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Immunology |
Institute of Immunology [R. B., M. L.], and Division of Urology of the Department of Surgery [W. R.], University of Heidelberg, 69120 Heidelberg; Department of Obstetrics and Gynecology, University of Heidelberg, 69115 Heidelberg [S. S.]; and University of Tübingen, 72076 Tübingen, [D. W., B. G.] Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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secreting cells, reduced interleukin 2 secretion, impaired proliferation, and a lack of CD154 re-expression on restimulation. In distinction, T-cell effector lysing capacity was not impaired by CD40-expressing tumor cell targets. The present results suggest that in marked contrast to antigen-presenting cells, CD40 expression on carcinoma cells suppresses T-cell activation. Our findings support the statement that CD40 functions are context dependent and imply a new function for CD40 expressed on nonantigen-presenting cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-8 and IL-12 (reviewed in Refs. 4
, 5
). Together, these costimulatory signals and cytokines are crucial for the development of type 1 TH cell response, which plays an important role in the priming of CTLs (6, 7, 8)
and the induction of antitumoral T-cell response (9
, 10)
. It was demonstrated recently that CD40 expression is not limited to cells of the immune system but is also present in several types of carcinoma, including those of the ovary, breast, bladder, lung, colon, prostate, and melanoma (11, 12, 13, 14, 15, 16) . Although the critical relevance of CD40 in APC/T-cell communication is already established, the functional role of CD40/CD154 interactions in nonhematopoetic cells is controversial discussed: whereas some studies postulated that CD40 expression on tumor cells serves as a possible receptor for mitogenic signals, others demonstrated that CD40 ligation on carcinoma cells results in growth inhibition and sensitization to apoptosis (reviewed in Ref. 11 ; Refs. 17 , 18 ). However, clinical studies evaluating lung cancer and melanoma patients demonstrated that CD40 expression may serve as a prognostic marker, which correlates with metastatic spread and poor prognosis (13 , 16) .
The present study addresses molecular aspects of the CD40/CD154 interaction in T-cell responses against malignant cells. Using constitutive CD40-expressing tumor cell lines of different entities, primary tumor material, as well as CD40-transfected tumor cell lines we demonstrated that CD154+ T lymphocytes may receive inhibitory signals from CD40-expressing tumors, subsequently leading to T-cell inactivation. This was confirmed by reduced IFN- and IL-2 secretion, and impaired proliferative responses after antigen dependent or Ab-mediated restimulation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PBMCs were prepared from the heparinized blood of healthy donors and patients by Ficoll-Hypaque (Pharmacia) density centrifugation. T cells were purified after an adherence step on plastic and subsequent rosetting with sheep erythrocytes (ICN, Meckenheim, Germany). Tumor cells, PBMCs, resting T cells, as well as TAL were cultured in RPMI 1640 (Life Technologies, Inc. GmbH, Karlsruhe, Germany) supplemented with 10% FCS, 4 mM L-glutamine, and 0.5% gentamicine (all Life Technologies, Inc.). As an exception, the KS breast carcinoma cell line was cultured in DMEM supplemented with 10% FCS, 4 mM L-glutamine, and 1% penicillin/streptomycin (all Life Technologies, Inc.).
The CD80 transfected sublines of KS (KS-CD80) and the melanoma cell line SkMel63 (SkMel63-CD80) have been described previously (19 , 20) and were maintained as their nontransfected parental counterparts, with the exception of G418 (Life Technologies, Inc.), which was supplemented at 0.5 mg/ml.
The EBV-transformed B-lymphoid cell line Laz509 expressing CD40 and other costimulatory molecules like CD80 was used as allogeneic APC and maintained in RPMI 1640.
DCs were propagated by cultivating plastic adherent PBMCs in the presence of granulocyte macrophage colony-stimulating factor and IL-4 for 7 days as described elsewhere (21) .
CD40 Subcloning and Transfection of SkMel63 and KS.
The CD40 fragment was isolated from the plasmid pCMV5-CD40 (W. R.) by HindIII/XbaI digestion and subsequently cloned into the HindIII/XbaI digested vector pcDNA3.1+ (Invitrogen, NV Leek, Netherlands). The correct orientation of the cDNA insert was confirmed by BamHI/XbaI digestion (all restriction enzymes Promega, Madison WI; the construct was done by T. Oehler, German Cancer Research Center, Heidelberg, Germany). SkMel63 or SkMel63-CD80 cells (2 x 106) were seeded in a 10-cm2 dish and lipotransfected with supercoiled pcDNA-3.1+-CD40 using 10 µg of DNA and 20 µg of lipofectin (Life Technologies, Inc.) according to the manufacturer’s protocol. KS cells were transfected by electroporation using a Gene Pulser (Bio-Rad, München, Germany) at 100 
, 500 µF, and 380 V. Selection was performed using 1 mg/ml G418. Growing cells were analyzed by immunofluorescence using a CD40-specific mAb (mAb89; Immunotech, Marseilles, France). Positive fractions were subcloned by limiting dilution and additionally expanded.
Flow Cytometry.
We incubated 3–5 x 105 each of tumor or lymphocytes with 50 µl of saturating mAb concentrations in PBS containing 10% FCS and 0.01% sodium azide (pH 7.2; 4°C). The following mAbs were used for indirect immunofluorescence: OKT3 (anti-CD3 mAb, 10 µg/ml; American Type Culture Collection), CD2.611F1, AICD2M1, and AICD2M2 (anti-CD2 mAbs, each 3 µg/ml, kindly provided by B. Schraven, Institute for Immunology, Heidelberg, Germany), mAb89 (anti-CD40 mAb, 2 µg/ml; Immunotech) and TRAP1 (anti-CD154 mAb, 2 µg/ml; Immunotech). A FITC-labeled goat-F(ab')2 antimouse immunoglobulin (immunoglobulin; Dako, Hamburg, Germany) was used as second-stage reagent, and secondary Ab staining alone served as negative control. Cells were analyzed on a Coulter Profile1 flow cytometer (Coulter Electronics, Hialeah, Finland) with logarithmic amplification (3-log scale).
T-Cell Activation.
When indicated, T cells were activated unspecifically either by overnight incubation with a combination of the calciumionophore A 23187 (250 nM; Sigma Chemical Co., Taufkirchen, Germany) and PMA (10-8 M; Sigma Chemical Co.) or by stimulatory mAbs. Hereby, an anti-CD28 mAb (IgM isotype; kindly provided by V. v. Fliedner, Ludwig Institute, Epalinges, Switzerland) acts comitogenic in soluble form without cross-linking; it was used as hybridoma supernatant at a final concentration of 25%.
For subsequent phenotypic analysis, preactivated T cells were cultured in medium without stimulus or were coincubated for 4 h with indicated -irradiated (200 Gy) tumor cells at a ratio of 10:1 at 37°C. The coincubation period was extended as indicated for subsequent functional T-cell assays. To block CD40/CD154 interactions the SkMel63-CD40 subline was preincubated with anti-CD40 mAb (mAb89; 5 µg/ml; 20 min; 37°C) before adding T cells.
To induce anergy, purified T cells were incubated for 48 h in culture dishes first precoated with rabbit antimouse immunoglobulin (100 µg/ml; Dako, Hamburg, Germany) for 2 h at room temperature followed by overnight incubation with OKT3 mAb (3 µg/ml) or TRAP1 mAb (3 µg/ml) at 4°C.
In restimulation experiments T lymphocytes were challenged for 72 h with stimulatory pairs of anti-CD2 mAbs (AICD2M1/AICD2M2, each 3 µg/ml, and CD2.611F1, 100 ng/ml) immobilized to culture dishes precoated with rabbit antimouse immunoglobulin or by -irradiated (30 Gy) allogeneic APC (Laz509; ratio 1:10).
Functional T-Cell Assays.
ELISpot assay was carried out as described before (22)
. In brief, multiscreen 96-well nitrocellulose plates (Millipore, Bedford, The Netherlands) were coated overnight at 4°C with antihuman IFN- mAb (1-D1K; 2 µg/ml; Mabtech, Stockholm, Sweden). After blocking, 1.5 x 105 T cells or TALs were cocultured with 1 x 104 tumor cells as indicated (18 h; 37°C; 5% CO2). Plates were washed extensively with PBS, 1% BSA, and 0.05% Tween 20 (Sigma Chemical Co.), and were additionally incubated with 100 µl/well anti-IFN-Ab (7-B6–1-biotin; 0.2 µg/ml; Mabtech). After incubation for 2 h at room temperature plates were washed and developed for additional 2 h with streptavidin-alkaline phosphatase (1 µg/ml; Mabtech). Spots were visualized by adding the substrate (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium; Sigma Chemical Co.) and counted under a stereomicroscope. The number of spots represents the frequency of activated T cells.
IL-2 secretion of activated T cells was determined in culture supernatants by a standard ELISA according to the manufacturer’s instructions (Pharmacia, Hamburg, Germany).
For assessment of proliferation, T cells were cultured at 1 x 105cells/well in 96-well round-bottomed microtiter plates (Nunc, Roskilde, Denmark) together with 1 x 104 -irradiated (200 Gy) stimulator cells or mitogenic Abs as indicated. Cell cultures were incubated for 2–4 days and pulsed for an additional 18 h with 37 KBq of [3H]thymidine (74.0 GBq/mM; New England Nuclear, Boston, MA) per well. Thymidine incorporation was determined by liquid scintillation counting and expressed as mean cpm of triplicates ± SE.
Cytolytic activity of T cells was assessed in a standardized [51Cr]chromate release assay. Briefly, 1 x 103 target cells labeled with sodium [51Cr]chromate together with various numbers of effector cells were incubated in a well of a 96-well plate for 4 h at 37°C at a final volume of 200 µl. At the end of the incubation, supernatants were harvested (100 µl/well) and counted in a gamma counter. The percentage of specific lysis was calculated as 100% x [(experimental release - spontaneous release)/(maximal release - spontaneous release)]. Spontaneous release and maximal release were determined in the presence of either medium alone or 1% SDS, respectively.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To ensure that decrease in CD154 expression was mediated by CD40-expressing tumors we transfected per se CD40- tumor cell lines with an expression vector encoding CD40. As shown in Fig. 1, C, D and F
, in marked contrast to the untransfected parental melanoma cell line SkMel63, cocultivation for 4 h with its CD40+ variant (SkMel63-CD40) causes a complete loss of CD154 expression on preactivated T cells. This was also true for the CD40-transfected breast carcinoma cell line KS-CD40 (Table 2)
. Moreover, preincubation of the CD40+ transfectant with a CD40-specific mAb blocked the SkMel63-CD40-mediated modulation of CD154 expression (Fig. 1E)
. Table 2
summarizes the ability of various tumor cell lines and fresh tumor material to modulate CD154 expression on preactivated T cells; on incubation with four different RCC cell lines an up to 3.5-fold reduction of CD154 expression in comparison to preactivated T cells cultivated in medium alone was observed. Furthermore, a panel of CD40+ FT-RCC, FT-ovarian, and FT-breast carcinoma cells down-modulated CD154 from the T-cell surface as well (1.5–6.6-fold), depending on the amount of CD40+ cells in the population. The expression density of other T-cell activation markers such as CD25 or CD69 was not impaired by coincubation with CD40+ tumor cells (data not shown). To exclude influences excited by soluble factors released by tumor cells the assays were repeated with paraformaldehyde-fixed tumor cells, receiving the same quantity of CD154 down-regulation (data not shown).
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. In contrast, T cells primed with SkMel63-CD40 showed impaired CD154 expression after CD2-mediated rechallenge (Fig. 2)
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. After short time stimulations (overnight) T lymphocytes activated by SkMel63-CD40 showed lower frequencies of IFN--secreting cells (3/300,000) compared with stimulations by the untransfected parental cell line (20/300,000) or the SkMel63-CD80 variant (44/300,000; Fig. 3A
). Just so, IL-2 secretion of T cells coincubated with SkMel63-CD40 was markedly reduced in contrast to wild-type or SkMel63-CD80 stimulations (Fig. 3B)
. Furthermore, the proliferative response induced by SkMel63-CD40 was decreased in comparison with T-cell proliferation stimulated by the parental cell line. T-cell proliferation on activation with a variant expressing the costimulatory molecule CD80 (SkMel63-CD80) was as high as expected (Fig. 3C)
. The T-cell proliferation in response to the SkMel63-CD80 variant was similar to proliferation rates after stimulation with the CD40/CD80-coexpressing double transfectant (SkMel63-CD40/CD80). These data indicate a dominance of CD80 on T-cell activation (data not shown). The inhibition of T-cell activation by CD40-expressing tumor variants was not attributable to apoptotic mechanisms as determined by both annexin-FITC/propidium iodide staining and trypan blue exclusion (data not shown). To exclude that tumor cell activities other than CD40 expression were responsible for the negative modulation of T-cell functions ELISpot assays were performed in the presence of neutralizing anti-CD40 mAb. Under these conditions suppression of T-cell activation was largely abrogated (Fig. 3D)
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It has been demonstrated that DC maturation is mediated via CD40 signaling, which enhances the induction of immunorelevant surface molecules like CD80. To investigate whether CD40 stimulation modulates the induction of CD80 on tumor cells as well as their immunostimulatory capacity, the SkMel63-CD40 cell line was incubated with immobilized anti-CD40 mAb. CD40 ligation did not induce any CD80 expression or modulation in MHC class I expression on CD40+ tumor cells as determined by flow cytometry nor had any influence on the activation of T lymphocytes as determined by T-cell proliferation assays toward CD40-triggered SkMel63 or SkMel63-CD40 cells (data not shown).
CD40 Expressed on Tumor Cells Inhibits T-Cell Priming but not Effector Functions of CTL.
To additionally assess whether phenotype and functional activity of T cells stimulated separately with SkMel63, SkMel63-CD40, SkMel63-CD80, or the double transfectant SkMel63-CD40/CD80 differs, long-term MLTCs were established. A phenotypic analysis of T cells stimulated either by the parental cell line or by SkMel63-CD40 on day 14 revealed no differences in the expression levels of CD2, CD8, CD25, CD69, and CD152 (data not shown). The cytotoxic activity of T cells cocultivated with different SkMel63 variants was also determined (Fig. 4)
. Even after four restimulations with SkMel63-CD40 T-cell lines did not show significant lysis of SkMel63 targets. Minor cytolysis was observed when the untransfected parental cell line was used as stimulators. However, the strongest cytolysis was detected when the CD80-expressing variant as well as the double-transfectant SkMel63-CD40/CD80 were used for stimulations (Fig. 4A)
. These results indicate that CD80 costimulation compensates CD40-mediated suppression of T-cell functions when expressed on the very same cell. However, CD40 expression on tumor cell targets could not inhibit their lysis by effector T-cell lines generated by SkMel63-CD80 stimulation (Fig. 4B)
. This illustrates that CD40+ tumor cells are susceptible to CTL effector function despite their inability to stimulate resting T cells.
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or DCs both expressing CD40 and other costimulatory molecules like CD80 (data not shown) were added after 24 h to the MLTCs. Cell cultures were incubated for an additional 5 days, and proliferation was assessed by [3H]thymidine incorporation. In marked contrast to SkMel63 and SkMel63-CD80-containing MLTCs the presence of a SkMel63-CD40 variant suppressed the activation of T cells by allogeneic APCs (Fig. 5A)
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shows that engagement of CD154 as well as CD3 led to T-cell unresponsiveness in terms of proliferation. Similar results could be achieved using allogeneic DCs instead of anti-CD2 mAbs for rechallenge of pretreated T cells (data not shown). |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To analyze the immunoregulative role of CD40 expressed by tumor cells during the initiation of an immune response, we compared T-cell functions after priming with CD40+or CD40- tumor cell variants. In contrast to primary stimulation of T cells using the melanoma cell line SkMel63 or its CD80-transfected variant as stimulator cells, SkMel63-CD40 led to down-regulation of CD154 expression and subsequently impaired T-cell activation. This was verified by reduced proliferation as well as decreased IFN- and IL-2 secretion of T cells. The effects were CD40 dependent, because the immunosuppression was abrogated in the presence of blocking anti-CD40 mAbs. Interestingly, experiments with the double-transfected SkMel63-CD40/CD80 led to similar results as with SkMel63-CD80 leading to the speculation that CD40 signaling is context dependent. These findings are supported by the fact that T cells are stimulated by CD40- and CD80-expressing DCs or B cells.
It was reported recently that receptor-mediated down-modulation and endocytosis of CD154 may regulate effector functions of activated T cells. By limiting the duration of CD154 expression, receptor-mediated modulation could help to minimize the risk of a runaway immune activation (23 , 24) . We speculate that CD40-expressing tumor cells mimic those immunoregulatory processes to down-regulate CD154 without providing costimulatory signals, thus inducing T-cell suppression. This hypothesis is supported by experiments where we determined whether CD40+ tumor cells could induce a long-lasting T-cell unresponsiveness. For this purpose we performed rechallenge experiments: T cells precultured with CD40+ tumor variants neither re-expressed CD154 after rechallenged with mitogenic pairs of anti-CD2 mAbs nor proliferated in response to allogeneic activation (DCs or B cells).
In several reports it was demonstrated that T-cell inactivation is induced by engagement of the T-cell receptor in the absence of costimulation (26)
. Here, we demonstrate that T-cell unresponsiveness can be induced by CD154 ligation mediated by CD40+ tumors. This is supported by our preliminary data comparing TALs and PBLs from patients with advanced CD40-expressing breast or ovarian carcinoma. TALs showed diminished CD154 expression on mitogenic stimulation and impaired IFN- secretion in response to allogeneic APCs. Thus, the expression of CD40 in a nonimmunogenic context on tumors might contribute to T-cell unresponsiveness and tumorescape.
In this context, a clinical study demonstrated down-regulation of CD154 expression on T cells by malignant CD40+ B cells in patients with chronic lymphocytic leukemia. It was suggested that CD154 down-modulation contributes to the generalized state of immunosuppression existing in these patients (27) . Although the situation for solid tumors might be different, retrospective clinical studies for human lung cancer and melanoma patients correlated CD40 expression on tumors with metastatic spread and poor prognosis (13 , 16) . Whether the clinical effects of CD40 expression are attributable to enhanced malignancy or poor immunogenicity of the tumor has yet to be answered. Several reports indicated that CD40 might influence diverse biological effects on tumor cells themselves, including adhesion, secretion of cytokines and proteases, differentiation, and regulation of apoptosis (reviewed in Refs. 11 , 12 , 28, 29, 30, 31, 32, 33, 34, 35 ). Because of the ability of CD40 ligation to provide survival signals for B cells it was suggested that CD40 in carcinoma may also serve as a receptor for mitogenic and/or survival signals. For example, stimulation of CD40 in human bladder carcinoma conferred protection against CD95 or chemotherapy-induced apoptosis (18) .
Also in vitro, differentiation and maturation of monocytes into DCs could be induced by triggering CD40 via specific Abs. In contrast to monocytes we could not induce up-regulation of MHC class I molecules or costimulative receptors on CD40-expressing melanoma or breast carcinoma cells by using anti-CD40 mAbs for cross-linking. We speculate that the response of tumor cells to CD40 stimulation may differ for diverse tumor entities, because mutations in different pathways might influence the outcome of such trigger (31, 32, 33, 34, 35)
. Furthermore, we did not detect any CD40-dependent influence on target susceptibility to already established CTLs as described by Leoprechting et al. (28)
. This discrepancy might be explained by the fact that in their report CD40-cross-linking was performed in the presence of IFN-. Although in our hands IFN- mediated up-regulation of CD40 and MHC class I expression on tumor cells and facilitated their lysis by effector cells (not shown) we could not enhance MHC class I molecule expression and target susceptibility solely by cross-linking CD40 on tumor cells. These results were similar for CD40+ transfectants as well as carcinoma cells constitutively expressing CD40. This indicates that our observation was not related to a lack of functionality of the transfected CD40 molecule. In our experimental settings the triggering of CD40 on tumor cells did not significantly affect on their proliferation rate and phenotype. Because our primary focus was on T-cell function modulated by CD154 triggering, the influence of CD40 signaling for the tumor cell faith has to be elucidated in additional studies.
Considering this complex balance between different CD40-mediated signals, it is necessary to additionally elucidate whether CD40 expression on carcinoma cells represents an independent prognostic marker for a more aggressive tumor growth and to define additional markers beside CD40 that can help to predict tumor behavior and immune response toward cancer cells.
In summary, we postulate that the molecular context in which a particular signal is delivered toward T lymphocytes is crucial for its functional consequences. On carcinomata, CD40 is obviously presented in a nonimmunogenic context and can contribute to T-cell unresponsiveness, providing a thus far unrecognized mechanism to evade an immune attack. The reversal of this CD40-dependent inactivation may be important for future immunotherapeutic approaches.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 These studies were supported by Grants from the Graduiertenkolleg experimentelle Nieren und Kreislaufforschung of the University of Heidelberg (to R. B.), the fortüne-Programm of the University of Tübingen (to S. S.), Deutsche Forschungsgemeinschaft Grant GU 511/1-1, and Deutsche Krebshilfe Grant 10-1529-Gü I (to B. G.). 
2 Present address: MTM Laboratories AG, 69120 Heidelberg, Germany.
3 To whom requests for reprints should be addressed, at Department of Obstetrics and Gynecology, University of Tübingen, Schleichstrasse 4, 72076 Tübingen, Germany. Phone: 49-70-71-29-77-62-6; Fax: 49-70-71-29-56-53; E-mail: brigitte.gueckel{at}uni-tuebingen.de
4 The abbreviations used are: DC, dendritic cell; APC, antigen-presenting cell; TRAP, telomeric repeat amplification protocol; ELISpot, enzyme-linked immunospot; FT, fresh tumor material; IL, interleukin; Ab, antibody; mAb, monoclonal antibody; MLTC, mixed lymphocyte tumor cell reaction; PBMC, peripheral blood mononuclear cell; RCC, renal cell carcinoma; TAL, tumor-associated lymphocyte; TH cell, T-helper cell; PMA, phorbol-12-myristate-13-acetate; PBL, peripheral blood lymphocyte.
Received 9/24/01. Accepted 1/30/02.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. J. Int. Immunol., 9: 853-860, 1997.
. J. Immunol., 162: 4140-4147, 1999.This article has been cited by other articles:
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  S. Coleman, A. Clayton, M. D. Mason, B. Jasani, M. Adams, and Z. Tabi Recovery of CD8+ T-Cell Function During Systemic Chemotherapy in Advanced Ovarian Cancer Cancer Res., August 1, 2005; 65(15): 7000 - 7006. [Abstract] [Full Text] [PDF]
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 C. Rentzsch, S. Kayser, S. Stumm, I. Watermann, S. Walter, S. Stevanovic, D. Wallwiener, and B. Guckel Evaluation of Pre-existent Immunity in Patients with Primary Breast Cancer: Molecular and Cellular Assays to Quantify Antigen-Specific T Lymphocytes in Peripheral Blood Mononuclear Cells Clin. Cancer Res., October 1, 2003; 9(12): 4376 - 4386. [Abstract] [Full Text] [PDF]
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R. Rieger and T. J. Kipps CpG Oligodeoxynucleotides Enhance the Capacity of Adenovirus-mediated CD154 Gene Transfer to Generate Effective B-Cell Lymphoma Vaccines Cancer Res., July 15, 2003; 63(14): 4128 - 4135. [Abstract] [Full Text] [PDF]
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), SkMel63-CD40 (
), SkMel63-CD80 (
), or SkMel63-CD40/CD80 (
) variants as stimulator cells. After four restimulations the cytolytic activities of the four different T-cell lines were tested using the SkMel63 parental melanoma cell line as target cells in a standard 51Cr-release assay. B, tumor cell lines are susceptible to CTL effector functions independently of CD40 expression on the target cell surface. An alloreactive T-cell line was established by repetitive stimulations with the SkMel63-CD80 variant. Cytolysis of SkMel63 parental melanoma cell line (