CD44 stimulation down-regulates Fas expression and Fas-mediated

International Immunology, Vol. 13, No. 10, pp. 1309–1319

© 2001 The Japanese Society for Immunology

CD44 stimulation down-regulates Fas expression and Fas-mediated apoptosis of lung cancer cells Manabu Yasuda, Yoshiya Tanaka1, Koichi Fujii1 and Kosei Yasumoto Second Department of Surgery and 1First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Japan, Kitakyushu 807-8555, Japan Keywords: apoptosis, CD44, cytotoxic T lymphocytes, Fas, lung cancer

Abstract Cytotoxic T lymphocytes (CTL) play a major role in the rejection of tumor cells, but tumor rejection does not always occur in vivo, indicating that defects in anti-tumor immune responses may be common. We here document a novel function for CD44—using lung cancer cells, we showed that stimulation of CD44 reduced Fas expression and Fas-mediated apoptosis: (i) lung cancer cells expressed high levels of CD44; (ii) engagement of CD44 on the cells by a specific antibody or fragmented hyaluronan reduced Fas expression; (iii) CD44 cross-linking reduced Fas-mediated apoptosis; (iv) stimulation of CD44 on lung cancer cells decreased IFN-γ production by autologous CTL; and (v) CD44 stimulation prevented killing of lung cancer cells by autologous CTL. Based on these findings, we postulate a new concept—that interaction of CD44 on lung cancer cells with fragments of extracellular hyaluronan present in the surrounding extracellular matrix reduces Fas expression as well as Fas-mediated apoptosis of cancer cells. This leads to reduced susceptibility of the cells to CTL-mediated cytotoxicity through the Fas–Fas ligand pathway. Introduction The role of the immune system in preventing tumor growth and the molecular requirements for effective function of cytotoxic T lymphocytes (CTL) have been emerging in recent years. It has also been demonstrated that tumor-bearing mice can be cured using a wide variety of approaches, some of which involve cytokine-mediated enhancement of CTL activity and specific molecular components of the immune system including Fas–Fas ligand (FasL) and CD28–CD80/CD86 (1–4). However, in contrast to the apparent success in experimental cancer models, clinically, CTL often fail to control tumor growth in vivo and thus only a minority of patients have so far benefited from CTL-based anti-tumor therapy. Many review articles have indicated that defects in the development or execution of anti-tumor immune responses are common (5–8). Several mechanisms have been suggested for these defects—tumors seem to protect themselves against CTL recognition or attack by various mechanisms. In this context, we have postulated that the failure of immune protection, the so-called immune escape mechanism, is due to intrinsic features of tumor cells which do not allow the induction of an effective immune response.

The Fas–FasL pathway, i.e. ligation of Fas, which is expressed on the surface of target tumor cells, and Fas ligand, on the surface of CTL, is the best-known mechanism that induces apoptosis of tumor cells (4,9). Fas is ubiquitously expressed in lymphoid and non-lymphoid tissues, and many primary tumors and tumor cell lines (10–12). However, recent data indicate that the induction of apoptosis by ligation of Fas is often markedly reduced in tumor cells, even in those expressing Fas (12,13), and in many cases the level of Fas on tumor cells appears to be reduced in vivo (14–16). These mechanisms may contribute to the evasion of tumor cells from immune surveillance. However, the immune escape mechanism through the regulation of Fas on tumor cells remains poorly understood. Tumor cells are surrounded in vivo by and encounter extracellular matrix components such as hyaluronan mainly through CD44 on the cell surface, indicating that the engagement of CD44 by extracellular matrix always occurs in tumor cells. CD44 is a transmembrane glycoprotein involved in various cell adhesion events, including lymphocyte migration, early hemopoiesis and tumor metastasis (17). Many

Correspondence to: Y. Tanaka Transmitting editor: T. Hamaoka

Received 14 May 2001, accepted 12 July 2001

1310 CD44 reduces Fas on cancer cells primary carcinoma tissues express high levels of CD44 (18). Since the initial description of the potential role of CD44 in tumorigenesis, several studies have investigated the pattern of CD44 distribution in tumors (17). Recently, the function of CD44 as a signaling molecule has been also demonstrated. We and others reported that stimulation of CD44 with mAb or hyaluronan transmits the signal into the cell, which leads to activation of T cells and cytokine or chemokine release from monocytes and synoviocytes (19–23). However, in tumor cells, the function of CD44 as a signaling molecule has not yet been demonstrated. In this study, we first detected a high expression level of CD44 on lung cancer cells. We also demonstrated that engagement of CD44 by a specific antibody or potent ligand hyaluronan reduced Fas expression and Fas-mediated apoptosis of lung cancer cells. We propose that continuous stimulation of tumor cells by hyaluronan, which is present abundantly around tumor cells in vivo, mainly via CD44, leads to immune escape from CTL-dependent killing in vivo.

Methods Tumor cell lines Eleven human lung cancer cell lines were used in the present study—A904L, C831L (lung large cell carcinoma), A110L, C422L (lung adenocarcinoma) and B1203L (lung squamous cell carcinoma). These cell lines were established in our laboratory as described previously (24,25). PC-9 and A549 were derived from lung adenocarcinoma (24) and QG56 was derived from lung squamous cell carcinoma (26). PC-1, PC-6 (27) and QG90 (28) were derived from lung small cell carcinoma. All cell lines were grown in RPMI 1640 (Nissui, Tokyo, Japan) with 10% FCS (Bio-Pro, Karlsruhe, Germany). Induction of CTL Regional lymph node lymphocytes (RLNL) from lung cancer patients were harvested at the time of surgery as described previously (25). RLNL were stimulated with solidphase anti-CD3 mAb (Ortho, Raritan, NJ) for 48 h, and expanded in RPMI 1640 containing 10% FCS and 50 JRU/ml recombinant human IL-2 (Takeda, Osaka, Japan) for 14 days. Subsequently, RLNL were stimulated weekly with irradiated autologous tumor cell lines for 2–4 weeks. Antibodies and reagents The following mAb were used as purified Ig in cell-surface analyses and functional assays; CD44 mAb NIH44-1, CD54 [intercellular adhesion molecule (ICAM)-1] mAb 84H10, (kindly provided by Dr S. Shaw, NIH, Bethesda, MD), CD44 mAb BU75 (Ancell, MN), CD29 (β1 integrin) mAb mAb13 (kindly donated by Dr K. M. Yamada, NIH, Bethesda, MD), CD95 (Fas) mAb CH11, FITC-conjugated Fas mAb UB2, CD95L (FasL) mAb 4H11 (Medical and Biological Laboratories, Nagoya, Japan), CD106 [vascular cell adhesion molecule (VCAM)-1] mAb 2G7 (kindly provided by Dr W. Newman, Otsuka America, Rockville, MD), CD11a [leukocyte function-associated antigen (LFA)-1α] mAb TS1/22, MHC class I mAb W6/32 and anti-glycophorin mAb 10F7 (ATCC,

Rockville, MD), and control murine IgG1 (Becton Dickinson, San Jose, CA). Stimulation of CD44 on lung cancer cells by CD44-specific mAb or hyaluronan Lung cancer cells were cultured to confluence and then incubated with NIH44-1 mAb and control mAb (10 µg/ml) for 30 min at 37°C. After washing the cells 3 times, 1 µg/ml of goat anti-mouse IgG Fc was added as the second antibody for CD44 cross-linking as descried previously (20,21). The cells were also incubated with fragmented or native hyaluronan (0.1 mg/ml) for 24 h at 37°C as described previously (20,21). FACS analysis Staining and flow cytometric analysis of lung cancer cells were conducted by standard procedures, as described previously (24,29), using a FACScan (Becton Dickinson, Mountain View, CA). Briefly, cells (2⫻105) were incubated with specific mAb and subsequent FITC-conjugated CD95 (Fas) mAb UB2 at saturating concentrations in FACS medium consisting of HBSS (Nissui, Tokyo, Japan), 0.5% human serum albumin (Green-Cross, Osaka, Japan) and 0.2% NaN3 (Sigma Aldrich, Tokyo, Japan) for 30 min at 4°C. After three washes in FACS medium, cells were analyzed with FACScan. Amplification of the mAb binding was provided by a three-decade logarithmic amplifier. Quantification of cell-surface antigens on one cell was performed using QIFKIT beads (Dako, Kyoto, Japan) as reported previously (30). Northern blot analysis For Northern blot analysis, total RNA was isolated from cultured cancer cells by a single-step isolation procedure. Total RNA (10 µg) was electrophoresed through a 1% agarose gel and blotted onto nylon filters (Amersham, Arlington Heights, IL). Fas cDNA was labeled with [32P]dCTP (DuPont NEN, Boston, MA) and Northern blot analysis was subsequently performed. Induction and detection of apoptosis For induction of apoptosis, CD44-stimulated lung cancer cells were cultured until confluence and then incubated with anti-Fas mAb CH11 (1 µg/ml) for 24 h at 37°C. Apoptosis was detected by Annexin V/FITC Kit (Immunotech, Marseille, France). Briefly, cells suspended in binding buffer were incubated with 0.25 µg/ml phosphatidylinositol (PI) and FITCconjugated Annexin V based on the method recommended by the manufacturer, and subsequently analyzed by flow cytometry. Cell proliferation assays Cell proliferation was determined by [3H]thymidine incorporation. After stimulation of CD44 on lung cancer cells for 24 h, cancer cells (1⫻105–1⫻103/200 µl) were incubated in flatbottomed 96-well plates in duplicate in the presence of either anti-Fas mAb CH11 (1 µg/ml) or control antibody for up to 24 h at 37°C, pulsed with 1 µCi of 3H-labeled thymidine (Amersham Chemical, Arlington Heights, IL) for 3 h, harvested on a Tomtec harvester (Orange, CT) and counted in a β-counter.

CD44 reduces Fas on cancer cells 1311 Table 1. Phenotypic analysis of multiple lung cancer cell lines Cell line

Origin

CD44

Fas

FasL

VCAM-1

LFA-1

A904L A110L QG90 C422L A549 PC-9 B1203L QG56 C831L PC-1 PC-6

(lung/largea) (lung/ad.b)

37,000 13,000 114,000 12,000 378,000 3,000 5,000 0 0 15,000 0

15,000 10,000 14,000 12,000 4,000 0 3,000 0 5,000 3,000 0

0 0 0 3,000 0 0 0 0 0 0 0

7,000 0 0 0 0 3,000 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

(lung/smallc) (lung/ad.) (lung/ad.) (lung/ad.) (lung/sq.d) (lung/sq.) (lung/large) (lung/small) (lung/small)

Eleven cancer cell lines were stained with CD44 mAb (NIH 44-1), Fas mAb (UB2), FasL mAb (4H9), VCAM-1 mAb (2G7) and CD11a mAb (TS1/22), and flow cytometric analyses were performed using FACScan. Data represent the number of molecules expressed per cell, calculated using standard QIFKIT beads. Representative data of five similar experiments. aLung large carcinoma. bLung adenocarcinoma. cLung small cell carcinoma. dLung squamous cell carcinoma.

ELISA of IFN-γ For detection of IFN-γ, autologous CTL (5⫻104/well) stimulated with phorbol myristate acetate (PMA) ⫹ ionomycin were added to the well containing tumor cells (5⫻104/well) in a final volume of 200 µl of RPMI 1640 with 10% FCS. After 24 h at 37°C of incubation, the supernatants were collected to measure IFN-γ by ELISA kit (Endogen, Woburn, MA) in a duplicate assay. Cytotoxicity assay The cytotoxicity of autologous CTL against tumor cells was determined by a standard 51Cr-release cytotoxicity assay as described previously (25). Tumor targets were labeled with sodium 51Cr for 1 h at 37°C and washed. Effector T cells stimulated with 25 ng/ml PMA (Sigma Aldrich) and 1 µg/ml ionomycin (Sigma Aldrich) for 2 h. Target cells (5⫻103) were incubated with effector T cells (E:T ratio ⫽ 1:1 to 20:1) in 200 µl of culture medium in a 96-well round-bottomed microtiter plate (Nunc, Roskilde, Denmark) for 8 h at 37°C. The supernatant (100 µl) was collected and samples were counted in a γ-counter. The percent specific lysis was calculated using the formula [(experimental 51Cr release – spontaneous 51Cr release)/(maximum 51Cr release – spontaneous 51Cr release)⫻100]. Results High expression of CD44 and Fas on lung cancer cell clones In the first step, we assessed the expression of various cellsurface functional molecules on 11 lung cancer cell lines using FACScan. Table 1 shows a summary of five representative molecules, CD44, Fas, FasL, VCAM-1 and LFA-1, on each type of cell. Figure 1 shows a histogram of cell-surface molecules expressed on representative A904L cell line and QG90 cell line. Among the screened molecules, CD44 was expressed on eight of 11 lung cancer cell lines, with the highest expression on A549 (adenocarcinoma), followed

by QG90 (small cell carcinoma) and A904L (large cell carcinoma). The level of CD44 on the cell surface was independent of the differentiation pattern of the cell lines. Fas was also expressed on six of 11 lung cancer cell lines and the number of Fas on four cell lines exceeded 1.0⫻104 molecules/cell. The expression was also independent of the differentiation pattern of cancer cells. In contrast, the expression of FasL and VCAM-1 was marginal on all 11 cell lines. We therefore postulated that CD44 and Fas might play an important role in lung cancer cells. To investigate the functional significance of these molecules, we mainly used the A904L cell line in the following experiments, as they highly expressed both CD44 and Fas as evident in the histogram shown in Fig. 1(A). Engagement of CD44 down-regulates Fas expression on lung cancer cells To characterize the function of CD44 on lung cancer cells, we assessed the effects of stimulation of CD44 on the expression of various cell-surface molecules. Among the screened molecules, flow cytometric analysis showed that the expression of Fas was markedly reduced by 24 h crosslinking of CD44 with NIH44-1, a specific mAb, on lung cancer cell line A904L (Fig. 2). CD44 engagement obviously reduced Fas expression on three lung cancer cell lines, A904L, A110L and QG90 cells, but did slightly or little on five lines including B1203L cells, among eight lines which expressed Fas (data not shown). These results suggest that some of lung cancer cells could lack the regulatory mechanism of Fas expression by CD44-mediated signaling. In contrast, CD44 stimulation failed to change the expression levels of VCAM-1, MHC class I and FasL antigen on the cells (Fig. 2). Furthermore, although the stimulation of A904L cells with CD44 induced a marked decrease in Fas protein expression on A904L cells, whereas stimulation of MHC class I, β1 integrin and ICAM-1 antigen by specific mAb did not (Fig. 3). Time-course experiments showed that Fas expression on A904L, A110L and QG90 cells reached low levels within

1312 CD44 reduces Fas on cancer cells

Fig. 1. Histogram of various cell-surface molecules expressed on A904L cells (A) and QG90 cells (B). A904L cells and QG90 cells were stained with CD44 mAb (NIH44-1), VCAM-1 mAb (2G7), CD29 mAb (mAb 13) and Fas mAb (UB2), MHC class I mAb (W6/32) or ICAM-1 mAb (84H10) and flow cytometric analyses were performed using a FACScan. Shadows represent profiles of the murine IgG used as a negative control. The ordinate represents the number of cells stained with the indicated mAb in each logarithmic scale of fluorescence amplifier. Representative data of five similar experiments are shown.

24 h of CD44 cross-linking. However, CD44 stimulation failed to reduce Fas expression on C831L cells which did not express CD44 as shown in Table 1. These results indicate that cross-linking of CD44 markedly reduces the expression of Fas on A904L lung cancer cells, although such expression does not occur without stimulation. Fragmented hyaluronan mediates CD44-induced Fas downregulation on A904L cells Hyaluronan is a major ligand for cell-surface CD44. We next assessed the biological activities of hyaluronan on the expression of Fas on A904L cells. As shown in Fig. 5, soluble full-length hyaluronan did not change Fas expression. Fragmented hyaluronan is thought to be relevant to inflammatory reactions with lung cancer (19–21). Fragmented hyaluronan, 1.7-, 6.9- and 40-kDa fragments, markedly reduced Fas expression within 24 h. Furthermore, following pretreatment of the cells with anti-CD44 blocking mAb BU75, no fragmented hyaluronan failed to reduce Fas (data not

shown). This suggests that hyaluronan, especially when fragmented, is a possible ligand involved in CD44-mediated Fas expression on A904L cells. CD44 stimulation does not reduce Fas mRNA transcription in A904L cells We also assessed whether the reduction of Fas protein on the cell surface is caused by reduced Fas mRNA transcription. After CD44 stimulation, expression levels of Fas transcripts in A904L cells did not change during the indicated time points (Fig. 6). These results imply that the reduced expression of Fas protein on CD44-stimulated cells does not attribute to inhibition of transcription of Fas genes and/or subsequent post-transcriptional events. Engagement of CD44 reduces Fas-mediated apoptosis of A904L cells Next, we investigated Fas-mediated apoptosis of A904L cells. Expression of Fas on A904L cells reached a minimum

CD44 reduces Fas on cancer cells 1313

Fig. 2. Cross-linking of CD44 reduced Fas expression on A904L cells. Down-regulation of Fas, but not VCAM-1, MHC class I or FasL, by CD44 on A904L cells. After cross-linking of CD44 on A904L cells with 10 µg/ml of CD44 mAb NIH44-1 for 0 h (upper) or 24 h (lower), the expression of Fas, VCAM-1, MHC class I and FasL antigen was analyzed by FACScan. Representative histogram of five similar experiments are shown.

Fig. 3. Down-regulation of Fas, but not control molecules, by CD44 stimulation on A904L cells. After cross-linking A904L cells with 10 µg/ml of CD44 mAb NIH44-1, MHC class I mAb W6/32, β1 mAb mAb13 or ICAM-1 mAb 84H10 for 24 h, Fas expression was analyzed by a FACScan. Each bar represents the number of molecules expressed per one cell, calculated using standard QIFKIT beads. Representative data of five similar experiments are shown.

at 24 h after the CD44 stimulation and at this time point the cells were stimulated by anti-Fas mAb in order to minimize Fas-mediated death signaling. After stimulation with anti-Fas mAb (1µg/ml) for 24 h, apoptosis was detected by staining with Annexin V and PI using flow cytometry (Fig. 7). Stimulation of A904L cells with anti-Fas mAb markedly induced both early apoptotic cells (Annexin VhighPIl°w) and late apoptotic cells or necrotic cells (Annexin VhighPIhigh). However, when the cells were pretreated with a 40-kDa fragment of hyaluronan for 24 h and subsequently treated with anti-Fas mAb, both Annexin VhighPIl°w cells and Annexin VhighPIhigh cells were reduced to the levels seen in control cells without stimulation. These results indicate that Fas on A904L cells is functional

and that CD44-induced down-regulation of Fas on A904L cells results in reduction of Fas-mediated apoptosis of the cells. CD44 stimulation prevents A904L cells from growth inhibition induced by anti-Fas mAb We next assessed the effects of CD44 stimulation on cell growth of A904L cells. As illustrated in Fig. 8, A904L cells spontaneously proliferated and engagement of CD44 by the 40-kDa fragment of hyaluronan did not change the proliferation of the cells, but anti-Fas mAb markedly suppressed the cell growth. However, it is noteworthy that pre-stimulation of A904L cells with the fragmented hyaluronan, but not with antiVCAM-1 mAb, significantly prevented the cells from growth

1314 CD44 reduces Fas on cancer cells inhibition induced by anti-Fas mAb. These results also indicate that CD44-induced down-regulation of Fas on A904L cells results in the reduction of growth arrest of the cells mediated by Fas stimulation. Stimulation of CD44 autologous CTL

modulates

IFN-γ

production

by

CD44-stimulated lung cancer cells were tested for their potential to stimulate the production of IFN-γ, a representative CTL-derived cytokine, by autologous CTL. Autologous CTL, A904L CTL and B1203L CTL, derived from patients with A904L cells and B1203L cells respectively, were examined

for IFN-γ production. We confirmed that the two lung cancer cell lines, A904L and B1203L, did not produce IFN-γ. Production of IFN-γ by A904L CTL and B1203L CTL against the respective cancer cells decreased following the addition of anti-FasL mAb and anti-MHC class I mAb, but not control IgG1. Stimulation of A904L cells with the 40-kDa fragment of hyaluronan reduced the production of IFN-γ by A904L CTL (Fig. 9A), whereas CD44 stimulation of B1203L cells neither changed Fas expression on the cells (data not shown) nor their production of IFN-γ by autologous CTL (Fig. 9B). These results indicate that stimulation of CD44 on A904L lung cancer cells modulates IFN-γ production by autologous CTL through

Fig. 4. Kinetics of Fas reduction by CD44 on lung cancer cell clones. After cross-linking of A904L, A110L, QG90 and C831L cells with 10 µg/ml of CD44 mAb NIH44-1 for the indicated duration (ordinate), the expression of Fas was analyzed by a FACScan. Each bar represents the number of molecules expressed per one cell, calculated using standard QIFKIT beads. Representative data of five similar experiments are shown.

Fig. 5. Fas down-regulation by fragmented hyaluronan on A904L cells. A904L cells were stimulated with fragmented hyaluronan (0.1 mg/ml) for 24 h and the expression of Fas was analyzed by a FACScan. The molecular masses of hyaluronan fragments were 1.7, 6.9, 40 kDa and native (~200 kDa). Each bar represents the number of molecules expressed per one cell, calculated using standard QIFKIT beads. Representative data of five similar experiments are shown.

CD44 reduces Fas on cancer cells 1315 Fas down-regulation apoptosis.

and

Fas–FasL

pathway-mediated

Stimulation of CD44 reduces lung cancer cell killing by autologous CTL Finally, we assessed the response of CD44-stimulated lung cancer cells to autologous CTL killing. Autologous, A904L and B1203L cells, derived from patients with A904L and B1203L cells respectively, were examined for autologous cytotoxicity. The addition of anti-FasL mAb, anti-MHC class I mAb, but not control IgG1, markedly decreased the cytotoxic activity of A904L CTL as well as B1203L CTL. It is noteworthy that stimulation of A904L cells with the 40-kDa fragment of

Fig. 6. CD44 stimulation did not reduce mRNA transcription of Fas in A904L cells. After stimulation of CD44 on A904L cells with 40-kDa fragments of hyaluronan for the indicated duration, expression levels of Fas transcripts were analyzed by Northern blotting. Representative data of five similar experiments are shown.

hyaluronan for 24 h reduced the cytotoxic activity of A904L CTL to the levels noted in the presence of anti-FasL mAb (Fig. 10A). In contrast, pretreatment of B1203L cells with hyaluronan neither changed Fas expression on the cells (data not shown) nor their killing by B1203L CTL (Fig. 10B). These results indicate that activation of CD44 on A904L lung cancer cells by cell binding to the extracellular matrix leads to escape from autologous CTL killing through Fas down-regulation. Discussion Although tumor-specific CTL are the best-defined biological anti-tumor weapons, they often fail to inhibit tumor growth in vivo and only a minority of patients benefit clinically from CTL-based anti-tumor therapy (5–8). Among many reasons for this lack of therapeutic effectiveness, tumor cells may protect themselves from CTL recognition or may actively inhibit CTL (31–33). For example, solid tumors express heterogeneously low levels of target antigens recognized by CTL (31). Many tumors show a decrease or loss of expression of HLA class I or class II molecules, which is required for antigen presentation, and co-stimulatory molecules such as CD28CD80/CD86 are down-regulated or even absent in some tumors (1,23). We here propose a new concept that both Fas expression and Fas-mediated apoptosis of tumor cells could be inhibited by interaction of cell-surface CD44 and extracellular matrix such as hyaluronan, surrounding the tumor cells in vivo, which results in evasion from Fas-mediated CTL

Fig. 7. CD44 stimulation reduced Fas-mediated apoptosis of A904L cells. CD44 on A904L cells was stimulated with or without fragmented hyaluronan (40 kDa; 0.1 mg/ml) for 24 h and apoptosis was induced by anti-Fas mAb (1 µg/ml) for 24 h. Apoptosis was detected by staining with FITC-conjugated Annexin V and PI, and analyzed them by flow cytometer. Annexin VhighPIhigh cells (upper right) represent late apoptotic or necrotic cells and Annexin VhighPIl°w cells (lower right) represent early apoptotic cells. Representative data of five similar experiments are shown.

1316 CD44 reduces Fas on cancer cells

Fig. 8. CD44 stimulation prevented A904L cells from growth inhibition induced by anti-Fas mAb. CD44-stimulated A904L cells with or without fragmented hyaluronan (40 kDa; 0.1 mg/ml) were treated with either anti-Fas or indicated mAb or antibody for 24 h and then pulsed with [3H]thymidine as described in Methods. Shown are the mean c.p.m. of [3H]thymidine incorporation into the cells. All experiments were done in duplicate and representative data of three similar experiments are shown.

Fig. 9. Stimulation of CD44 on lung cancer cells modulates IFN-γ production by autologous CTL. Autologous CTL (A904L and B1203L CTL) were induced in patients with A904L (A) and B1203L (B) cells respectively. A904L or B1203L cells (stimulators) were incubated with (closed bars) or without (open bars) fragmented hyaluronan (40 kDa) for 24 h. PMA ⫹ ionomycin-activated CTL (responders) were added to the culture in the presence or absence of the indicated mAb for 24 h at 37°C. The supernatant of the mixed cultures were collected and assayed for IFN-γ. Data represent IFN-γ production from responders in a representative of three experiments. *Not detected.

killing. This conclusion stems from the following results. (i) Lung cancer cells expressed high levels of CD44. (ii) Engagement of CD44 on the cells by a specific mAb or fragmented hyaluronan reduced Fas expression. (iii) CD44 stimulation reduced Fas-mediated apoptosis or growth

inhibition of the cells. (iv) Stimulation of CD44 on lung cancer cells reduced IFN-γ production by autologous CTL. (v) Engagement of CD44 prevented the cells from autologous CTL-mediated killing. Adhesion molecules that modulate the interaction between

CD44 reduces Fas on cancer cells 1317

Fig. 10. CD44 stimulation prevented lung cancer cells from killing by autologous CTL. Autologous CTL (A904L and B1203L CTL) were induced in patients with A904L cells (A) and B1203L (B) cells respectively. A904L or B1203L cells (target) were incubated with (closed bars) or without (open bars) fragmented hyaluronan (40 kDa) for 24 h. After labeling cells with 51Cr, PMA- ionomycin-activated CTL (effector) were added to the culture in the presence or absence of anti-FasL mAb 4H11 (1 µg/ml), anti-hamster IgG1 and anti-MHC class I mAb W6/32 (1 µg/ml), for 8 h at 37°C. Cytotoxic activity of CTL against tumor target cells was assessed by 51Cr release from target cells stimulated by effector CTL at an E:T ratio of 20:1. Data are expressed as the mean percentage of 51Cr release from targets in a representative of five experiments.

tumor cells and various host cells or extracellular matrix are known to be involved in the enhancement of survival, arrest and invasiveness of tumor cells in vivo (34–37). Among various adhesion molecules, high levels of CD44 are expressed on a variety of primary tumor cells, and the variant isoforms of CD44 (CD44v) confer the metastatic potential of tumor cells in vitro and in vivo (17,18,38,39). Thus, the importance of CD44 expression for tumorigenesis as well as metastasis has been emerging in recent years. However, in tumor cells, the function of CD44 as a signaling molecule remains poorly understood. Recently, we and others reported that stimulation of CD44 transmits the signal into the cells, which leads to activation of T cells and production of cytokines from monocytes and synoviocytes (19–23). We here report that lung cancer cells highly expressed CD44 and engagement of CD44 on the cells by a specific mAb or fragmented hyaluronan lowered Fas expression and reduced Fas-mediated apoptosis, resulting in protection of tumor cells against CTL killing. In vivo, tumor cells are surrounded by and encounter extracellular matrix such as hyaluronan mainly through their receptors including CD44, indicating that the engagement of CD44 by matrix protein always occurs in tumor cells. Thus, our results imply that in vitro culture steps without extracellular matrix may introduce major biases; in vitro tumor rejection by CTL is efficiently induced by specific molecular components of the immune system during the interaction of tumor cells

and CTL, although, in vivo, tumor cells may protect themselves against CTL recognition and active immune evasion mechanisms are acquired by tumor cells through interaction with extracellular matrices. Fas–FasL pathway and ligation of Fas, which is expressed on the surface of tumor cells, by FasL on the surface of CTL play an important role in the induction of apoptosis of tumor cells specifically and directionally (12,13). However, previous immunohistochemical studies demonstrate that Fas levels on tumor cells appear to be reduced in vivo (14–16). Our results showed that engagement of CD44 by a specific mAb or fragmented hyaluronan markedly reduced Fas expression in lung tumor cells and that the reduced levels of Fas were gradually recovered within 48–72 h. Because the amounts of cytoplasmic Fas were not altered by CD44 engagement (data not shown), we rather postulated that the CD44-mediated signaling might induce proteolysis of Fas glycoproteins from the following reasons: when A904L cells were stimulated with CD44 for 24 h, the level of Fas mRNA transcripts did not change (Fig.6); CD44 engagement did not alter the amounts of cytoplasmic Fas (data not shown); by Western blotting using anti-Fas mAb, a 25-kDa protein was detected in the culture supernatant of CD44-stimulated A904L cells (data not shown). CTL play a major role in the rejection of tumor cells, but tumor rejection does not always occur spontaneously in vivo,

1318 CD44 reduces Fas on cancer cells indicating that defects in the generation or execution of the anti-tumor immune response may be common. We here propose an alternate immune evasion mechanism, based on the interaction between CD44 on lung cancer cells and extracellular hyaluronan, which induced the reduction of both Fas expression and Fas-mediated apoptosis of the cells, resulting in less susceptibility of the cells to CTL-mediated cytotoxicity through Fas–FasL pathway. Several clinical studies are underway to test various strategies to induce or strengthen anti-tumor immune responses in cancer patients and the rational design of future therapeutic strategies for lung cancer may thereby include the exploitation of CD44 and Fas death pathway in order to directly reduce tumor growth in vivo.

Acknowledgements We thank Ms T. Adachi for the excellent technical assistance. We also thank Drs S. Shaw, W. Newman and K. M. Yamada for providing mAb and reagents. The authors also thank Dr F. G. Issa (Word-Medex, Sydney, Australia) for the careful reading and editing of the manuscript. This work was supported in part by a Grantin-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

Abbreviations CTL FasL ICAM LFA PI PMA RLNL TNF VCAM

cytotoxic T lymphocytes Fas ligand intercellular adhesion molecule leukocyte function-associated antigen phosphatidylinositol phorbol myristate acetate regional lymph node lymphocytes tumor necrosis factor vascular cell adhesion molecule

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