| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Immunology |
Laboratory for Immunological Research, Schering-Plough Research Institute, Dardilly, France
Requests for reprints: Christophe Dercamp, Institut National de la Sante et de la Recherche Medicale U404, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France. Phone: 33-4-37-28-23-98; Fax: 33-4-37-28-23-91; E-mail: dercamp{at}cervi-lyon.inserm.fr.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production and cytotoxicity by tumor-associated antigen (TAA)–specific CD8 T cells; (c) the lack or loss of expression of relevant class I MHC tumor antigen peptides by tumor cells; and (d) the suppressive activity of cytokines such as interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) and, more recently, regulatory T cells (Treg). Treg cells encompass several populations of regulatory T cells, defined by their distinct ontogeny, phenotype, or regulatory mechanism (2, 3). "Natural" Treg refers to a population of CD4+CD25+ T cells selected in the thymus and involved in peripheral tolerance. Depletion of natural Treg using CD25 antibody before tumor cell inoculation was able to enhance antitumor responses (4, 5), suggesting an antigen-independent inhibition mechanism. In contrast, other Treg subsets such as Tr1 cells seem induced in an antigen-specific fashion to control ongoing immune responses (6, 7). Treg can be associated with human tumors, although their precise origin in that case remains unclear (8, 9). Cytokines, and in particular IL-10 and TGF-ß, play major roles in the generation and function of Treg activity, although again their particular contributions to different Treg subsets is still a matter of debate. An important recent finding was that either immature classic (10), or other less classic dendritic cell subsets referred as tolerogenic dendritic cells (11) could induce Treg. In turn, CD25+ Treg cells can inhibit dendritic cell functions (12).
Dendritic cells might play a central role in the induction of tumor immunity, and especially in the initiation of CD8 T-cell responses, because they are able to cross-present MHC class I–restricted antigens captured from apoptotic bodies (13–15). Solid human tumors are frequently infiltrated by dendritic cells (16, 17) and a few animal studies have shown that tumor-infiltrating dendritic cells (TIDC) could de novo present TAA to naive CD8+ T cells (18, 19). We and others have hypothesized that impaired TIDC functions could be responsible for the absence of an efficient antitumor response. In particular, the tumor milieu lacks strong dendritic cell–activating stimuli such as those delivered by microbes. Indeed, intratumoral delivery of Toll-like receptor (TLR) ligands can improve antitumor CD8 T-cell responses (20–22). We have described, however, that in several mouse models, TIDC could not produce IL-12 in response to the TLR-9 ligand CpG unless endogenous IL-10 was neutralized using IL-10R (19). Subsequently, anti-IL-10R antibody treatment in vivo allowed for a robust, CD8-dependent antitumor response following intratumoral CpG injection (19). We could not define, however, whether IL-10 was acting solely and directly at the level of TIDCs or through other regulatory mechanisms.
Given the roles of IL-10 in dendritic cells and Treg biology, we wanted to further analyze the relative contribution of IL-10 and CD25+ Treg on tumor antigen–specific CD8 T-cell response. We show herein that IL-10 and Treg act in concert, in particular at the level of TIDC, to impair antitumor CD8 T-cell effector differentiation and induce tumor antigen–specific suppressor CD8 T cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß-TCR reactive with the H-2Kb-restricted 257-264 peptide of OVA (24), and C57BL/10 RAG KO mice were purchased from Charles River Laboratories (St. Germain sur l'Arbresle, France). All mice were housed in our pathogen-free facility and used between 5 to 10 weeks of age. The experiments were done in compliance with relevant European Economic Community laws and institutional guidelines. Culture medium. Splenic CD11c+ cells, TIDCs, and T cells were cultured in RPMI 1640 (Invitrogen, Cergy Pontoise, France) supplemented with 10% FCS (Invitrogen), 1 mmol/L HEPES (Invitrogen), 2 mmol/L L-glutamine (Invitrogen), Gentallin (Schering-Plough, Union, NJ), and 2 x 10–5 M ß2-mercaptoethanol (Sigma, St. Louis, MO). Tumor cells were cultured in DMEM (Invitrogen) with the same additives.
Tumor model and in vivo procedures. EL-4 H-2b or EG-7 (105, EL-4 stably transfected to express ovalbumin) thymoma cells (American Type Culture Collection, ATCC, Rockville, MD) were s.c. injected in the right flank in 100 µL of RPMI 1640. Tumor growth was monitored by palpation and mice sacrificed when tumors reached 1 cm in diameter. For in vivo activation, mice were injected intratumorally with 10 µg of the TLR-9 ligand CpG 1668 (5'-TCCATGAGGTTCCTGATGCT-3', MWG Biotech, Ebersberg, Germany) in 50 µL PBS (Invitrogen) and i.p. with 250 µg of endotoxin-free purified anti-IL-10R antibody (1B13A, DNAX Research Institute of Molecular and Cellular Biology Research Institute, Palo Alto, CA) in 200 µL PBS. When indicated, some groups of mice were i.p. injected with anti-CD25 supernatant (PC61-5-3, ATCC) at the day of tumor cell inoculation and then twice a week until adoptive transfer. The latter treatment allowed for the depletion of CD4+CD25+ splenocytes from 3% to <0.2% of CD4+ splenocytes, as monitored by fluorescence-activated cell sorting (FACS) analysis using another anti-CD25 antibody (7D4).
CFSE staining. Naive T cells or splenocytes (1 x 107) in 1 mL HBSS (Invitrogen) were labeled with CFSE (Molecular Probes, Eugene, OR) at 1 to 10 µmol/L according to different assays for 10 minutes at 37°C (25). Cells were washed twice in medium containing 10% FCS to block staining.
Flow cytometry analyses. Fc receptors were blocked using Fc-Block (BD Biosciences PharMingen, San Diego, CA). All antibodies used in this study were from BD Biosciences PharMingen: CD4 (L3T4), CD8ß (Ly-3.2), CD11b (M1/70), CD11c (HL3), CD25 (7D4), CD62L (MEL-14), and CD28 (37.51). For intracellular detection of IL-12 p40/p70, cells incubated for 2 hours in Brefeldin A (Sigma) and stained with CD11c were processed with the Cytofix/Cytoperm Kit (BD Biosciences PharMingen) and stained with PE-labeled anti-IL-12 (C15.6). Variables were acquired on a FACSCalibur (BD Biosciences, San Jose, CA).
Naive T-cell and tumor-infiltrating dendritic cell isolation. Naive T cells were sorted from spleen cells on a FACSVantage based on the coexpression of CD8 and CD62L to >95% purity. In some experiments, CD8+ T cells were enriched by negative selection using a CD8+ T-cell isolation kit (Miltenyi Biotec, Bergisch, Gladbach, Germany). TIDCs, or splenic dendritic cells as control, were purified using CD11c+ Microbeads (Miltenyi Biotec), at a purity >92% (19).
Tumor-infiltrating dendritic cell induction of naive T-cell proliferation in vitro. Naive OT-I T cells were labeled with 7.5 µmol/L CFSE and cultured in 96-well plates at 1 x 106 cells/mL with 2 x 105/mL irradiated TIDC or splenic CD11c+ cells in culture medium, with or without OVA peptide (10 nmol/L SIINFEKL peptide, Neosystem, Strasbourg, France), and/or activation stimulus. Activation consisted of 5 µg/mL of CpG 1668 ± 10 µg/mL anti-IL-10R antibody. After 5 days of culture, cell division was analyzed based on CFSE labeling intensity using the Modfit software (BD Biosciences).
Adoptive transfer of naive T cells. CFSE-labeled OT-I naive T cells (8-10 x 106) were transferred into tumor-bearing mice via i.v. injection. Cell division and surface phenotype was analyzed in tumor-draining and contralateral lymph nodes by flow cytometry at 3 and 7 days following transfer.
Tumor-infiltrating dendritic cell induction of naive T-cell functions in vivo. Naive C57BL6/ mice were adoptively reconstituted with 8 to 10 x 106 CFSE-labeled OT-I naive T cells. One day later, 5 x 105 TIDC recovered from resting animals or animals treated 2 hours before with CpG 1668 ± anti-IL-10R were injected in the right footpad. Popliteal draining lymph nodes were harvested 5 days following TIDC injection and IFN--secreting CD8 T cells numbered by ELISPOT. Briefly, 96-well nitrocellulose-bottom plates (Millipore, Molsheim, France) coated with rat anti-IFN- (clone R4-6A2, BD Biosciences PharMingen) were seeded with 2 x 105 lymph node cells and incubated with or without 10 nmol/L OVA class I peptide for 48 hours. Cytokine-producing cells were revealed by sequential incubations with anti-IFN- biotinylated antibody (clone XMG1.2, BD Biosciences PharMingen), peroxidase-streptavidin (Southern Biotechnology Associates, Inc., Birmingham, AL) and AEC substrate (Vector Laboratories, Burlingame, CA). Spots were quantified using an ELISPOT reader (Microvision Instrument, Evry, France).
In vivo CTL assay. Spleen cells from C57BL/6 mice were pulsed with 10 nmol/L OVA class I peptide and labeled with CFSE at a final concentration of 1 µmol/L; unpulsed spleen cells were labeled at a final concentration of 10 µmol/L. The two cell populations were mixed at 1:1 ratio and 2 x 107 total cells were injected i.v. into tumor-bearing or naive mice. The percentage of antigen-specific lysis was determined 18 hours following target cell injection by analyzing the relative proportion of unpulsed versus pulsed targets based on CFSE labeling intensity in tumor-draining lymph node (TDLN) cells. FACS acquisition was done over 1 minute and the percentage of specific lysis calculated as follows: [(M2 – M1) / M2] x R x 100, where M2 = total unpulsed target events, M1 = total pulsed target events, and R = the mean ratio of unpulsed/pulsed targets from three naive recipients.
Sorted CFSE+ T-cell induction of naive T-cell proliferation in vitro. Three days following adoptive transfer, CFSE+ cells were sorted from TDLNs. Proliferation assays were done by culturing 5 x 104 CD8+CD62L+-sorted naive T cells (OT-I mice) per well for 72 hours with various concentrations of CFSE+-sorted T cells in flat-bottomed 96-well plates with irradiated APC (T cell–depleted splenocytes) and 10 nmol/L OVA class I peptide. In some experiments, cells were stimulated with 5 µg/mL plate-bound anti-CD3 (145-2C11, BD Biosciences). In some experiments, 30 µg/mL anti-TGF-ß (1-D11, R&D Systems, Minneapolis, MN), or isotype control antibodies were added to the culture. Cells were pulsed with 1 µCi per well [3H]thymidine (Amersham, Les Ulis, France) for the last 8 hours of culture, and proliferation was measured in a TopCount apparatus (Perkin-Elmer, Courtaboeuf, France).
Statistical analysis. Statistical analysis was done with Student's t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Three days following adoptive transfer in EL-4 tumor-bearing mice, we detected approximately the same percentage of CFSE+ CD8+ cells in TDLNs and in the contralateral lymph nodes (CLN; Fig. 2B). In contrast, antigen-driven local accumulation of CD8 T cells was observed in EG-7 tumor-bearing mice in which 2.5 times more CFSE+ CD8+ T cells on average were retrieved at day 3 from TDLN than in CLN. At this time, only a very low percentage of cells showed decreased CFSE labeling, indicative of cell division, in C57BL/6 mice (Fig. 2A). At day 7 after transfer, the analysis of CFSE intensity indicated a high level of cell division and an even greater accumulation of CFSE+CD8+ cells in the TDLN of EG-7 tumor-bearing animals than EL-4 tumor-bearing animals. These results strongly suggested that cell division was at least partly responsible for the accumulation (Fig. 2A).
|
CD25 antibody treatment also resulted in a greater antigen-specific CD8 T-cell accumulation in the TDLN at day 3 (P = 0.03) in C57BL/6 mice (Fig. 2B). Treatment of IL-10 KO mice with CD25 antibody did not show an additive effect (Fig. 2B). These data suggest that both IL-10 and Treg cells prevent the optimal proliferation of tumor antigen–specific CD8 T cells in vivo in a redundant fashion.
Interleukin-10 and CD25+ Treg cells decrease in vivo tumor-specific CD8 T-cell functions. We observed a modest but significant (P < 0.01) increased frequency of OVA antigen-specific IFN--producing cells in the TDLN of EG-7 tumor-bearing IL-10 KO mice compared with C57BL/6 mice (Fig. 3B, right). Much more evident results were observed after adoptive transfer of OT-I CD8 T cells in EG-7 tumor-bearing mice. Three days following transfer, the frequency of OVA-specific IFN--secreting cells in TDLN was significantly higher in IL-10 KO mice and in mice treated with CD25 antibody than in untreated WT C57BL/6 mice (Fig. 3A, left). The absence of IFN--producing cells in TDLN from EL-4 tumor-bearing mice or when OVA peptide was omitted during in vitro restimulation confirmed the antigen specificity of the response (Fig. 3B). Treatment of IL-10 KO mice with CD25 antibody did not significantly increase the frequency of IFN--producing cells (Fig. 3A, left).
|
-producing cells was still significantly (P = 0.01) increased in IL-10 KO mice and CD25 antibody–treated mice, compared with untreated C57BL/6 recipients (Fig. 3A, right). Intriguingly, the frequency of tumor antigen–specific IFN--producing cells at day 7 was lower than that observed at day 3 in IL-10 KO mice or CD25 antibody–treated WT C57B/6 mice but strikingly higher than at day 3 in IL-10 KO mice with CD25 antibody (Fig. 3A, right).
We also investigated the generation of tumor-specific CTL activity as measured in vivo against OVA class I peptide-pulsed fluorescently labeled spleen cells. Unlike IFN- secretion, in vivo CTL activity was measured in tumor-bearing mice in the absence of OT-I cell–adoptive transfer. Tumor antigen–specific lysis was increased only modestly and not in a statistically significant way in IL-10 KO compared with C57BL/6 recipient mice and significantly (P = 0.03) following CD25 antibody treatment of C57Bl/6 mice (Fig. 3C). Furthermore, treatment of IL-10 KO mice with CD25 antibody yielded the highest lysis rate (Fig. 3C). The presence of antigen-expressing tumor was required because only background lytic activity was observed in EL-4 tumor-bearing mice (Fig. 3C). Collectively, these data strongly suggest that both IL-10 and Treg prevent the optimal early development of tumor antigen–specific CD8 T-cell functions, in a nonredundant fashion.
Interleukin-10 and CD25+ Treg cells are required for in vivo generation of tumor-induced antigen-specific regulatory CD8 T cells. Although we could not detect significant CD8 effector functions at the steady state, EG-7 but not EL-4 tumors were able to trigger modest OT-I CD8 T-cell proliferation. This suggested that antigen-specific priming had occurred. We further investigated whether these primed CD8 T cells would have acquired regulatory properties in vivo, characterized by an anergic state and the capacity to inhibit naive T-cell proliferation. To do so, we retrieved, 3 days following adoptive transfer, OT-I cells from TDLN using cell sorting based on CFSE labeling.
We first tested the capacity of the sorted cells to proliferate in vitro in response to anti-CD3 stimulation (Fig. 4A). We observed that OT-I cells parked in EL-4 tumor-bearing mice proliferated in response to anti-CD3 even to a greater extent than freshly isolated naive OT-I CD8 T cells or total C57BL/6 CD8 T cells. In marked contrast, OT-I cells parked in EG-7 tumor-bearing mice poorly proliferated in response to anti-CD3. Although we could not completely rule out that the inability of proliferate was due to clonal exhaustion, this result as well as the poor proliferation of OT-I cells in vivo (Fig. 2) argues in favor of a tumor-induced, antigen-specific anergy in these CD8 T cells.
|
Endogenous interleukin-10 suppresses the ability of tumor-infiltrating dendritic cell to produce interleukin-12 while CD25+ Treg cells do not. We addressed the functional status of TIDC. We had previously shown that dendritic cells infiltrating mouse tumors were impaired in their capacity to produce IL-12 in response to CpG (19). This was reversed by the addition of anti-IL-10R antibody during stimulation. Here, we observed a similar inability to produce IL-12 in TIDC from EG-7 tumor-bearing WT C57BL/6 mice (Fig. 5). However, TIDC from EG-7 tumors implanted in IL-10 KO mice displayed robust IL-12 production in response to CpG alone (Fig. 5). The vast majority of TIDC were CD11clowCD11b+ and this dendritic cell subset was responsible for IL-12 production in response to CpG, as previously described (19). We also examined whether the impairment of TIDC to produce IL-12 in C57BL/6 mice could be mediated by Treg cells. We observed that TIDC from RAG-KO mice, deficient for all T cells including Treg, required anti-IL-10R treatment to achieve optimal CpG-induced IL-12 secretion (Fig. 5). Thus, the ability of the tumor environment to inhibit TIDC IL-12 production requires endogenous IL-10 but it is not mediated by Treg.
|
|
Following injection in naive mice reconstituted with OT-I cells, only TIDC isolated from WT C57B/6 mice treated in vivo with CpG 2 hours before isolation induced IFN- production. The ability to induce IFN- production was enhanced in IL-10 KO mice or CD25 antibody–treated WT mice and was highest with TIDC isolated from CD25 antibody–treated IL-10 KO mice (Fig. 6D).
Thus, both endogenous IL-10 and Treg cells impaired the ability of TIDC to induce antigen-specific CD8 T-cell responses. Whereas IL-10 and Treg cells were redundant in suppressing the ability of TIDC to induce CD8 T-cell proliferation, they seemed to act synergistically in suppressing the ability of activated TIDC to induce IFN- production in vivo. Although the level of IL-12 production is likely to be one of the factors determining the ability of TIDC to induce IFN- production, the mechanism(s) through which TIDC induced a better proliferative effect remain(s) to be determined because the expression of costimulatory molecules such as CD40, CD80, and CD86 by TIDC was not significantly modified (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the absence of IL-10, tumor-specific CD8 T-cell proliferation and function as well as the ability of TIDC to prime naive CD8 T cells were dramatically enhanced. Indeed, better proliferation or function was observed even in the absence of activation. This was ascribed to the artificially high frequency of TAA-specific CD8 T cells in adoptively transferred animals. Our observations are in line with the broad immunosuppressive functions of IL-10 (reviewed in ref. 30). However, the remarkable restoration of TIDC and antitumor CD8 T-cell functions in IL-10 KO mice suggests that IL-10 has a much more central and unique role than previously hypothesized. The reversion of the suppression in IL-10 genetically deficient mice bearing IL-10-sufficient tumors suggests that endogenous and not tumor-derived IL-10 is responsible for immunosuppression, at least in our model.
Depletion of Treg cells had beneficial effects similar to IL-10 deficiency or neutralization when treating tumor-bearing mice with CpG. Because IL-10 was shown to allow the development and support the functions of the Tr1 subset of Treg cells (reviewed in ref. 7), it was tempting to hypothesize that IL-10 was playing a major role in Treg immune suppression in our model. However, our data showed that the immunosuppressive functions of Treg and IL-10 were not superimposable. This was best exemplified by the fact that endogenous IL-10 was able to inhibit TIDC IL-12 secretion in a Treg-independent way, whereas both IL-10 and Treg depletion increased the capacity of TIDC to prime CD8 effector T cells. Furthermore, results obtained in RAG-KO mice suggest that non-T cells such as tumor-associated macrophages or TIDC could be the main source of immunosuppressive IL-10 (reviewed in refs. 30, 31). Intriguingly, we observed lower numbers of IFN- secreting cells and, possibly, lower accumulation of transferred OT-I CD8 T cells at day 7 than at day 3 in IL-10 KO mice. Although this could be related to a migration of tumor antigen–specific cells outside the TDLN, we did not observe an increase of the transferred OT-I CD8 T cells within the tumor (data not shown). Another possible explanation is that IL-10, whereas detrimental to the initiation of the CD8 T-cell response, would help to maintain it at later times, as shown elsewhere (32).
Although not affecting TIDC capacity to produce IL-12, we showed that Treg suppress the capacity of TIDC to induce effector CD8 T cells in vitro and in vivo. Indeed, human CD25+ Treg have been shown to decrease the capacity of dendritic cells to stimulate allogeneic naive T cells (12). The induction of the expression of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) via CTLA4/B7 interactions in dendritic cells has been proposed as a mechanism to explain the propagation of Treg activity, including in the tumor setting. Indeed, CTLA4 provided by Tr1 cells can up-regulate IDO expression in dendritic cells, whereas IDO-expressing dendritic cell–induced Treg (33–35). We have observed by immunohistochemistry that TIDC express IDO, in WT as well as in IL-10 KO mice (data not shown), and we are currently investigating IDO expression in TIDC according to the different treatments.
Depletion of CD25+ cells, including CD4+ and possibly CD8+ Treg, increased both the in vivo proliferation of tumor antigen–specific CD8 T cells and their capacity to differentiate into effector cells producing IFN- and lysing tumor cells. Besides inhibiting TIDC functions, Treg cells could directly act on both the proliferation and functional activation of CD8 T cells through mechanisms distinct from IL-10 such as TGF-ß and CTLA4 (reviewed in refs. 2, 36), although it has been suggested that part of the tumor immunosuppressive effects of CTLA4 could be attributed to IL-10 (37).
Our results identified a novel mechanism of tumor escape: the de novo generation of tumor-induced regulatory CD8 T cells that are specific for an antigen expressed only in the tumor. The suppressive effect of these CD8 T cells was mediated by TGF-ß but not by IL-10 or CTLA4. Both endogenous IL-10 and CD25+ Treg cells were involved in the differentiation of these CD8 regulatory cells. Although most studies on Treg cells are restricted to CD4 T cells (reviewed in ref. 2), there are also several examples of regulatory CD8 T cells. (38). CD8+ Treg cells expressing the Foxp3 transcription factor thus resembling "natural" CD4+ Treg cells, were described in rats (39), but we did not observe Foxp3 expression in our suppressor CD8 T cells by PCR (data not shown). In humans, non-antigen-specific regulatory CD8 T cells can be generated from CD28– CD8 T cells in a process involving IL-10 (40). Interestingly, we have observed that the nonproliferating fraction of OT-I cells parked in EG-7 tumor-bearing mice were mostly CD28–. Antigen-specific CD8 Treg cells can also be induced, as it was shown following injection of antigen-loaded immature dendritic cells into human volunteers (41). Our tumor model is also reminiscent of chronic hepatitis C virus infection, in which virus-specific liver CD8 T cells can suppress T-cell responses via IL-10.
Thus, our tumor therapy model points to the multiplicity of mechanisms through which immune tolerance to tumors may arise and propagate. The initiation of effector CD8 T cells by TIDC may be impeded by the presence of "natural" Treg and/or the presence of IL-10 in the tumor milieu. Consequently, TIDC in the presence of IL-10 may induce regulatory CD8 T cells and possibly other Treg such as Tr1 that will further inhibit the response, leading to infectious immune tolerance. Particular subsets of dendritic cells have been suggested to be able to induce Tr1 cells (42), but those cells were characterized by high CD45RB expression, unlike TIDC in our system (data not shown). We are currently investigating the possible differentiation of Tr1 cells using TIDC.
Toll-like receptor-mediated signals to dendritic cells, at least in part acting through IL-6 release, have been shown to be able to prevent the T suppressive activity of Treg (43) and break Treg-mediated CD8 tolerance in vivo (44). Moreover, CpG activation, as well as CD40L ligation, can release dendritic cells from the suppressive activity of Treg to some extent (45). In the tumor setting, however, Toll-like receptor signals may not be sufficient to fully alleviate Treg activity (31). Our results show that whereas CpG alone has little antitumor activity, it can induce tumor rejection when IL-10 or Treg are blocked. However, even when IL-10 was blocked and Treg deleted, treatment with CpG was still needed to achieve tumor rejection. We observed that CpG activation was required to detect some level of IFN--secreting CD8 T cells following transfer of TIDC into WT mice, whereas these cells were readily detectable in the TDLN of IL-10 K0 or CD25-depleted EG-7 tumor-bearing mice. TIDC activated in vivo with CpG were, however, consistently more active than nonactivated TIDC in inducing IFN--secreting CD8 T cells when adoptively transferred. This is in part explained by the fact that CpG activation is required to obtain high levels of IL-12 secretion by TIDC. In other tumor models we have shown that the antitumor effect of the CpG plus anti-IL-10R antibody combination could be ascribed to both CD8 T cells and natural killer cells and that mechanisms of innate resistance and adaptive immunity are sequentially playing an essential role in the tumor eradication obtained with this treatment (19, 46). Thus, we conclude that Treg and anti-IL-10, through both distinct and overlapping mechanisms, prevent the development of an effective CD8 T-cell antitumor response in tumor-bearing mice, as well as the CpG-induced antitumor innate and adaptive immune responses. Such results have strong implications for the design and implementation of successful cancer immune therapies.
|
Acknowledgments |
|---|
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.
We thank I. Durand for cell sorting and Drs. D. Kaiserlian and F. Powrie for helpful discussions.
|
Footnotes |
|---|
G. Trinchieri is currently at the Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Building 50, Room 6150, 50 South Drive, MSC 8003 NIH, Bethesda, MD 20892-8003.
The authors have no financial conflict of interest.
Received 4/17/05. Revised 6/ 7/05. Accepted 6/17/05.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-dependent antiangiogenic activity, as well as long-lasting tumor immunity elicited by peptide vaccination. J Immunol 2003;171:5931–9.-chain pairing determines the specificity of residue 262 within the Kb-restricted, ovalbumin 257-264 determinant. Int Immunol 1992;4:861–7. antibody in vivo induces localized autoimmune disease. Eur J Immunol 1996;26:1608–12.[Medline]
) monoclonal antibody. Cancer Res 1999;59:3128–33.This article has been cited by other articles:
|
|
 |
|
  W. Wang, R. Lau, D. Yu, W. Zhu, A. Korman, and J. Weber PD1 blockade reverses the suppression of melanoma antigen-specific CTL by CD4+CD25Hi regulatory T cells Int. Immunol., September 1, 2009; 21(9): 1065 - 1077. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Bauer and E. A. Rondini REVIEW PAPER: The Role of Inflammation in Mouse Pulmonary Neoplasia Vet. Pathol., May 1, 2009; 46(3): 369 - 390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ahmadi, D. C. Emery, and D. J. Morgan Prevention of Both Direct and Cross-Priming of Antitumor CD8+ T-Cell Responses following Overproduction of Prostaglandin E2 by Tumor Cells In vivo Cancer Res., September 15, 2008; 68(18): 7520 - 7529. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Chiron, I. Bekeredjian-Ding, C. Pellat-Deceunynck, R. Bataille, and G. Jego Toll-like receptors: lessons to learn from normal and malignant human B cells Blood, September 15, 2008; 112(6): 2205 - 2213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Piconese, B. Valzasina, and M. P. Colombo OX40 triggering blocks suppression by regulatory T cells and facilitates tumor rejection J. Exp. Med., April 14, 2008; 205(4): 825 - 839. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yotsumoto, K. Saegusa, and Y. Aramaki Endosomal Translocation of CpG-Oligodeoxynucleotides Inhibits DNA-PKcs-Dependent IL-10 Production in Macrophages J. Immunol., January 15, 2008; 180(2): 809 - 816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Zhou, L. L'italien, D. Hodges, and X. M. Schebye Pivotal Roles of CD4+ Effector T cells in Mediating Agonistic Anti-GITR mAb-Induced-Immune Activation and Tumor Immunity in CT26 Tumors J. Immunol., December 1, 2007; 179(11): 7365 - 7375. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Andreani, G. Gatti, L. Simonella, V. Rivero, and M. Maccioni Activation of Toll-like Receptor 4 on Tumor Cells In vitro Inhibits Subsequent Tumor Growth In vivo Cancer Res., November 1, 2007; 67(21): 10519 - 10527. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-X. Chen, K. Man, G. S. Ling, Y. Chen, B.-S. Sun, Q. Cheng, O. H. Wong, C.-K. Lo, I. O.-L. Ng, L. C. Chan, et al. A Crucial Role for Dendritic Cell (DC) IL-10 in Inhibiting Successful DC-Based Immunotherapy: Superior Antitumor Immunity against Hepatocellular Carcinoma Evoked by DC Devoid of IL-10 J. Immunol., November 1, 2007; 179(9): 6009 - 6015. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Dobrzanski, J. B. Reome, J. C. Hylind, and K. A. Rewers-Felkins CD8-Mediated Type 1 Antitumor Responses Selectively Modulate Endogenous Differentiated and Nondifferentiated T Cell Localization, Activation, and Function in Progressive Breast Cancer J. Immunol., December 1, 2006; 177(11): 8191 - 8201. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
), 10 µg CpG 1668 injected intratumorally (
), 250 µg anti-IL-10R antibody injected i.p. (
), or combination of both (
). B, EG-7 tumor-bearing IL-10 KO mice were treated with nothing (
). Two to three experiments were done with similar results. D, CD28 versus CFSE expression from TDLN 7 days following adaptive transfer of CFSE-labeled OT-I cells. Left, sorted CFSE+ cells from EG-7 TDLN; right, gated CFSE+ cells from EL-4 TDLN.
) or EL-4 (
) TIDC and increasing proportions of irradiated EG-7 tumor cells. C-D, TIDC recovered from CpG-treated (open columns) or not (black columns) C57BL/6 or IL-10 KO EG-7 tumor-bearing mice, with or without in vivo CD25 depletion (as indicated), were analyzed for their capacity to prime antigen-specific T-cell responses. CpG treatment was done 2 hours before TIDC isolation. C, analysis of naive OT-I cell proliferation in vitro based on CFSE expression as described in Materials and Methods. Columns, mean percentage of CFSE+ cells in generation 4 to 6 from three individual experiments; bars, ±SE. D, analysis by ELISPOT of the capacity of TIDC to induce OVA-specific IFN-