF-β may induce repressing signals within the cells, leading to the negative modulation of their function [61 ]. The balance between these pro- and anti-inflammatory signals governs the activation status of these cells. The data depicted in our current study provide a demonstration of this unstable, activation-suppression equilibrium. Depending on the concentration of LPS, the ability of Tregs to hinder target cell function differed significantly. Indeed, cells exposed to low concentrations of LPS were sensitive to Treg inhibition but became resistant when exposed to higher doses of the TLR4 agonist. This finding suggests that the negative signaling triggered by Tregs may be counteracted by increasing the strength of the proinflammatory signals. We reasoned that a similar phenomenon may explain the observed resistance of CRCL-treated cells to Treg-mediated inhibition. However, Tregs failed to hamper target cell activation, even at low doses of CRCL. This indicates that CRCL concentration is not a factor that would contribute to the refractory character of the treated cells.
One could argue that CRCL may directly compromise the immunosuppressive function of Tregs, thus preventing them to impair DC or macrophage activation. We therefore examined whether Tregs pretreated with CRCL retained their potency to suppress effector T cells. We clearly documented that CRCL had no detectable effect on Treg survival, FoxP3 expression, and immunosuppressive function. Therefore, a direct inhibition of Tregs by CRCL does not occur.
Our results thus highlight CRCL as a powerful adjuvant endowed with the capacity to overcome tumor-induced Treg-inhibitory effects on APC. The mechanism(s) responsible for the resistance of CRCL-treated DC and macrophages to Treg suppression remain to be determined. The molecular basis underlying CRCL signaling in DC and macrophages is currently under investigation in our laboratory. The elucidation of these APC signaling events would provide fundamental information for the development of more effective anti-tumor vaccines that could overcome Treg-immunosuppressive effects.
ACKNOWLEDGEMENTS
This work was supported in part by National Institutes of Health grant R01 CA104926, the Leukemia and Lymphoma Society Fellow Award 5188-07 (N. L.), and the Tee Up for Tots and Raise a Racquet for Kids Funds.
Received September 17, 2007; revised November 16, 2007; accepted December 2, 2007.
REFERENCES
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