Functional CD8⁺ T cells infiltrate into nonsmall cell lung carcinoma

Lung cancer patient

The primary tumor of a 63-year-old male patient KLP11 (HLA-A11, HLA-A24, HLA-B7, HLA-B57, HLA-Cw6, HLA-Cw7) with a squamous cell carcinoma of the lung was resected and a stable cell line, designated KLP11, was established by mincing the tumor sample into small pieces of approximately 1 mm³ and culturing the adherent cells in DMEM (Life Technologies, Breda, The Netherlands) with 8% heat inactivated FCS, penicillin (100 μg/ml), streptomycin (100 μg/ml), and l-glutamine (2 mM). Peripheral blood mononuclear cells (PBMC) were isolated from venous blood after Ficoll-gradient separation. HLA typing of the patient’s PBMC and tumor cell line KLP11 was performed by standard serological techniques as well as by PCR/oligotyping as described elsewhere [44]. Appropriate informed consent and approval by the local Medical Ethical Committee was obtained.

Cell lines and culture

Lung carcinoma cell lines GLC-2, GLC-8, GLC-36 were kindly provided by Dr. L. de Leij (Pathology and Laboratory Medicine, University Hospital Groningen, The Netherlands). Breast carcinoma cell lines MDL-13E, MDA-MB-134, MDA-MB-231, MCF-7, ZR75.1, BT549, and the natural killer target cell line K562 were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). The renal cell carcinoma (RCC) cell line MZ1774–RCC was provided by Dr. B. Seliger (Johannes Gütenberg University, Mainz, Germany). The melanoma cell line MEWO was provided by Mrs. M. Wilke (Immunohaematology, Leiden University Medical Center, The Netherlands) and the melanoma cell line 93.03A, the RCC cell line RCC90.03, and the ovarian carcinoma cell line COV504 were established in our laboratory. All cell lines were cultured in DMEM with 8% heat inactivated FCS, antibiotics and l-glutamine, as described above.

EBV-transformed B-lymphoblastic cell lines (EBV) were generated from PBMC of the lung cancer patient (EBV-KLP11) and normal healthy donors (EBV-OU, EBV-RU, EBV-RO). These newly generated EBV, the previously established EBV-JY and the Fas-positive EBV SKW6.4 and the TNF-sensitive WEHI-164 clone 13 cells (obtained from P. Coulie, University of Louvain, Brussels, Belgium) were all cultured in RPMI 1640 medium with 5% heat inactivated FCS and l-glutamine and antibiotics as described above. HLA typing of all cell lines was obtained by PCR/oligotyping.

Flow cytometric analysis

Cell surface expression of HLA class I antigens, tumor-associated Ag, and apoptosis-related molecules was studied by FACS analysis using the following primary antibodies: anti-HLA-class I murine mAb W6/32 (anti-HLA-A, anti-HLA-B, and anti-HLA-C) [25], the human anti-HLA-A11 mAb OK2H12 (IgM, anti-HLA-A11, anti-HLA-A3, anti-HLA-A36, anti-HLA-A32, kindly provided by Dr. A Mulder, Leiden University Medical Center). Antibodies against tumor associated Ag: anti-Her-2 mAb 9G6 [42], Muc-1 mAb 214D4 (a gift from Dr. J. Hilken, NKI Amsterdam, The Netherlands), EpCAM-1 (Ber-Ep4, Dako) and antibodies against apoptosis-related molecules anti-Fas/CD95 mAb APO-1 (mouse IgG1, a kind gift from Dr. P. Krammer, German Cancer Research Center, Heidelberg, Germany) and SM1/23 (Fab-fragments mouse IgG2b, Alexis, Lausanne, Switzerland) an apoptosis blocking anti-Fas mAb.

MAbs against HLA-A11 (0284HA) and HLA-A24 (0041HA) (One Lambda Inc., Canoga Park, CA, USA) were used for immunohistochemical staining as described [13] after confirmation of their specificity by FACS analysis.

The CTL clone was phenotyped with FITC-conjugated anti-CD3 mAb and PE-conjugated anti-CD4 or anti-CD8 mAb (BD Bioscience, Alphen aan den Rijn, The Netherlands), respectively, and TCR analysis was performed using the TCR Vβ repertoire kit (Beckman Coulter), according to the manufacturer’s instructions. Expression of NK inhibitory and activating receptors on CTL21/74 was studied by FACS analysis using a panel of PE-conjugated mouse-anti-human mAb including GL183 (IgG1, anti-CD158b1/b2,j, Beckman Coulter), EB6B (IgG1, anti-CD158a,h, Beckman Coulter), DX-9 (IgG1, anti-NKB1, BD Pharmingen), FES172 (IgG2a, anti-CD158i, Beckman Coulter), HP-3D9 (IgG1, anti-CD94 /Kp43, BD Pharmingen), and NKG2D (IgG1, anti-NKG2D, R&D Systems, Abingdon, UK).

COBRA-FISH

To describe the karyogram of the lung cancer cell line KLP11 we prepared slides with cells treated with colcemid and KCl in order to get metaphase spreads. The cells were harvested and fixed according to standard procedures. We performed COBRA-FISH as previously described by Wiegant et al. [45] with a hybridization time of 2- days. Three fluorochromes were used to ratio-label pair wise two sets of 12 whole chromosomal paints (WCP). A fourth binary fluorochrome was added to distinguish the two sets of ratio-labeled WCP specific for either the even or the odd chromosomes. A fifth binary fluorochrome was used to label chromosome arm-specific probes (PQ-COBRA-FISH). Fluorescence filters and imaging and image processing were used as described previously [45]. Per passage of the cell line 15 metaphases were analyzed.

RNAse protection assay

Messenger RNA expression for apoptosis-related proteins was determined by the RiboQuant RNAse protection assay (Pharmingen/BD Bioscience) using the hAPO-3 and hAPO-5c multi-probe sets, according to the manufacturer’s instructions.

Immunohistochemistry

Immunohistochemistry was performed on formalin fixed, paraffin embedded sections (unless stated otherwise) following standard procedures and after Ag retrieval if appropriate by boiling for 2 h in citrate buffer (10 mM, pH 6.0) in a microwave. For detection the ABC system (Dako) was used, i.e. biotinylated secondary mAb and streptavidin-HRP followed by incubation with 3,3-diaminobenzidine/H₂O₂ (DAB) substrate to visualize positive staining. For staining of HLA class I molecules, HCA2 and HC10 mAb (generous gift of Jacques Neefjes, NKI, Amsterdam, The Netherlands) were used. HCA2 recognizes β2m-free HLA-A (except A24), HLA-B7301 and HLA-G and HC10 recognizes all β2m-free HLA-B, HLA-C and in addition HLA-A10, A28, A29, A30, A31, A32, and A33 [17, 26, 37, 38]. MAbs against HLA-A11 (0284HA) and HLA-A24 (0041HA) (One Lambda Inc.) were used to determine HLA-A24 loss in KLP11 primary tumor tissue. For Ag retrieval these sections were incubated in 0.1% trypsin in CaCl₂ (0.1%, pH 7.4) for 10 min at 37°C in an incubator with 5% CO₂ as described by Hiraki et al. [13]. As a positive control expression of the epithelial cell marker cytokeratin (Mon3003, Monosan, Uden, The Netherlands) was studied in KLP11 tumor tissue and as a negative control mouse IgG1 (Dako) was used.

CD2 and CD3 mAb (Dako) were used to detect T-cell infiltration, and PI9-17 mAb (kindly provided by A. Kummer, University Medical Center Utrecht, The Netherlands) was used to detect expression of the serine protease inhibitor PI-9 in tumor tissue of KLP11, as previously described [3]. Apoptosis of KLP11 tumor cells in vivo was studied in KLP11 tumor tissue using mAb M30 (Roche Molecular, Indianapolis, IN, USA) as described [20].

Tumor-associated antigens anti-Her-2 mAb 9G6, anti-Muc-1 mAb 214D4, anti-EpCAM-1 mAb Ber-Ep-4, and p53 mAb DO-7 (Dako), apoptosis-related factors, caspase-3 pAb 67341A (BD Pharmingen, San Diego, CA, USA), caspase-7 pAb Asp198 (rabbit pAb, Cell Signaling Technology Inc., Beverly, MA, USA) and apoptosis inhibitory factors XIAP mAb H62120 (mouse IgG1, Transduction Labs, Lexington, KY, USA), cIAP-1 pAb AF818 (rabbit pAb, R&D Systems, Minneapolis, MN, USA), c-IAP-2 pAb AF817 (rabbit pAb, R&D Systems), survivin (rabbit pAb, R&D Systems) were analyzed on acetone fixed KLP11 cells grown on glass cover slips.

Detailed investigation of tumor infiltrating immune cells was performed on formalin fixed, paraffin embedded sections by multiparameter confocal laser microscopy essentially as described by Sandel et al. [34]. For the immunogold staining of keratin anti-cytokeratin AE1/AE3 (IgG1, Dako) and Ultra Small Gold-labeled goat-anti-mouse (IgG, Aurion) were used. Polyclonal rabbit-anti-human-CD3 Ab ab828 (Abcam, Cambridge, UK), mouse-anti-human-CD8 mAb 4B11 (IgG2b, Novo Castra, Newcastle Upon Tyne, UK), and mouse-anti-human-CD57 mAb HNK-1 (IgM, hybridoma supernatant, ATCC, Manassas, VA, USA), and as secondary Abs goat-anti-rabbit IgG-Alexa-546, goat-anti-mouse-IgG2b-Alexa-647, and goat-anti-mouse-IgM-Alexa-488 (Molecular Probes, Leiden, The Netherlands) were used. The presence of granzyme B positive T cells was analyzed after staining of tumor sections with a cocktail of mouse-anti-human granzyme B (GrB7, IgG2a, Sanbio, Uden, The Netherlands), mouse-anti-human-CD3 and mouse-anti-human-CD-8 mAb and fluorescence-labeled secondary Abs consisting of goat-anti-rabbit IgG-Alexa-546, goat-anti-mouse-IgG2a-Alexa-647, and goat-anti-mouse-IgG2b-Alexa-488. Each fluorochrome was given an artificial color: Alexa-488-green, Alexa-546-red, and Alexa-647-blue.

Mixed lymphocyte tumor cell cultures and CTL cloning

Mixed lymphocyte tumor cell cultures (MLTC) were performed as described [43]. Lung carcinoma cell line KLP11 was incubated with 100 U/ml recombinant human IFN-γ (IFN-γ, Bender Medsystems GmbH, Vienna, Austria) for 48 h at 37°C to obtain maximal HLA class I expression, as determined by FACS analysis after staining with mAb W6/32 (1 μg/ml) followed by staining with the detecting goat-anti-mouse-FITC mAb. These cells were lethally irradiated (100 Gy) before use as stimulator cells in the MLTC. Reactivity of the bulk cultures was assessed weekly, starting from day 21, in a cytotoxicity assay. Reactive CTL clones were obtained from lytic bulk cultures by limiting dilution performed at week 6 as described [43].

Cytotoxicity and apoptosis assay

Cytotoxicity was measured with a standard ⁵¹Cr-release assay. When mentioned, IFN-γ-stimulated, ⁵¹Cr-labeled target cells were incubated with blocking mAb for 30 min at 37°C and further diluted prior to incubation with effector cells for 4 h at 37°C. Blocking mAb was used in previously determined optimal concentrations of approximately 10 μg/ml. Residual NK cell activity was blocked in all cytotoxicity assays by addition of a 50-fold excess unlabelled K562 cells to the labeled target cells. For inhibition of the Fas-dependent cytotoxicity, tumor cells were preincubated with an apoptosis-inhibiting anti-human Fas mAb SM1/23 (IgG2a Fab-fragments, Alexa) 10 μg/ml for 30 min at 37°C. The mAb remained present although diluted during the assay. Perforin-dependent cytotoxicity was inhibited by preincubation of CTL21/74 with Concanamycin A (CMA, Sigma) [16] 200 nM for 2 h at 37°C before addition to either IFN-γ-stimulated as well as nonstimulated KLP11 target cells. The CMA remained present in the assay and after the total incubation period of 6 h the viability of CMA treated CTL was assessed using trypan blue and was more than 95% in all cases.

Analysis of Fas-mediated apoptosis was studied after cross-linking of the Fas receptor using a complex of anti-Fas mAb (10 ng/ml) and protein-A (2 μg/ml). KLP11 cells or positive control cells SKW6.4 were incubated with this complex for 20 h at 37°C before DNA fragmentation was determined by FACS analysis after PI staining according to Nicoletti et al. [24].

TNF assay

As a more sensitive alternative for the cytotoxicity assay, we used the WEHI bioassay for TNF-α release (TNF assay) as described [43]. In the case of antibody blocking, the target cells were preincubated with mAb for 30 min at 37°C prior to addition of the CTL.

Enzyme-linked immunoSPOT (ELISPOT) assay and granzyme B ELISA

Effector cells were seeded in precoated 96-well nylon plates (Nalgene Nunc International) at a concentration of 1 × 10⁴ cells per well together with 2 × 10⁴ target cells in 100 μl of IMDM + 5% HS overnight at 37°C. The IFN-γ production of effector cells was measured by an IFN-γ ELISPOT as described elsewhere [10]. The granzyme B release was measured in serial dilutions of the culture supernatant by a granzyme B-ELISA kit (CLB, Amsterdam, The Netherlands), according to the manufacturer’s instructions.

Blocking studies

Recognition of target cells in the TNF-assay, ELISPOT assay, and Granzyme B ELISA was blocked using mAb W6/32, mAb B1.23.2 (anti-HLA-B and anti-HLA-C) [30] mAb 4E (anti-HLA-A29, anti-HLA-B, and anti-HLA-C) [46] and the human mAbs OK2H12 and OK6H12 (IgM, anti-HLA-B21, anti-HLA-B56, anti-HLA-B72, anti-HLA-B35, anti-HLA-B53, weak anti-HLA-B15) (kindly provided by Dr. A. Mulder, Leiden University Medical Center, Leiden, The Netherlands).

Statistical analysis

The IHC evaluation of infiltrating immune cells was statistically analyzed using the Student’s paired t test. Fas/APO-1 induced apoptosis of KLP11 and SKW6.4 cells was analyzed by the Student’s unpaired t test and correlation between HLA class I expression and immune infiltrate was analyzed using the Pearson’s test.

Intratumoral T cell infiltration in situ in a panel of lung carcinomas

We analyzed a panel of 16 NSCLC tumors, both squamous and adenocarcinoma, utilizing CD3- and CD8-specific mAb and multiparameter confocal microscopy for the presence of immune cells. The amount of immune infiltrate, present either in the stroma, in the tumor epithelium, or in both, varied greatly among the 16 NSCLC tumors studied (Table 1). There did not seem to be a major difference in the number of infiltrating immune cells between adenocarcinomas or adenosquamous tumors versus squamous carcinomas. The majority (63%) of the infiltrating cells are CD3⁺ T cells and 37% are CD3⁺CD8⁺ cytotoxic T cells. Although marked immune infiltrate was observed in some tumor samples only a minority of these cells (17%) migrated into the tumor epithelium. When corrected for the surface area of stroma versus tumor, in all tumors a proportionally lower number of immune cells were observed in the tumor epithelium (P ≤.001, Table 1, ratio of cells infiltrating the tumor epithelium versus the stroma always being less than 1.0). Interestingly, the cells that do migrate into tumor epithelium consist of a significantly (P = 0.018) higher percentage of CD3⁺CD8⁺ double positive cells (43%) as compared to that in stroma (33%) suggesting that either cytotoxic T cells tend to preferentially migrate into the tumor epithelium or that non-CD3⁺CD8⁺ cells have a comparatively poorer ability to infiltrate the tumor epithelium. The mean number of tumor epithelium-infiltrating cells was 185 cells/mm² (range 21-92 cells/mm²) for CD3⁺ T cells and 134 cells/mm² (range 15-68 cells/mm²) for CD3⁺CD8⁺ T cells. In contrast, the mean number of stroma-infiltrating cells was 758 cells/mm² (range 57-,093 cells/mm²) for CD3⁺ T cells and 423 cells/mm² (range 25-,059 cells/mm²) for CD3⁺CD8⁺ T cells.

Lung carcinoma KLP11 with highest number of tumor-infiltrating immune cells

The absolute number of CD3⁺ and CD3⁺/CD8⁺ tumor epithelium-infiltrating cells as well as the percentage of CD3⁺/CD8⁺ cells were much higher, both in the tumor (768 CD3⁺CD8⁺ T cells/mm²) as well as in the stroma (1,059 CD3⁺CD8⁺ T cells/mm²) than in all other tumors (Table 1). T cell infiltrate in KLP11 and six representative squamous and adenocarcinomas is shown in Fig. 1a, c, d and Fig. 1e–j, respectively. Furthermore, KLP11 tumor epithelium infiltrating cells were mainly (84%) CD3⁺CD8⁺ of which a minority co-express CD57⁺, i.e. effector cells with a memory phenotype [approximately 67% were CD3⁺CD8⁺CD57^- (pink), 17% were CD3⁺CD8⁺CD57⁺ (white/magenta), 13% were CD3⁺CD8^- (red), and only 2% were CD3^-CD57⁺, i.e. NK cells (green)] (see representative picture in Fig. 1c).

More than 90% of the tumor-infiltrating CD3⁺CD8⁺ cells were in an activated state as demonstrated by their expression of granzyme B, a marker of effector function (Fig. 1b). In stromal areas dense infiltration with CD8 positive cells was observed of which approximately 50% were positive for granzyme B, as well.

HLA class I expression in NSCLC tumors

The HLA class I expression was studied in all tumor samples by IHC using mAb HCA2 and HC10. The panel of NSCLC tumors showed a very heterogeneous expression of HLA class I molecules, ranging from a very prominent expression to a very weak or no expression. Interestingly, some tumor tissues showing clear HLA class I expression also contained large areas of cells that lost their HLA class I molecules almost completely. Infiltrating lymphoid cells and stroma stained positive in these areas thus serving as internal positive control for HLA class I expression.

HLA class I expression was normal, i.e. all tumor cells were positive after staining with both HCA2 as well as HC10, in 4/16 (25%) and aberrant, i.e. reduced (HLA class I negative and HLA class I positive tumor cell areas or no membrane staining) in 11/16 (69%), or completely absent (no HLA class I-expressing tumor cells) in 1/16 (6%) of tumors studied. HLA-class I molecule staining by HCA2 (preferentially staining HLA-A) correlated with staining by HC10 (preferentially staining HLA-B/C) in the majority of tumors except for number 14, 15 and KLP11. Downregulation of HLA class I expression did not significantly correlate with immune infiltration although a trend of more immune infiltrate in tumors with higher HLA class I expression is observed as indicated by the ranking assigned to each tumor sample based on positivity after staining with HCA2 and HC10, and number of infiltrating cells/mm², respectively (Table 2).

Characterization of NSCLC cell line and tumor KLP11: loss of HLA haplotype

FACS analysis and IHC of lung carcinoma cell line KLP11 established from the tumor of patient KLP11 demonstrated that KLP11 cells express the epithelial cell marker cytokeratin, the carcinoma-related Ags Muc-1, EpCAM-1, Her-2/neu, and overexpress p53. Expression of the same antigens could also be demonstrated in paraffin sections of the primary tumor (data not shown). With respect to proteins involved in apoptosis, KLP11 cells express CD95/Fas/Apo-1 as assessed by FACS analysis using CD95-specific mAb. The RNAse protection assay revealed that KLP11 cells express mRNA for the anti-apoptotic proteins XIAP, c-IAP-1, c-IAP-2, and survivin. Expression of these anti-apoptotic molecules was confirmed at the protein level by IHC using specific mAb (data not shown). Other important modulators of apoptosis, including caspase-8, FADD and TRADD, involved in receptor-mediated apoptosis, were expressed in KLP11 cells at the mRNA level as well. IHC or WB revealed expression of caspase-8 and caspase-9, weak expression of caspase-3 and absence of caspase-7 and of the protease inhibitor PI-9 (data not shown).

The obtained cell line KLP11 expresses HLA class I molecules, as determined by FACS analysis using anti-HLA-A, anti-HLA-B and anti-HLA-C mAb (W6/32), anti-HLA-A11 and anti-HLA-A24 mAb, and upregulation of total HLA class I and more specifically HLA-A11, but not HLA-A24, could be obtained by incubation of KLP11 cells with IFN-γ (Fig. 2).

Comparison of the PCR/oligotyping results of tumor cells and PBMC confirmed the loss of a complete HLA class I haplotype; KLP11 cells were HLA-A*1101, B*5701, Cw*0602, DRB1*0701, DRB4*01030102N, DQB1*0303, whereas the patient’s PBMC were HLA-A*1101, A*2402/01, B*0702, B*5701, Cw*0602, Cw*0702, DRB1*0701, DRB1*1302, DRB3*0301, DRB4*01030102N, DQA1*0102, DQA1*0201, DQB1*0303, DQB1*0604, DPB1*0301, DPB1*0402. FACS analysis indicated that KLP11 tumor cells express only HLA-A11, whereas the patient’s corresponding EBV-B cells were shown to express both HLA-A11 and HLA-24 (Fig. 2, upper panel), underscoring HLA class I haplotype loss.

In addition, paraffin-embedded tissue samples of KLP11 tumor were immunohistochemically stained with HLA-A11 and HLA-A24-specific mAb (0284HA and 0041HA, respectively) and revealed expression of HLA-A11, but not of HLA-A24 in the KLP11 tumor tissue, whereas normal lung epithelium present in the same tissue section expressed both HLA-A11 as well as HLA-A24 (Fig. 2, lower panel).

Karyotyping of KLP11 lung carcinoma cells

Karyotyping of lung carcinoma cell line KLP11 was performed by COBRA-FISH analysis to confirm the haplotype loss already observed by PCR/oligotyping for HLA class I and II molecules. The obtained karyogram showed that for most chromosomes three copies were present in the cell line (Fig. 3). Moreover, multiple aberrant chromosomes were observed in the karyogram including a nonreciprocal translocation in the p-arm of one copy of chromosome 6 with an unclassified chromosome (repeat sequences) and in addition a translocation of the p-arm of chromosome 6 with chromosome 22, while another copy of chromosome 6 is still intact (Fig. 3). Translocation in the 6p21 region that encodes HLA molecules seems likely, which explains the genetic loss of one HLA haplotype observed by HLA PCR/oligotyping. The complete karyogram description is as follows: modal range 60-5 <3n>XX, der(1)t(1;15), i(2)q(10), del(2p), i(3)(p10), i(3)(q10), der(3)t(3;20)x2, der(5)t(5;15), -, der(6)t(6;?),+7,+8, -9x2,+10,+11, i(11)(q10), -3, -5x2, i(15)(q10), -7, -8, der(18)t(9;18), -9, -0, -1, -2, der(22)t(6;22), der(22)t(19;22). Analysis of 15 metaphases per passage of various early, sequential passages, i.e. passage 3, 7, 9, and a later passage 35 of the primary tumor cell cultures of KLP11 showed that the translocation at chromosome 6 was present in all passages examined, suggesting that the translocation already occurred in vivo. Aberrations that were stable throughout all karyotyped passages include der(1)t(1;15), i(3)(p10), i(3)(q10), der(3)t(3;20)x2, der(6)t(6;?), i(11)(q10), der(18)t(9;18), and der(22)t(6;22).

Generation of HLA class I-restricted antitumor cytotoxic T-cells

Specific lysis of unstimulated and IFN-γ-stimulated KLP11 lung carcinoma cells by bulk lymphocyte cultures of 40 and 70%, respectively, was detected at an E/T ratio of 30. The specific lysis could be blocked by anti-HLA class I mAb W6/32, indicating that recognition is HLA class I restricted. The responder cells did not lyse autologous EBV-B cells of KLP11 indicating that lytic activity was tumor antigen specific. After limiting dilution 17 individual clones could be selected and one CD3⁺CD8⁺ clone (CTL 21/74) expressing TCR-Vβ1, as determined by FACS analysis (data not shown), was studied in more detail and shown to release TNF-α, IFN-γ, and granzyme B (Fig. 4a–c) upon recognition of the autologous tumor cells. CTL reactivity against KLP11 lung carcinoma cells was inhibited by mAb W6/32 and by OK2H12 indicating HLA class I- and HLA-A11-restriction (Fig. 4). MAb 4E that is of the same isotype as W6/32 did not block recognition of KLP11 cells nor did the HLA antigen-specific control mAb OK6H12 that does not bind to HLA-A11 (Fig. 4a).

Reactivity of this CTL was tested against a broad panel of epithelial tumor cell lines consisting of three other cell lines that match with KLP11 cells only for HLA-A11, two cell lines that match for HLA-B57, one cell line that matches for HLA-Cw6, and one tumor cell line that matches for HLA-A24, as well as B57 and Cw6. Reactivity of CTL21/74 was also tested against melanoma, lung, and other epithelial tumor cell lines without matching HLA class I alleles. No HLA-A11 positive lung carcinoma cell lines were available to test for recognition by this CTL clone. Two of the three tumor cell lines that match with KLP11 cells for HLA-A11, i.e. one breast carcinoma ZR75.1 and one ovarian carcinoma COV504 cell line, were recognized by CTL21/74. In a cytotoxicity assay specific lysis of ZR75.1 as well as COV504 by CTL21/74 was approximately 20% at an E/T ratio of 30, whereas at the same E/T ratio lysis of KLP11 by CTL21/74 was about threefold higher. TNF-α release by CTL21/74 in response to ZR75.1 and COV504 was almost 75% of that found in response to KLP11. All other target cell lines were not recognized by CTL 21/74 (i.e. specific lysis at an E/T ratio of 30 was in all cases less than 5% and CTL21/74 did not produce TNF-α in response to these targets). By FACS analysis CTL 21/74 was shown to express NKG2D (mean fluorescence intensity was 9.6 times higher than that of the negative control) but none of the NK inhibitory receptors studied (data not shown).

Mechanism of lysis of KLP11 tumor cells following interaction with CTL

The Fas-mediated apoptosis pathway was functional in KLP11 as indicated by the fact that incubation of KLP11 cells with anti-Fas mAb/protein-A, resulted in 47% apoptosis after 20 h of incubation compared to 15% of spontaneous apoptosis (Fig. 4d), measured by PI staining of total DNA content and FACS analysis (Nicoletti assay [24]). As a control, apoptosis was induced in 67.0% of the Fas-expressing SKW6.4 cells after 20 h of incubation with anti-Fas mAb/protein-A as compared to 14.8% of spontaneous apoptosis (Fig. 4d). However, preincubation of KLP11 cells with anti-Fas mAb SM1/23 that blocks apoptosis induction did not inhibit CTL-mediated lysis of KLP11 cells in a cytotoxicity assay (data not shown). Lysis of IFN-γ stimulated (Fig. 4e) as well as nonstimulated (data not shown) KLP11 cells could almost completely be blocked by preincubation of CTL with the perforin inhibitor CMA [16] indicating that most if not all cytolytic action of the CTL against KLP11 could be attributed to the exocytosis pathway-mediated apoptosis (Fig. 4e).

In vivo killing of KLP11 cells could be detected by IHC using the M30 mAb that recognizes a neo-epitope of cytokeratin-18 that appears upon induction of apoptosis in epithelial cells (Fig. 4f).