Regulates Effector T Cell Functions Functional

Functional Dichotomy in CD40 Reciprocally Regulates Effector T Cell Functions This information is current as of March 1, 2017.

Subscriptions Permissions Email Alerts

J Immunol 2006; 177:6642-6649; ; doi: 10.4049/jimmunol.177.10.6642 http://www.jimmunol.org/content/177/10/6642

This article cites 37 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/177/10/6642.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

Downloaded from http://www.jimmunol.org/ by guest on March 1, 2017

References

Gopal Murugaiyan, Reena Agrawal, Gyan C. Mishra, Debashis Mitra and Bhaskar Saha

The Journal of Immunology

Functional Dichotomy in CD40 Reciprocally Regulates Effector T Cell Functions1 Gopal Murugaiyan, Reena Agrawal, Gyan C. Mishra, Debashis Mitra, and Bhaskar Saha2 Activation of T cells requires signals through Ag-specific TCR and costimulatory molecules such as CD40L. Although the use of defined tumor Ags for the induction of protective T cells met with limited success, the CD40-CD40L interaction that was proposed to induce antitumor T cells did not prevent tumor growth completely. Using a model for prostate tumor, a leading cause of tumor-induced mortality in men, we show that the failure is due to a novel functional dichotomy of CD40 whereby it self-limits its antitumor functions by inducing IL-10. IL-10 prevents the CD40-induced CTL and TNF-␣ and IL-12 production, Th1 skewing, and tumor regression. Priming mice with tumor lysate-pulsed IL-10-deficient dendritic cells (DCs) or wild-type DC plus anti-IL-10 Ab establishes antitumor memory T cells that can transfer the protection into syngenic nude mice. Infusion of Ag-pulsed IL-10-deficient but not wild-type DCs back into syngenic mice results in successful therapeutic autovaccination. Thus, we demonstrate the IL-10-sensitive antitumor T cell memory formulating a novel prophylactic and therapeutic principle. The Journal of Immunology, 2006, 177: 6642– 6649. induces IL-12 from bone marrow-derived DC. IL-10 prevents CD40-induced IL-12 production, generation of CTL response, and tumor regression. Priming mice with IL-10-deficient DC results in complete prevention of the tumor growth. Transfer of tumor Agloaded syngenic DC, as a model for therapeutic autovaccination, into tumor-bearing mice resulted in significant reduction in tumor load. Our results not only demonstrate the functional dichotomy of CD40 functions, but also suggest completely preventative prophylactic strategies and a model for therapeutic autovaccination.

Materials and Methods RM-1 cell line and development of tumor in mice RM-1 cells (17) were gifted by Dr. T. R. Thompson (Baylor College of Medicine, Houston, TX). These cells were maintained in Ham’s F12K complete medium supplemented with 10% FCS and antibiotics. A total of 2 ⫻ 105 cells were transferred s.c. into C57BL/6 or other gene-deficient mice of C57BL/6 background (The Jackson Laboratory) as indicated. Unless otherwise mentioned, the mice were sacrificed 3 wk after RM-1 cell injection; the tumor growth and T cell functions were assessed. All the experiments were performed according to the animal use protocols approved by the Institutional Animal Care and Use Committee following the guidelines framed by the Committee for the Purpose of Control and Supervision of Experiments on Animals, a central authority that regulates animal experimentation.

DC cultures DCs were cultured from bone marrow progenitor cells using a modified protocol of a previously described method (18). In brief, bone marrow cells were harvested from femurs and tibias. Washed bone marrow cells were prepared in DC culture medium (RPMI 1640 medium, 10% FCS, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 50 ␮M 2-ME, 20 ng/ml GM-CSF, and 10 ng/ml IL-4) at a density of 1 ⫻ 106 cells/ml, then plated in 24-well plates at 1 ml/well. Culture medium was replaced with fresh medium every 3 days. At day 6, dislodged cells were used as bone marrow-derived DCs.

National Centre for Cell Science, Ganeshkhind, Pune, India

Preparation of RM-1 cell Ag

Received for publication March 3, 2006. Accepted for publication August 25, 2006.

The Ags were prepared by rapid freeze-thaw cycles for seven cycles, followed by sonication and clarification by microfuging. The protein content was assayed by the BCA protein assay kit (Pierce).

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 The work was supported by the Department of Biotechnology, Government of India. R.A. is supported by an Indian Council of Medical Research fellowship. 2

Address correspondence and reprint requests to Dr. Bhaskar Saha, National Centre for Cell Science, Ganeshkhind, Pune 411007, India. E-mail address: sahab@ nccs.res.in 3 Abbreviations used in this paper: DC, dendritic cell; TNP, trinitrophenyl; KLH, keyhole limpet hemocyanin.

Copyright © 2006 by The American Association of Immunologists, Inc.

Tumor Ag pulsing of DCs and immunization Bone marrow-derived DCs were pulsed with irradiated tumor cells (10,000 rad) at a ratio of 3:1 for 12 h. Later the DCs were harvested and washed extensively with sterile PBS and used for in vivo priming. For prophylactic experiments, mice were immunized s.c. thrice at 7-day intervals with 3 ⫻ 106 DCs. In some experiments, mice were injected i.p. with anti-IL-10 neutralizing Ab together with s.c. injections of tumor Ag-pulsed DCs. One 0022-1767/06/$02.00


A

ctivation of T cells requires two signals through Agspecific TCR and costimulatory molecules such as CD28 and CD40L (1). The APC such as dendritic cells (DC)3 process and present a protein Ag to generate the peptideMHC complex that is recognized by the Ag-specific TCR, triggering the first signal (2). The APC also provides the second signal through costimulatory molecules such as CD80/CD86-CD28 and CD40-CD40L interactions (1– 4). Although the use of defined tumor Ags for the induction of protective T cells met with limited success (5–7), CD40-CD40L interaction that was proposed to induce antitumor T cells did not prevent tumor growth completely (8, 9). The antitumor immune response that results in the killing of tumor cells is mediated by CTL (10). The elicitation of an efficient antitumor CTL response that regresses tumor, albeit incomplete, is dependent on CD40-CD40L interaction (10 –12). The CD40mediated elicitation of CTL response is IL-12-dependent, thus injection of IL-12 or expression of IL-12 in the adoptively transferred DCs also induces significant antitumor CTL responses (13, 14). In contrast, CD40 signaling in macrophages induces not only IL-12 but also IL-10 (15). Although IL-12 is required for the induction of IFN-␥ (16), an important surrogate marker of a CTL response, IL-10 inhibits CD40-induced IL-12 expression (15), suggesting a plausible functional dichotomy in the CD40 regulation of an antitumor CTL response. Therefore, we tested whether manipulation of the circuitry that regulates the CD40-induced IL-12 production by differential induction of IL-10 resulted in complete prevention of tumor growth. The results from our experiments demonstrated that low-dose CD40 ligation induces IL-10, whereas high-dose CD40 ligation


6643

week after the last immunization, mice were challenged s.c. with live tumor cells (2 ⫻ 105). For therapeutic experiments, mice were first injected with 2 ⫻ 105 RM-1 cells. After tumor challenge the mice received three s.c. injections of tumor

Ag-pulsed DCs (2 ⫻ 106) at a 5-day interval. Animals were followed for survival, and sacrificed before the control animals died of increased tumor burden. Tumor weight has been taken from the tumor samples collected from every individual animal.


FIGURE 1. IL-10 limits the protective antitumor immune response. B6 wild-type (B6-WT), IL-10⫹/⫺, and IL-10⫺/⫺ mice were injected s.c. with RM-1 cells (2 ⫻ 105/mouse). Tumor growth was monitored and mice were sacrificed when the control mice showed adverse effects from tumor, which happened 3 wk after the tumor cell injection. A, The collected tumor sample from every individual animal (n ⫽ 6 mice) of each group has been weighed, and the average of tumor weight in each group has been shown. Error bars represent mean ⫾ SD. IL-10 production by tumor Ag exposed splenocytes in vitro (inset). Data are representative of three independent experiments. B, Splenic T cells from five individual mice from the same group were cultured in 96-well plates for 3 days at 2 ⫻ 105 cells/well in presence or absence of RM-1 cell Ags. In the last 16 h, cells were pulsed with 1 ␮Ci of [3H]thymidine to determine tumor Ag-specific T cell proliferation. Data represent the mean of triplicate assays. C, Splenic T cells from five individual mice from the same group were cultured in 96-well plates with RM-1 Ags in triplicate for 48 h. Cell-free supernatants were harvested and assayed for IFN-␥ production by ELISA. D, Splenocytes were plated at 1.5 ⫻ 107 cells/well in 6-well dishes with 1.5 ⫻ 106 irradiated RM-1 cells. After 5 days of coculture, viable CD8⫹ T cells were harvested and plated against [3H]thymidine-incorporated target cells (RM-1) and tested for their cytolytic activity in a standard 4 h JAM test. The E:T ratio was as shown. Each data point is the mean of triplicate samples. The results represent three individual experiments and error bars represent the mean ⫾ SD of a given group. Student’s t test was performed to ascertain the significance of difference between the means of the control group and the experimental group or between two experimental groups. E, IL-10 modulates the antitumor Ab response. B6 wild-type and IL-10⫺/⫺ mice were injected s.c. with RM-1 cells (2 ⫻ 105/mouse). Three weeks after injection of the tumor cells, the sera from these mice were assayed for tumor Ag-reactive Ig isotypes (IgM, IgG1, IgG2a) on RM-1 Ag-coated ELISA plates. The plates were coated with 10 ␮g/ml RM-1 soluble Ags. The Ags were prepared by rapid freeze-thaw cycles for seven cycles, followed by sonication and clarification by microfuging. The protein content was assayed by the BCA protein assay kit. The data shown represent one of three individual experiments having a minimum of three animals per group. F–H, IL-10 regulates the B cell and T cell response in response to a hapten-carrier conjugate. B6 wild-type and IL-10⫺/⫺ mice were injected s.c. with TNP-KLH in CFA (100 ␮g/mouse). Ten days later the sera (primary response sera) were drawn from these mice and were used for anti-TNP Ab isotyping on TNP-BSA coated ELISA plates. Four weeks later, the mice were given a boost with TNP-KLH in IFA. Seven days after the boost, the sera (secondary response sera) were collected and the mice were sacrificed. F, Splenocytes of four individual mice from the same group were cultured in 96-well plates for 3 days at 2 ⫻ 105 cells/well in presence or absence of KLH (5–10 ␮g/ml). In the last 16 h, cells were pulsed with 1 ␮Ci of [3H]thymidine to determine KLH-specific T cell proliferation. The resultant culture supernatants from the experiment were used for determining secreted IFN-␥. The anti-TNP isotypes (IgM, IgG1, IgG2a) in the primary (G) and secondary (H) response sera were assayed. The experiments were repeated three times and representative data from one experiment are shown.

6644

DUALITY IN CD40 FUNCTION DICTATES ANTITUMOR T CELL RESPONSE

Tumor models and Ab treatment A total of 2 ⫻ 105 RM-1 cells were injected s.c. in the right flank of mice in 100 ␮l of PBS. Tumor growth was monitored and mice were sacrificed when the control mice just started to show adverse effects (difficulty in movement or any other visible distress) from tumor. Mice were supervised daily by the resident veterinarian. For in vivo activation, mice were injected i.p. with different doses of endotoxin-free anti-CD40 mAb in 200 ␮l on day 5, 7, and 9 after tumor cell inoculation. As indicated, some mice were coadministered with 200 ␮g of anti-IL-10 Ab (BD Pharmingen).

T cell purification and DC-T cell coculture assay T cells were isolated from the spleen and lymph nodes of mice (15). In brief, the collected spleen and lymph node were dissected into single cell suspensions, and after removing red cells by hypotonic lysis, the T cells were isolated using a nylon wool column. The CD8⫹ T cells were isolated by using the CD8⫹ T cell enrichment mixture from StemCell Technologies. Six-day-old DCs were cultured with purified T cells from naive mice in a 96-well round-bottom plate at a ratio of 1:3 in the presence or absence of anti-CD3 Ab (BD Pharmingen) for 72 h. The resultant culture supernatants were assayed for the secreted cytokines.

Adoptive T cell transfer experiment

Tumor Ag-specific T cell proliferation and IFN-␥ production The collected spleens from control and DC-immunized mice were used for preparing single cell suspension of splenocytes by homogenizing spleens between frosted glass slides (Fisher Scientific) and removing RBC with buffered ammonium chloride lysis buffer. T cells were purified as described. The T cells were cultured in 96-well plates for 3 days at 2 ⫻ 105 cells/well with RM-1 Ags with irradiated splenocytes as the APCs. In the last 16 h, cells were pulsed with 1 ␮Ci of [methyl-3H]thymidine (Board of Radiation and Isotope Technology). Cells were then harvested onto a membrane filter using a filter-mate harvester (Packard Instruments), and incorporation of [3H]thymidine was used as a measure of DNA synthesis using liquid scintillation counter (Packard Instruments). Assays were performed in quadruplicate. Supernatants from parallel cultures were harvested 48 h after the initiation of the cultures. IFN-␥ in the supernatants were assayed by IFN-␥ ELISA kits (BD Pharmingen) following the manufacturer’s instructions.

Cytotoxic T cell assay For CTL assay, splenocytes were plated at 1.5 ⫻ 107 cells/well in 6-well dishes with 1.5 ⫻ 106 irradiated RM-1 cells. After 5 days of coculture, viable CD8⫹ T cells were isolated and plated against thymidine-incorporated target cells (RM-1) and tested for their cytolytic activity in a standard 4-h JAM test (19).

Flow cytometry Cells were incubated at 4°C in 0.1% (w/v) BSA in PBS and surface stained with FITC-conjugated or PE-conjugated Abs for 45 min before washing three times with ice-cold PBS. Cells were then fixed in 0.1% (w/v) paraformaldehyde in PBS and samples were analyzed using a FACSVantage flow cytometer (BD Biosciences) with CellQuest software.

Cytokine ELISA Cytokine levels in culture supernatants were detected by a standard twosite sandwich ELISA for cytokines as described in the BD Pharmingen manual. In brief, ELISA plates were coated with 50 ml of anti-mouse cytokine mAb in 0.1 M NaHPO4 (pH 9.0) overnight at 4°C. Plates were washed three times with wash buffer (PBS with 0.05% Tween 20) and blocked with 200 ␮l of blocking buffer (PBS with 1% BSA, 0.05% Tween 20, and 0.05% NaN3) for 1 h at room temperature. Plates were washed three times with wash buffer before adding 100 ␮l of murine recombinant cytokine standard (BD Pharmingen) or culture supernatants in binding buffer (PBS, 1% BSA, 0.05% Tween 20) before incubating overnight at 4°C. Plates were washed four times with wash buffer before adding 100 ml of biotin-conjugated, anti-mouse cytokine mAb for 1 h at room temperature. Plates were again washed four times with wash buffer before addition of 100 ␮l of peroxidase-conjugated streptavidin, and incubated for 45 min at room temperature. Plates were then washed six times, after which 100 ␮l of tetramethylbenzidine substrate (BD Pharmingen) was added to each well

FIGURE 2. IL-10 restricts the CD40-induced antitumor immune response. B6 mice were injected s.c. with RM-1 cells (2 ⫻ 105/mouse). The mice were injected i.p. with the indicated doses of endotoxin-free anti-CD40 mAb in 200 ␮l of saline on day 5, 7, and 9 after tumor cell inoculation. As indicated, some mice were coadministered with 200 ␮g of anti-IL-10 Ab. The mice were sacrificed 3 wk after the tumor cell injection. A, The collected tumor sample from every individual animal (n ⫽ 8 mice) of each group has been weighed and the average tumor weight in each group has been shown. Error bars represent the mean ⫾ SD of the readings from the mice in an individual group. Cytokine expression in the tumor tissue (inset); total RNA was isolated from the collected tumor tissue of individual animal and used for first strand cDNA synthesis. The cDNA was then used as a template for PCR amplification of mouse IL-10, IL-12, IFN-␥ using gene specific primers. Results are expressed as individual data. B, Splenic T cells from five individual mice from the same group were cultured in 96-well plates with RM-1 cell Ags in triplicate for 48 h. Cell-free supernatants were harvested, and assayed for IFN-␥ production by ELISA. C, Splenocytes were plated at 1.5 ⫻ 107 cells/well in 6-well dishes with 1.5 ⫻ 106 irradiated RM-1 cells. After 5 days of coculture, viable CD8⫹ T cells were harvested and plated against [3H]thymidine-loaded target cells (RM-1) and tested for their cytolytic activity in a standard 4 h JAM test. The E:T ratio was as shown. Each point is the mean of triplicate samples. The data shown are mean ⫾ SD from one of three experiments.

and allowed to develop for 10 min at room temperature before stopping the reaction with the addition of 50 ml of 1 N H2SO4 in double distilled H2O. Absorbance at 450 nm was measured using an automated microplate absorbance reader (Bio-Tek Instruments).

Tumor-reactive Ab ELISA To detect tumor reactive Ig, 96-well Nunc Maxisorb microtiter plates were coated overnight with tumor lysate protein (10 ␮g/ml) at 4°C. The plates were blocked with 1% BSA in PBS for 2 h at room temperature and the collected sera from the naive and tumor-bearing animals were added at different dilutions, as indicated, and prepared in binding buffer overnight at 4°C. Plates were washed four times with wash buffer, and bound Abs were detected using biotin-conjugated goat anti-mouse IgG1, IgG2A, IgM (all biotin-conjugated Abs were from BD Pharmingen).


T cells were purified from spleen of naive and DC-immunized mice, washed extensively in PBS, and then injected i.v. (107 cells in 200 ␮l of PBS/mouse) into recipient 6- to 8-wk-old C57BL/6 nu/nu mice.


6645

RT-PCR

Results

Total RNA was isolated from tumor cells, DCs, and T cells using TRIzol (Sigma-Aldrich) according to manufacturer’s instructions, and used for first strand cDNA synthesis using the Thermoscript RT-PCR system (Invitrogen Life Technologies). The cDNA was then used as template for PCR amplification of mouse IL-10, IL-12, TNF-␣, T-bet, GATA-3, IFN-␥, and IL-4 using gene-specific primers. The primers were for IL-10 forward 5⬘TCA CTC TTC ACC TGC TCC AC-3⬘, reverse 5⬘-CTA TGC TGC CTG CTC TTA CTC-3⬘; IL-12 forward 5⬘-AAA CAA GAC CCG CCC AAG AAC-3⬘, reverse 5⬘-AAA AAG CCA ACC AAG CAG AAG ACA G-3⬘; TNF-␣ forward 5⬘-GCG ACG TGG AAC TGG CAG AAG-3⬘, reverse 5⬘-GGT ACA ACC CAT CGG CTG GCA-3⬘; IFN-␥ forward 5⬘-CAT TGA AAG CCT AGA AAG TCT G-3⬘, reverse 5⬘-CTC ATG AAT GCA TCC TTT TTC G-3⬘; T-bet forward 5⬘-ACT TGT ACC ACA CAG GTG GTG G-3⬘, reverse 5⬘-ATG GGC ATC GTG GAG CCG GGC T-3⬘; GATA-3 forward 5⬘-GAA GGC ATC CAG ACC CGA AAC-3⬘, reverse 5⬘-ACC CAT GGC GGT GAC CAT GC-3⬘; and ␤-actin forward 5⬘-TGG AAT CCT GTG GCA TCC A-3⬘, reverse 5⬘-TAA CAG TCC GCC TAG AAG CA-3⬘. Each sample was amplified for mouse DHFR (dihydrofolate reductase) or ␤-actin to ensure equal cDNA input.

IL-10 limits the protective antitumor T cell response

Statistical analysis Each individual experiment was repeated a minimum of three times. The error bars represent the mean ⫾ SD of triplicate cultures in vitro. For in vivo experiments, error bars represent the mean ⫾ SD of the readings from the number of mice, which is a minimum of five mice per group. In some experiments we used even eight mice per group. The significance of the difference between the means of the control group and an experimental group or between two experimental groups as indicated was deduced by Student’s t test.

We injected RM-1 cells, a C57BL/6 mouse prostate-derived cell line (17), to induce tumor in C57BL/6 (B6), IL-10-deficient (IL10⫺/⫺; B6 background), and B6 ⫻ IL-10⫺/⫺ (F1 or IL-10⫹/⫺) mice. The B6 mice had significantly bigger tumors than those developed in IL-10⫹/⫺ mice ( p ⬍ 0.001) (Fig. 1A). IL-10⫺/⫺ mice did not develop any tumor at all (Fig. 1A). When compared with the B6 mice, in accordance with the tumor growth, T cell proliferation (Fig. 1B), IFN-␥ production (Fig. 1C), and CTL responses (Fig. 1D) were higher in IL-10⫺/⫺ mice ( p ⬍ 0.001), suggesting that IL-10 plays a tumor-promoting function perhaps by downregulation or differential modulation of the host’s antitumor T cell responses. Indeed, the production of Ab isotypes against RM-1 Ags was differentially regulated: higher IgM and IgG1 in B6 mice, whereas IgG2a was higher in IL-10⫺/⫺ mice (Fig. 1E). To test whether the role of IL-10 observed in the tumor model also holds in a different model, we examined the anti-trinitrophenyl (TNP) Ab response and the carrier-specific recall T cell response in the hapten-carrier conjugate trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH)-injected B6 and IL-10⫺/⫺ mice. It was observed that TNP-KLH priming of IL-10⫺/⫺ mice resulted in higher recall antiKLH T cell proliferation and higher IFN-␥ production (Fig. 1F) but differential regulation of Ig isotype production-less IgM and IgG1 in both primary and memory responses but higher IgG2a only in


FIGURE 3. IL-10 regulates CD40-induced DC cytokine production and the Th subset cytokine-inducing ability of DC. Bone marrow-derived DC from B6 wild-type (B6-WT) and IL-10⫺/⫺ mice were stimulated with the indicated doses of CD40-specific Ab for 4 h. A, Expression of IL-12 and TNF-␣ mRNA was detected by PCR. Data are representative of three separate experiments. B, B6 wild-type-derived DCs were stimulated with varying doses of CD40-specific Ab for 4 or 48 h; ELISA (from 48 h culture) and RT-PCR (from 4 h culture) (inset) detected the IL-10 expressions. C, IL-10 neutralization enhances CD40-induced IL-12 production from B6-derived DCs, as assessed by RT-PCR (inset) and ELISA. D, IL-10 suppresses the CD40-induced IL-12 production. Cells were pretreated with the indicated doses of IL-10 for 8 h and they were stimulated with anti-CD40 Ab for another 4 h. Total RNA was isolated and used for cDNA synthesis. The cDNA was then used as template for PCR amplification of mouse IL-12 gene specific primers. The experiment was repeated thrice, and results from one experiment were shown. E, IL-10 deficiency resulted in significantly less IL-4 but higher IFN-␥ productions; 6-day-old DC from B6 wild-type and IL-10⫺/⫺ mice were cultured with purified T cells from naive B6 wild-type and IL-10⫺/⫺ mice in a 96-well round-bottom plate at a ratio of 1:3 in the presence or absence of anti-CD3 Ab for 72 h in triplicates. The resultant culture supernatants were assayed for the secreted cytokines. The data shown represents one of three individual experiments. Error bars represent the mean ⫾ SD. F, Splenic T cells from B6 and IL-10⫺/⫺ mice were stimulated with anti-CD3⫹ anti-CD28 with or without rIL-10. Twelve hours into cultures, the cells were assessed for IFN-␥, T-bet, IL-4, and GATA-3 expression by RT-PCR. Data shown are representative of two separate experiments. G, B6 mice were injected with the RM-1 cells as mentioned, followed by i.p. treatment with anti-IL-10 (200 ␮g/mouse) or anti-IL-12 (100 ␮g/mouse) Abs or with rIL-12 (200 ng/mouse) for 5 consecutive days from the day of tumor cell injection. The tumor weight was measured 3 wk later. The results represent three individual experiments having a minimum of four animals per group. Error bars represent the mean ⫾ SD. Student’s t test was performed to ascertain the significant difference between the mean values.

6646


memory responses (Fig. 1, G and H), suggesting that the IL-10 effect was imposed during the priming phase but implemented during the memory response. These observations indicate that IL-10 does regulate the nature of the recall T cell response in vivo. IL-10 limits the CD40-induced antitumor immune response It was proposed that CD40 plays an important role in antitumor immune responses (8 –12). However, in most cases, the investigators used CD40⫹ tumor cell lines and examined the effects of anti-CD40 Ab administration. In contrast, the RM-1 cells do not express CD40 (data not shown). Therefore, we tested the effect of anti-CD40 Ab administration in B6 mice with or without IL-10 neutralization. It was observed that low doses of anti-CD40 Ab administration augmented tumor growth in B6 mice; IL-10 neutralization prevented the tumor growth (Fig. 2A). In contrast administration of a higher dose of the anti-CD40 Ab resulted in significantly less tumor growth than that observed in the control mice (Fig. 2A). Corroborating to the tumor growth profile with low and high anti-CD40 Ab, we observed that the IFN-␥ production (Fig. 2B) and CTL response (Fig. 2C) were significantly higher in the recipients of the high-dose anti-CD40 Ab. Also, we observed that IL-10 production in low-dose anti-CD40 Ab-treated mice was much higher in comparison to the level of IL-10 produced in the high-dose anti-CD40 Ab recipients (G. Murugaiyan and B. Saha, unpublished observations). Coadministration of anti-IL-10 Ab in low-dose anti-CD40 Ab recipients reduced the tumor growth but increased the IFN-␥ production and RM-1 Ag-pulsed target cell lysis, as compared with those mice that were treated with antiCD40 Ab alone ( p ⬍ 0.001) (Fig. 2). These results suggest that CD40 has a dichotomous effect on the antitumor immune response.

FIGURE 5. T cells from IL-10⫺/⫺ DC-primed mice transfer protection against tumor in nude mice. A total of 5 ⫻ 104 RM-1 cells were injected s.c. into nude mice. T cells (1 ⫻ 107) from wild-type DC-primed (WT-DC-T) and IL-10⫺/⫺ DC-primed (IL-10-DC-T) mice were adoptively transferred into nude mice. The mice were monitored for tumor growth. Mice were sacrificed 17 days after tumor injection and the tumor weight (A), tumor Ag-specific T cell proliferation (B), and IFN-␥ production (C) were assessed as described. D, Cytotoxicity assay was performed by culturing splenocytes from the same group with irradiated tumor cells for 5 days, viable CD8⫹ T cells were tested for their cytolytic activity against live tumor cells in a 4 h JAM test, as described. The E:T ratio was as shown. Each point is the mean of triplicate samples. The results represent two individual experiments and the error bars represent the mean ⫾ SD. Student’s t test was performed to ascertain the significant difference between the mean values.


FIGURE 4. IL-10 deficiency induces host-protective antitumor memory and IL-10 neutralization during priming prevents tumor growth completely. A, B6 mice were primed with tumor Ag-pulsed DCs (3 ⫻ 106 cells/mouse) derived from the bone marrow of wild-type and IL-10⫺/⫺ mice. Seven days after the last immunization, mice were challenged with live tumor cells. Three weeks later the mice were sacrificed. A, The collected tumor sample from every individual animal (n ⫽ 5 mice) of each group has been weighed, and the average of tumor weight in each group has been shown. Error bars represent thr mean ⫾ SD. B, Splenocytes from these mice were plated at 1.5 ⫻ 107 cells/well in 6-well dishes with 1.5 ⫻ 106 irradiated RM-1 cells. After 5 days of coculture, viable CD8⫹ T cells were harvested and plated against [3H]thymidine incorporated target cells (RM-1) and tested for their cytolytic activity in a standard 4h JAM test. The E:T ratio was as shown. Each point is the mean of triplicate samples. The data shown are the mean ⫾ SD from one of three experiments. C, RM-1 tumor Ag-specific T cell proliferation was assessed by the standard [3H]thymidine incorporation assay, as described. Data present the mean of triplicate assays. D, The cell-free supernatants from the cultures described (culture set up as in C) were assayed for IFN-␥ production by ELISA. E, In some of the experiments, B6 mice were treated with anti-IL-10 Ab (200 ␮g/mouse) for 5 consecutive days during the priming with tumor Ag-pulsed wild-type DCs. The mice were primed four times. Seven days after the last priming, mice were challenged with live tumor cells. Three weeks later, the mice were sacrificed and the following assays were performed. E, The mean tumor weight of tumor samples collected from five mice per group is shown. F, Tumor Ag-specific T cell proliferation was performed by culturing the cells with RM-1 tumor Ag for 72 h as described. Data represent the mean of triplicate assays. G, The 48-h culture supernatants from the described experiment in F were assayed for the secreted IFN-␥. H, Cytotoxicity assay was performed by culturing splenocytes with irradiated tumor cells for 5 days. Later on, viable CD8⫹ T cells were tested for their cytolytic activity against live tumor cells in a 4 h JAM test. The E:T ratio was as shown. Each point is the mean of triplicate samples and the data shown represent one of three individual experiments.


6647

At low dose, the Ab induces IL-10 that nullifies the antitumor effect of CD40 stimulation. On the contrary, at higher doses, the Ab induces most probably less IL-10 and therefore, a protective antitumor immune response. In addition, the anti-IL-10 Ab coadministration reverses the effect of low-dose anti-CD40 Ab, demonstrating the therapeutic benefit of IL-10 neutralization. IL-10 limits the CD40-induced TNF-␣ and IL-12 production Because the antitumor immunity in B6 mice was compromised due to CD40-induced IL-10 production and IL-10 neutralization in B6 mice significantly reduced the tumor burden and heightened the CTL response to the level observed in IL-10⫺/⫺ mice (G. Murugaiyan and B. Saha, unpublished observation), it is possible that there exists an association between CD40-CD40L interaction and IL-10 in determining tumor growth. Indeed, CD40 induced TNF-␣ expression, an important determinant of tumor regression (20), and IL-12 expression in DCs in an IL-10-sensitive manner (Fig. 3, A–D) This observation indicates that CD40 induces not only the host-protective cytokines such as IL-12 and TNF-␣ (Fig. 3A) but also an anti-inflammatory cytokine such as IL-10 (Fig. 3B). Because IL-12 induces but IL-10 inhibits IFN-␥ production (21, 22), the CD40-induced IL-10 may alter the balance between Th1-type and Th2-type cytokines as well. Indeed, in a DC-T cell coculture system, IL-10 deficiency resulted in significantly less IL-4 but higher IFN-␥ productions (Fig. 3E), accompanied by a differential modulation of Th1- and Th2-specific transcription factors, T-bet and GATA-3, respectively (Fig. 3F). These observations suggest that IL-10 reduces the Th1 to Th2 ratio eventuating

in tumor growth. However, the stability of the altered T cell responses remained to be tested. Because IL-12 is required for IFN-␥ induction, it is possible that IL-10 impairs memory T cell generation by reducing the production of IL-12 (Fig. 3G), a cytokine implied in the process. Indeed, in RM-1 challenge experiments, rIL-12 enhanced while anti-IL-12 abrogated the antitumor effect of anti-IL-10 treatment during priming (Fig. 3G). Therefore, these observations suggest that IL-10 regulates both T cell effector functions and the generation of memory T cell response. IL-10 deficiency induces host-protective antitumor memory Next, we investigated whether priming B6 mice with IL-10deficient DC would result in a stable host-protective antitumor T cell response during tumor challenge. We observed that the B6 mice, which were primed a minimum of four times with IL-10deficient DC, did not develop any tumor after tumor challenge (Fig. 4A). As compared with the T cells from the mice that received wild-type DC, the T cells from the recipients of IL-10⫺/⫺ DC exhibited a higher CTL response ( p ⬍ 0.005) (Fig. 4B), enhanced proliferation ( p ⬍ 0.001) (Fig. 4C), and increased IFN-␥ production ( p ⬍ 0.001) (Fig. 4D). Although the priming with B6derived DC resulted in significantly less tumor growth compared with growth seen in unprimed controls ( p ⬍ 0.001) (Fig. 4A), the complete prevention of tumor growth in mice primed with IL10⫺/⫺ DC confirms that DC-expressed IL-10 self-limits the efficacy of the DC priming-induced antitumor T cell effector functions. Therefore, we tested whether IL-10 neutralization during DC-mediated priming would result in a similar protection against


FIGURE 6. Therapy with IL-10-deficient DCs imparts significant antitumor T cell response. B6 mice were inoculated with 2 ⫻ 105 RM-1 cells, followed by three injections of tumor Ag-pulsed IL-10⫺/⫺ DCs, beginning on the day of RM-1 injection (D0) or 3 days (D3) or 7 days (D7) after RM-1 cell injection. Some groups of mice received unpulsed DC alone. Twenty-one days later, the mice were sacrificed and the following assays were performed. A, The collected tumor sample from every individual animal (n ⫽ 6 mice) of each group has been weighed and the average of tumor weight in each group has been shown. Error bars represent the mean ⫾ SD. B, Tumor Ag-specific T cell proliferation was assessed by a standard [3H]thymidine incorporation assay. Data present the mean ⫾ SD of triplicate assays. C, Cell-free supernatants from parallel cultures as described in B were assayed for IFN-␥ production by ELISA. D, Cytotoxicity assay was performed by culturing splenocytes of the mice with irradiated tumor cells for 5 days. Later on, viable CD8⫹ T cells were tested for their cytolytic activity against live tumor cells in a 4 h JAM test. The E:T ratio was as shown. Each point is the mean of triplicate samples. The results represent three individual experiments and the error bars represent the mean ⫾ SD. Student’s t test was performed to ascertain the significant difference between the mean values.

6648


Discussion the tumor challenge. We observed that the mice primed with DC alone had significantly less tumor growth after the tumor challenge ( p ⬍ 0.001) (Fig. 4E), but simultaneous IL-10 neutralization during DC-mediated tumor Ag priming resulted in complete protection of the mice against the tumor challenge (Fig. 4E). The protection was accompanied by a significantly higher T cell proliferation in response to tumor Ags ( p ⬍ 0.001) (Fig. 4F), higher IFN-␥ production ( p ⬍ 0.001) (Fig. 4G), and higher CTL response ( p ⬍ 0.001) (Fig. 4H). In fact, the latter group of survivors remained resistant to the second rechallenge with the tumor cells (Fig. 4E, inset). In an adoptive transfer experiment, the nude mice that received T cells from the IL-10-deficient DC-primed B6 mice had significantly smaller tumor (Fig. 5A and inset) than the nude mice that received T cells from the wild-type DC-primed B6 mice ( p ⬍ 0.001); the T cells from the former group of mice showed higher proliferation ( p ⬍ 0.01) (Fig. 5B) IFN-␥ production ( p ⬍ 0.001) (Fig. 5C), and CTL responses ( p ⬍ 0.001) (Fig. 5D) than the latter group of mice. Thus, these data indicate for the first time that IL-10 plays a significant role in the regulation of memory T cells and that its neutralization during priming heightens the efficacy of the antitumor functions of the memory T cells. Therapy with IL-10-deficient DCs imparts significant antitumor T cell response leading to a model for therapeutic autovaccination Next, we tested whether transfers of IL-10-deficient DCs could alter the function of these antitumor T cells in tumor-bearing B6 mice. We observed that i.v. transfer of tumor Ag-pulsed IL-10deficient DC lastin 3 consecutive days prevented tumor growth completely (Fig. 6A). However, delaying the DC administration as late as 7 days after RM-1 cell injection resulted in tumor growth but still significantly less than that observed in the untreated con-

The CD40-CD40L interaction is proposed to be crucial for IL-12dependent, IFN-␥- and CTL-mediated immunity against various pathogens and tumors (10 –12, 20 –24). The critical role of the CD40-CD40L interaction in tumor immunology was first suggested by studies of protective immunity against syngenic tumors in mice demonstrating that blockade of the pathway with antiCD40L Abs inhibited the generation of protective immune response against potent tumor vaccines, and that CD40-deficient mice were unable to generate an antitumor immune response (25). The other studies on the CD40-induced antitumor immunity attributed the function to CD40-induced DC maturation (26) or linked it to the enhanced Ag processing and presentation (27, 28). Although all these studies examined the indirect effect of CD40 on the T cell-mediated antitumor immunity, there were several studies that looked into the direct cytotoxic effects of CD40 Ab (29), soluble CD40L (30), or CD40L-expressing cells (31) on the CD40expressing tumors. Altogether, all these studies indicate that the CD40 has antitumor effects. In contrast, our data suggest a duality in the CD40 function as a function of its extent of cross-linking. At high doses, CD40 Ab elicits antitumor T cell responses whereas at low doses, the Ab elicits a counteractive T cell response that promotes tumor growth. Because RM-1 cells do not express CD40 (G. Murugaiyan and B. Saha, unpublished observation), our observations suggest indirect effects through the modulation of DC functions. The nature of the counteractive T cells and the mechanism of their regulation by CD40 signaling in DC need to be examined in detail. As an alternative, it is quite possible that there are CD8⫹ T regulatory cells that may suppress the functions of active CD8⫹ CTLs. However, such a hypothesis is currently under investigation. Our accompanying data have proposed how modulation of IL-10 to IL-12 ratio may play a crucial role in such CD40directed tumor promotion or regression.


FIGURE 7. Therapeutic autovaccination regresses the tumor significantly. B6 mice were inoculated with 2 ⫻ 105 RM-1 cells. Once the tumor developed to a palpable size, some of these mice were sacrificed, tumor cells isolated, and Ag prepared; other mice were treated with either wild-type DC or IL-10⫺/⫺ DCs pulsed with the tumor lysate extracted from the tumor excised from the first group of B6 mice. Tumor growth was monitored and mice were sacrificed after 3 wk. A, The collected tumor sample from every individual animal (n ⫽ 4 mice) in each group was weighed, and the average of tumor weight in each group has been shown. T cell proliferation (B) and IFN-␥ production (C) were assessed as described. D, Splenocytes from the mice were plated at 1.5 ⫻ 107 cells/well in 6-well dishes with 1.5 ⫻ 106 irradiated RM-1 cells. After 5 days of coculture, viable CD8⫹ T cells were harvested and plated against [3H]thymidine-labeled target cells (RM-1) and tested for their cytolytic activity in a standard 4 h JAM test. The E:T ratio was as shown. Each point is the mean of triplicate samples. The results represent two individual experiments and error bars represent the mean ⫾ SD.

trol group ( p ⬍ 0.002) (Fig. 6A) or the unpulsed DC-treated group ( p ⬍ 0.001) (Fig. 6A). The protection was associated with higher T cell proliferation in response to tumor Ags ( p ⬍ 0.001) (Fig. 6B), higher IFN-␥ production ( p ⬍ 0.01) (Fig. 6C), and higher CTL response ( p ⬍ 0.01) (Fig. 6D). The results thus identify IL-10 as a crucial factor that significantly limits the antitumor functions of T cells in both therapeutic and prophylactic modes of tumor intervention strategies. Because tumor Ags may vary among patients due to host genetic factors, the success of a defined tumor Ag for establishing a protective antitumor T cell response appears to be limited (5–7). The other important limitations of using defined tumor Ag for vaccination or therapy include mobilization of only a small proportion of the possible antitumor T cell repertoire, HLA polymorphism, and a possible shift in T cell reactivity toward the Ag during an ongoing immune response. To bypass these shortcomings, we also examined whether the IL-10-deficient DCs, pulsed with the tumor Ag extracted from the tumor excised from the same individual, might control tumor growth. Therefore, tumor-bearing B6 mice were treated with the B6-derived or IL-10-deficient DCs pulsed with Ags extracted from the tumor excised from B6 mice. We observed that the mice treated with IL-10-deficient DCs developed a significantly smaller tumor as compared with the recipient of B6-derived DCs ( p ⬍ 0.01) (Fig. 7A). Compared with the T cells from the wild-type DC-treated mice, the T cells from the IL-10⫺/⫺ DC-treated mice showed higher proliferation ( p ⬍ 0.05) (Fig. 7B), higher IFN-␥ production ( p ⬍ 0.01) (Fig. 7C), and higher CTL activity ( p ⬍ 0.01) (Fig. 7D) in response to tumor lysate Ag. These results indicate that the DCs turned IL-10-deficient can be used for successful “therapeutic autovaccination” against a tumor.


14.

15.

16.

17.

18. 19. 20.

21.

22.

23.

24.

25.

Disclosures The authors have no financial conflict of interest.

26.

References 1. Steinman, R. M., and J. W. Young. 1991. Signals arising from antigen-presenting cells. Curr. Opin. Immunol. 3: 361–372. 2. Germain, R. N., and I. Stefanova. 1999. The dynamics of T cell receptor signaling: complex orchestration and the key roles of tempo and cooperation. Annu. Rev. Immunol. 17: 467–522. 3. Quezada, S. A., L. Z. Jarvinen, E. F. Lind, and R. J. Noelle. 2004. CD40/CD154 interactions at the interface of tolerance and immunity. Annu. Rev. Immunol. 22: 307–328. 4. Grewal, I. S., and R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16: 111–135. 5. Dunn, G. P., L. J. Old, and R. D. Schreiber. 2004. The three Es of cancer immunoediting. Annu. Rev. Immunol. 22: 329 –360. 6. Houghton, A. N., J. S. Gold, and N. E. Blachere. 2001. Immunity against cancer: lessons learned from melanoma. Curr. Opin. Immunol. 13: 134 –140. 7. Houghton, A. N. 1994. Cancer antigens: immune recognition of self and altered self. J. Exp. Med. 180: 1– 4. 8. Alexandroff, A. B., A. M. Jackson, T. Paterson, J. L. Haley, J. A. Ross, D. L. Longo, W. J. Murphy, K. James, and D. D. Taub. 2000. Role for CD40CD40 ligand interactions in the immune response to solid tumors. Mol. Immunol. 37: 515–526. 9. Diehl, L., A. Th. den Boer, S. P. Schoenberger, E. I. van der Voort, T. N. Schumacher, C. J. Melief, R. Offringa, and R. E. Toes. 1999. CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy. Nat. Med. 5: 774 –779. 10. Stumbles, P., A. R. Himbeck, J. A. Frelinger, E. J. Collins, R. A. Lake, and B. W. Robinson. 2004. Cutting edge: tumor-specific CTLs are constitutively crossarmed in draining lymph nodes and transiently disseminate to mediate tumor regression following systemic CD40 activation. J. Immunol. 173: 5923–5928. 11. Chiodoni, C., P. Paglia, A. Stoppacciaro, M. Rodolfo, M. Parenza, and M. P. Colombo. 1999. DCs infiltrating tumors co-transduced with granulocyte/ macrophage colony-stimulating factor (GM-CSF) and CD40 ligand genes take up and present endogenous tumor-associated antigens, and prime naive mice for a cytotoxic T lymphocyte response. J. Exp. Med. 190: 125–133. 12. Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, and C. J Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393: 480 – 483. 13. Mortarini, R., A. Borri, G. Tragni, I. Bersani, C. Vegetti, E. Bajetta, S. Pilotti, V. Cerundolo, and A. Anichini. 2000. Peripheral burst of tumor-specific cytotoxic

27.

28.

29.

30.

31.

32. 33.

34.

35.

36. 37.

T lymphocytes and infiltration of metastatic lesions by memory CD8⫹ T cells in melanoma patients receiving IL-12. Cancer Res. 60: 3559 –3568. Sanford, M. A., Y. Yan, S. E. Canfield, W. Hassan, W. A. Selleck, G. Atkinson, S. H. Chen, and S. J. Hall. 2001. Independent contributions of GR-1⫹ leukocytes and Fas/FasL interactions to induce apoptosis following IL-12 gene therapy in a metastatic model of prostate cancer. Hum. Gene Ther. 12: 1485–1498. Mathur, R. K., A. Awasthi, P. Wadhone, B. Ramanamurthy, and B. Saha. 2004. Reciprocal CD40 signals through p38MAPK and ERK-1/2 induce counteracting immune responses. [Published erratum appears in 2004 Nat. Med. 7: 755.] Nat. Med. 10: 540 –544. Chan, S. H., B. Perussia, J. W. Gupta, M. Kobayashi, M. Pospisil, H. A. Young, S. F. Wolf, D. Young, S. C. Clark, and G. Trinchieri. 1991. Induction of interferon ␥ production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers. J. Exp. Med. 173: 869 – 879. Baley, P. A., K. Yoshida, W. Qian, I. Sehgal, and T. C. Thompson. 1995. Progression to androgen insensitivity in a novel in vitro mouse model for prostate cancer. J. Steroid Biochem. Mol Biol. 52: 403– 413. Ahuja, S. S. 2001. In vitro generation of functional human and murine DCs. Methods Mol. Biol. 156: 67–77. Matzinger, P. 1991. The JAM test: a simple assay for DNA fragmentation and cell death. J. Immunol. Methods 145: 185–192. Havell, E. A., W. Fiers, and R. J. North. 1988. The anti-tumor function of tumor necrosis factor (TNF). I. Therapeutic action of TNF against an established murine sarcoma is indirect, immunologically dependent, and limited by severe toxicity. J. Exp. Med. 167: 1067–1085. Kelsall, B. L., E. Stuber, M. Neurath, and W. Strober. 1996. Interleukin-12 production by DCs: the role of CD40-CD40L interactions in Th1 T-cell responses. Ann. NY Acad. Sci. 795: 116 –126. Mosmann, T. R., and R. L. Coffman. 1989. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7: 145–173. Schmidt, C. S., and M. F. Mescher. 1999. Adjuvant effect of IL-12: conversion of peptide antigen administration from tolerizing to immunizing for CD8⫹ T cells in vivo. J. Immunol. 163: 2561–2567. Gollob, J. A., C. P. Schnipper, E. Orsini, E. Murphy, J. F. Daley, S. B. Lazo, D. A. Frank, D. Neuberg, and J. Ritz. 1998. Characterization of a novel subset of CD8⫹ T cells that expands in patients receiving interleukin-12. J. Clin. Invest. 102: 561–575. Mackey, M. F., J. R. Gunn, P. P Ting, H. Kikutani, G. Dranoff, R. J. Noelle, and R. J. J. Barth. 1997. Protective immunity induced by tumor vaccines requires interaction between CD40 and its ligand, CD154. Cancer Res. 57: 2569 –2574. van Mierlo, G. J. D., A. Th. den Boer, J. P. Medemea, E. I. van der Voort, M. F. Fransen, R. Offringa, C. J. Melief, and R. E. Toes. 2002. CD40 stimulation leads to effective therapy of CD40-tumors through induction of strong systemic cytotoxic T lymphocyte immunity. Proc. Natl. Acad. Sci. USA 99: 5561–5566. von Leoprechting, A., P. van der Bruggen, H. L. Pahl, A. Aruffo, and J. C Simon. 1999. Stimulation of CD40 on immunogenic human malignant melanomas augments their cytotoxic T lymphocyte-mediated lysis and induces apoptosis. Cancer Res. 59: 1287–1294. Khanna, R., L. Cooper, N. Kienzle, D. J. Moss, S. R. Burrows, and K. K. Khanna. 1997. Engagement of CD40 antigen with soluble CD40-ligand up-regulates peptide transporter expression and restores endogenous processing function in Burkitt’s lymphoma cells. J. Immunol. 159: 5782–5785. Funakoshi, S., D. L. Longo, M. Beckwith, D. K. Conley, G. Tsarfaty, I. Tsarfaty, R. J. Armitage, W. C. Fanslow, M. K. Spriggs, and W. J. Murphy. 1994. Inhibition of human B-cell lymphoma growth by CD40 stimulation. Blood 83: 2787–2794. Ghamande, S., B. L. Hylander, E. Oflazoglu, S. Lele, W. C. Fanslow, and E. A. Repasky. 2001. Recombinant CD40 ligand therapy has significant anti-tumor effects on CD40-positive ovarian tumor xenografts grown in SCID mice and demonstrates an augmented effect with cisplatin. Cancer Res. 61: 7556 –7562. Chu, P., D. Deforce, I. N. Pedersen, Y. Kim, S. Kitada, J. C. Reed, and T. J. Kipps. 2002. Latent sensitivity to Fas-mediated apoptosis after CD40 ligation may explain activity of CD154 gene therapy in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 99: 3854 –3859. Urosevic, M., and R. Dummer. 2003. HLA-G and IL-10 expression in human cancer– different stories with the same message. Semin. Cancer Biol. 13: 337–342. Chattergoon, M. A., V. Saulino, J. P. Shames, J. Stein, L. J. Montaner, and D. B. Weiner. 2004. Co-immunization with plasmid IL-12 generates a strong T-cell memory response in mice. Vaccine 22: 1744 –1750. Adris, S., E. Chuluyan, A. Bravo, M. Berenstein, S. Klein, M. Jasnis, C. Carbone, Y. Chernajovsky, and O. L. Podhajcer. 2000. Mice vaccination with interleukin 12-transduced colon cancer cells potentiates rejection of syngeneic nonorgan-related tumor cells. Cancer Res. 60: 6696 – 6703. Monti, P., B. E. Leone, A. Zerbi, G. Balzano, S. Cainarca, V. Sordi, M. Pontillo, A. Mercalli, V. Di Carlo, P. Allavena, and L. Piemonti. 2004. Tumor-derived MUC1 mucins interact with differentiating monocytes and induce IL-10hiIL-12lo regulatory dendritic cell. J. Immunol. 172: 7341–7349. Steinbrink, K., M. Wolfl, H. Jonuleit, J. Knop, and A. H. Enk. 1997. Induction of tolerance by, IL-10-treated DCs. J. Immunol. 159: 4772– 4780. Jonuleit, H., E. Schmitt, H. Kakirman, M. Stassen, J. Knop, and A. H. Enk. 2002. Infectious tolerance: human CD25⫹ regulatory T cells convey suppressor activity to conventional CD4⫹ T helper cells. J. Exp. Med. 196: 255–260.


Although controlled Th1 and CTL responses can exert a significant antitumor immunity, the same responses, if exaggerated, may result in host-tissue destruction. Therefore, as a part of immune homeostasis, these T cell responses need to be counter-regulated. In this study, we demonstrate the CD40-centered built in control system. The CD40 stimulation induces not only antitumor cytokines such as TNF-␣ and IL-12 but also IL-10, a protumor cytokine that may act in an autocrine or a paracrine manner to counterregulate the possible host-tissue destruction by hyperactive Th1 cells or CTLs or TNF-␣. Therefore, the growth of tumor cells may exaggerate IL-10 production as a possible modus operandi for its immune evasion (32) exploiting the immune homeostatic mechanism. IL-10 limits the antitumor immunity, at least, at two different stages of the immune response. First, during the priming of the tumor Ag-specific T cells, IL-10 may directly impair the generation of memory T cells either directly by abrogating the clonal expansion of the Ag-specific T cells or indirectly, by reducing the production of IL-12 (15, 20), a cytokine implied in the generation of memory T cells (33, 34). Second, because IL-10-secreting DC are known to tolerize the T cells (35, 36), the T cells primed in the presence of IL-10 may already be anergized preventing them from responding to antigenic challenge in vitro and in vivo in the effector phase. Alternatively, the memory T cells generated in the presence of IL-10 may actively suppress the other Ag-specific T cells, preventing them from exerting the antitumor immune response, a phenomenon reminiscent of infectious tolerance (37). Therefore, IL-10 neutralization during priming or therapy using CD40-CD40L interaction is proved to be a completely preventative antitumor strategy, which may have even broader implications in many infectious and autoimmune diseases.

6649