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Originally published online as doi:10.1189/jlb.0604360 on June 3, 2005

Published online before print June 3, 2005
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(Journal of Leukocyte Biology. 2005;78:359-371.)
© 2005 by Society for Leukocyte Biology

Receptors and lytic mediators regulating anti-tumor activity by the leukemic killer T cell line TALL-104

Clara Brando*,1, Sunil Mukhopadhyay*,2, Eniko Kovacs*, Rosa Medina*,3, Pritesh Patel*,4, Tracey L. Catina{dagger}, Kerry S. Campbell{dagger},5 and Daniela Santoli*

* The Wistar Institute, Philadelphia, Pennsylvania; and
{dagger} Fox Chase Cancer Center, Philadelphia, Pennsylvania

5 Correspondence: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497. E-mail: kerry.campbell{at}fccc.edu


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ABSTRACT
 
The major histocompatibility complex nonrestricted cytotoxic leukemic T cell line T acute lymphoblastic leukemia (TALL)-104 is being pursued as a therapeutic agent for cancer. However, the receptors and effector mechanisms responsible for its broad tumoricidal function remain undefined. Here, we examined the roles played by natural cytotoxicity receptors (NCR), killer cell immunoglobulin-like receptors, cytolytic granule components, and tumor necrosis factor (TNF) family members in tumor recognition and lysis by TALL-104 cells. The perforin-granzyme pathway, TNF-related apoptosis-inducing ligand (TRAIL), and Fas were each involved in the lysis of particular tumor targets by TALL-104. Furthermore, phorbol 12-myristate 13-acetate/ionomycin treatment induced surface expression of Fas-L and TRAIL. In addition, supernatants from CD3-stimulated TALL-104 cultures exhibited antiproliferative activity, which was blocked 50–90% by anti-TNF-{alpha} monoclonal antibody (mAb). Although negative for the NCR natural killer (NK)p44, this cell line was found to express NKp46. An anti-NKp46 antibody strongly blocked TALL-104-mediated lysis of certain targets and directly induced cytokine production, granule release, and redirected lysis responses. Anti-NKG2D and anti-2B4 also stimulated redirected cytotoxicity by TALL-104. By contrast, anti-NKG2A mAb did not stain the cells or inhibit killing responses. Alternatively, KIR3DL2 was detected on TALL-104, and expression of its reported ligand, human leukocyte antigen (HLA)-A, on target cells provided protection from cytotoxicity. Thus, NKp46, NKG2D, and 2B4 are activating receptors, and KIR3DL2 is an inhibitory receptor on TALL-104. The data demonstrate the ability of TALL-104 cells to recognize a wide variety of tumors with NK cell receptors and kill them with a broad arsenal of cytolytic effector mechanisms, including cytolytic granules and TNF family ligands.

Key Words: cancer therapy • cytotoxic mechanisms • regulatory receptors


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INTRODUCTION
 
Tumor target recognition varies among different types of cytotoxic cells. Antigen-specific CD8+ cytotoxic T lymphocytes lyse tumors upon T cell receptor (TCR) engagement with the antigenic peptide in the context of the appropriate major histocompatibility complex class I (MHC-I) molecules on the membrane of the target cells (reviewed in refs. [1 , 2 ]). Natural killer (NK) cells kill primarily when the Fc portions of an immunoglobulin G (IgG) trigger their Fc receptor for IgG (Fc{gamma}R)IIIA (CD16); natural cytotoxicity receptors (NCR), NKp44, NKp46, and NKp30, recognize determinants on target cells to initiate spontaneous cytotoxicity; and/or MHC-I deficiency is recognized on target cells (reviewed in ref. [3 ]). Additional activating receptors on NK cells, such as NKG2D, 2B4, and NKp80, also contribute to spontaneous cytotoxicity responses [3 ]. Inhibitory signals are delivered to NK cells mostly through a subset of killer cell Ig-like receptors (KIR) that bind MHC-I molecules on target cells, thus protecting normal endogenous cells from lysis (reviewed in ref. [4 ]). Another type of effector cell, designated NKT, shares the expression of CD161 and NKG2D with NK cells but also bears a nonpolymorphic TCR expressing the V{alpha}24 chain; NKT cells lyse tumors upon receptor recognition of a nonproteic antigen in the context of CD1 molecules (reviewed in ref. [5 ]).

Cytotoxic cells can lyse tumors using perforin-dependent or death ligand-mediated mechanisms. Cytotoxicity mediated by perforin and granzymes requires effector-target cell contact and involves activation of caspases [6 ]. The tumor necrosis factor (TNF) ligand family, which includes TNF-{alpha}, lymphotoxin {alpha} (LT-{alpha}), Fas ligand (Fas-L), and TNF-related apoptosis-inducing ligand (TRAIL) [7 8 9 10 ], is also involved in the tumoricidal action of cytotoxic lymphocytes. Receptors for all of these ligands have an intracellular death domain; thus, their triggering results in activation of caspases and ultimately, in apoptotic death [11 , 12 ].

The interleukin (IL)-2-dependent MHC-nonrestricted T acute lymphoblastic leukemia (TALL)-104 cell line, generated in our laboratory from the blood of a child with TALL [13 ], represents a promising new tool for adjuvant therapy of cancer. TALL-104 cells are CD3+, CD8+, CD56+, CD16–, CD161+, thus sharing phenotypic features with NKT cells; however, they do not express the V{alpha}24 chain (unpublished data), indicating that these cells are not CD1-restricted. Also, unlike NKT and classical lymphokine-activated killer (LAK) cells, the TALL-104 cell line can be expanded in vitro for several months maintaining stable tumoricidal activity [14 15 16 ]. Lethally irradiated TALL-104 cells have been used successfully to treat advanced tumors in experimental animals [17 18 19 20 21 22 23 24 25 26 ] and in pet dogs with spontaneous cancers [27 28 29 30 ]. In addition, phase I clinical trials in adult and pediatric cancer patients (ref. [31 ] and unpublished data) have shown that irradiated TALL-104 cells are well-tolerated at high doses. It is important that irradiated TALL-104 cells have also been shown to be capable of killing tumors in vitro [32 ], which enhances their potential clinical usefulness.

Despite the extensive information that has been accumulated on the anti-tumor efficacy and safety of the TALL-104 cell line in a clinical setting, its antigen specificity and mechanisms of tumor recognition remain unclear. The identification of activating and inhibitory receptors and of lytic mediators used by this cell line to recognize and kill a wide variety of tumors is essential for effective planning of future clinical investigations. In the present study, we analyzed the differential roles of some NK cell-activating receptors (NKG2D, 2B4, NKp44, and NKp46), inhibitory receptors (KIR3DL2 and NKG2A/CD94), perforin, and TNF mediators (Fas, TNF-{alpha}, and TRAIL) in TALL-104 activation and tumoricidal function, as compared with T cell-enriched LAK cells from healthy donors and the established NK cell line, NK-92 [33 ].


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MATERIALS AND METHODS
 
Cell lines
TALL-104 cells were maintained at 37°C in 10% CO2 in Iscove’s modified Dulbecco’s medium (Life Technologies, Grand Island, NY), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologics, Norcross, GA; complete medium) and 100 U/ml recombinant human (rh)IL-2 (Chiron, Emeryville, CA). The NK-92 cell line [33 ] was cultured in complete medium in the presence of rhIL-2 (100 U/ml). All the human tumor cell lines used as targets, as well as the Fc{gamma}R+ mouse mastocytoma cell line P815, were cultured in complete medium at 37°C in 10% CO2. Hela cells, mock-transfected and transfected with the CD1a, CD1b, CD1c, and CD1d molecules [34 , 35 ], were provided by Dr. Steven Porcelli (Albert Einstein College of Medicine, Bronx, NY). The parent 721.221 target cell line [human leukocyte antigen (HLA–); referred to as Phebo] and transfectants expressing HLA-B*2702 (HLA-B+), HLA-A0301 (HLA-A3+), and HLA-Cw3 (HLA-C+) were provided by Dr. Marco Colonna (Washington University, St. Louis, MO). The human tumor target cell lines K562 (erythroleukemia), U937 (monocyte-like histiocytic lymphoma), RPMI8866 (B lymphoma), Daudi (B lymphoma), and Jurkat (T cell lymphoma) were purchased from American Type Culture Collection (Manassas, VA) and maintained in RPMI-1640 medium supplemented with 10% FBS.

T-LAK cell preparations
Peripheral blood lymphocytes were derived from the blood of healthy volunteers by centrifugation on Ficoll gradients and depleted of adherent CD14+ cells. T cell enrichment was achieved using a human T cell enrichment column (R&D Systems, Minneapolis, MN), following the vendor’s instructions. The resulting population was 95% CD3+, ≤1% CD16+, and ≤1% CD14+. T-LAK cells were generated by culturing the T cell-enriched population in 200 U/ml rhIL-2 for 7–21 days. After 11 days of culture, the cell population (averaged from three donor cultures) was 69% CD3+, 84% CD8+, and 34% CD56+, indicating a mix of T cells and NK cells.

Antibodies and reagents
Mouse monoclonal antibodies (mAb) against human CD3 (UCHT1), Fas-L (NOK-1), biotinylated anti-mouse IgG, and phycoerythrin (PE)-conjugated streptavidin were purchased from PharMingen (San Diego, CA); anti-NKp44 (3.43.13), anti-NKp46 (9E2), anti-KIR (116.1, 5.133 and DX31), and anti-MHC-I (W6/32) antibodies were provided by Dr. Marco Colonna; anti-KIR3DL1 antibody (DX9) was purchased from BioSource (Camarillo, CA); anti 2B4 (C1.7) antibody [36 ] was a gift from Dr. Giorgio Trinchieri (Schering-Plough, Dardilly, France); and anti-NKG2D and anti-NKG2A antibodies were purchased from R&D Systems; enzyme-linked immunosorbent assay (ELISA) kit for detection of TNF-{alpha} was purchased from Endogen (Woburn, MA). The anti-Fas mAb ZB4 and the anti-Fas-L mAb B-R17 were purchased from Upstate Biotechnology (Lake Placid, NY). Blocking goat polyclonal antibodies anti-human TNF-{alpha} and mAb anti-TRAIL/TNFSF10 were purchased from R&D Systems. Fluorescein-conjugated secondary antibody directed against the Fc portion of mouse IgG was obtained from Organon Teknika Corp. (Durham, NC). 3H-Thymidine was purchased from Amersham (Arlington Heights, IL), and Na351CrO4 was from Perkin-Elmer (Boston, MA). Mouse serum, concanamycin A (CMA), phorbol 12-myristate 13-acetate (PMA), ionomycin, and propidium iodide were purchased from Sigma Chemical Co. (St. Louis, MO).

Cytotoxicity assay
Cell-mediated cytotoxicity was measured in standard 4 h and 18 h 51Cr release assays, as described previously [14 , 16 ]. TALL-104 and T-LAK cells were tested at four different concentrations against 51Cr-labeled Jurkat, U937, RPMI-8866, MDA-MB231, and Daudi cells (104 target cells/well). When indicated, effector cells were preincubated with 400 nM CMA for 2 h at 37°C. CMA was left present at the concentration of 200 nM for the duration of the 51Cr release assay. Neutralizing mAb against Fas, TRAIL, TNF-{alpha}, NKp44, NKp46, and KIR3DL2 were added to the effector/target mixture at the concentration of 1 µg/ml. Redirected lysis of the Fc{gamma}R+ P815 cell line was performed as described previously [14 , 16 ].

Immunofluorescence analysis
TALL-104 and LAK cells were resuspended in phosphate-buffered saline (PBS), supplemented with 10% FBS at 107/ml. Primary antibodies were added at the concentration indicated by the vendor, and cells were incubated at 4°C for 20 min. After washing in ice-cold PBS, cells were resuspended at 107/ml and 2 µg/ml fluorescein isothiocyanate (FITC)-conjugated secondary antibody directed against the primary antibody, which was added to each sample. Cells were incubated for 20 min at 4°C, washed twice, resuspended in 500 µl PBS, and analyzed on a FACScan flow cytometer (Becton Dickinson, Lincoln Park, NJ). For detection of membrane-bound TRAIL and Fas-L, a three-step staining method was used. Cells were stained with primary antibody, followed by staining with appropriate biotinylated secondary antibody. After incubation and washing, nonspecific binding was blocked with mouse serum. Next, PE-conjugated streptavidin was added; after 20 min of incubation, cells were washed and analyzed by flow cytometry. To avoid membrane shedding of Fas-L by metallo-proteases, cell stimulation and staining procedures were performed in the presence of 1 µM KB8301 protease inhibitor (PharMingen).

PMA/ionomycin stimulation
TALL-104 and T-LAK cells (5x106) were resuspended in 1 ml complete medium, 10 nm PMA and 1 µm ionomycin were added, and the cells were incubated at 37° for 2 h, washed twice, and resuspended in PBS for fluorescein-activated cell sorter analysis.

Generation of TALL-104 and T-LAK supernatants
TALL-104 and T-LAK cells, at the concentration of 5 x 106/ml, were incubated overnight in the presence of 2.5 µg/ml-immobilized mouse IgG (control) or UCHT1 mAb (referred to as anti-CD3-stimulated cells). Supernatants were collected, filtered through a 0.2-µm filter (Becton Dickinson), and used in proliferation and apoptosis assays on tumor targets, as described below.

Proliferation assay
Jurkat, Daudi, RPMI-8866, and U937 cells were seeded in 96-well plates (Becton Dickinson) at 5 x 104/well in 50 µl complete medium. Medium or various supernatant dilutions from unstimulated and anti-CD3-stimulated effectors were added to the wells (100 µl/well). After 24 h in culture, 10 µCi 3H-thymidine was added to each well; isotope incorporation was measured after an overnight incubation using a ß-counter (Packard Instrument, Downer Grove, IL). Determinations were performed in triplicates. In some experiments, neutralizing mAb against Fas, TNF-{alpha}, or TRAIL (1 µg/ml) were included in the proliferation assay.

Apoptosis assay
Jurkat, U937 (2.5x106ml), and Daudi cells (1x106/ml) were incubated overnight in complete medium or in supernatants from unstimulated or anti-CD3-stimulated effectors (1:2 final dilution). Cells were collected, DNA was extracted, and apoptosis was measured as DNA ladder (R&D Systems), according to the manufacturer’s instructions.

ELISA
The presence of TNF-{alpha} in the supernatant of anti-CD3-activated TALL-104 and T-LAK cells and the production of lymphokines [interferon-{gamma} (IFN-{gamma}), granulocyte macrophage-colony stimulating factor (GM-CSF), TNF-{alpha}] upon cross-linking of CD3 and NKp46 were measured by ELISA using a kit from Endogen, as described previously [19 , 31 ].

Granule release assay
Granule secretion was assayed by measuring release of ß-hexosaminidase into culture supernatants from TALL-104 cells stimulated with plate-bound antibodies, as described by Darmon et al. [37 ]. p-Nitrophenyl N-acetyl-ß-D-glucosaminide (Sigma Chemical Co.) was used as substrate in assays from triplicate cultures, and percent release was calculated as 100 x [optical density (O.D.)415 of supernatant–O.D.415 supernatant from unstimulated cells]/(O.D.415 of total Triton X-100 lysate extract–O.D.415 supernatant from unstimulated cells).


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RESULTS
 
Molecular mediators of tumor cell lysis by TALL-104 cells
CMA treatment enhances the degradation of perforin in cytolytic granules of effector cells by raising the granule pH [38 ]. Thus, a residual cytotoxicity after CMA treatment is independent of the release of perforin from these lytic granules. The ability of TALL-104 and T-LAK cells to lyse tumor targets before and after treatment with 400 nM CMA was compared in a 51Cr release assay. To assess the roles of Fas, TRAIL, and TNF-{alpha} on the killer response, parallel samples were also treated with 1 µg/ml neutralizing mAb against these proteins or mouse IgG control in the effector/target mixture. Perforin and granzymes are the lytic mediators responsible for rapid killing responses. Conversely, Fas-L/TNF family receptor ligands are mainly responsible for late-term target killing by cytotoxic cells. Therefore, we performed in vitro cytotoxicity assays of 4 h or 18 h duration to assess both cytolytic responses.

CMA treatment substantially reduced the ability of TALL-104 cells to lyse the RPMI-8866 tumor target and totally abrogated Daudi cell killing in the 18-h assays (Fig. 1 ). Lysis of both of these targets was also affected by the presence of an anti-TRAIL mAb; conversely, anti-Fas mAb slightly reduced U937 cell killing and inhibited, to a greater extent, the ability of CMA-treated TALL-104 cells to lyse Jurkat cells (Fig. 1) . It is not surprising that inclusion of CMA in the 4-h assays eliminates 51Cr release completely, confirming that the earlier killing response is mostly mediated by perforin/granzyme (data not shown).



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  		Figure 1. TALL-104 cells were preincubated in medium alone or in medium supplemented with 400 nM CMA. The 51Cr release assay was performed for 18 h in the presence of control IgG or neutralizing antibodies (1 µg/ml) against Fas, TRAIL, or TNF-{alpha}. Results are the mean ± SD of three determinations. SDs ≤5 are not shown.

For comparative purposes, T-LAK cells were also tested in parallel experiments to assess the mechanisms by which they perform tumor cell lysis (data not shown). In contrast to TALL-104, T-LAK cells appeared to exert their tumoricidal function, mostly through a granzyme-perforin-dependent pathway, as their killing was abrogated (in the case of RPMI-8866, U937, and Daudi) or strongly reduced (in the case of Jurkat) by CMA treatment; and the anti-TRAIL and TNF-{alpha} antibodies were totally ineffective, and anti-Fas antibody further reduced cytotoxicity of Jurkat cells (data not shown). Cytotoxicity at 4 h by TALL-104 and T-LAK was abolished by CMA treatment (results not shown).

Fas-L and TRAIL are up-regulated in activated TALL-104 cells
A three-step immunofluorescence analysis was performed to detect the presence of Fas-L and TRAIL on the membrane of stimulated TALL-104 and T-LAK cells. Treatment with PMA/ionophore was chosen over antibody stimulation, as exposure to immobilized antibodies interferes with the three-step fluorescence analysis necessary for detection of Fas-L and TRAIL. Figure 2 shows that Fas-L and TRAIL were not expressed on resting TALL-104 cells, but treatment with PMA and ionophore resulted in an increased level of staining of both proteins. Conversely, Fas-L and TRAIL expression on T-LAK cells increased only marginally after PMA/ionophore treatment (Fig. 2) . These data correlate well with the cytotoxicity results shown in Figure 1 , confirming the capacity for TALL-104 cells to up-regulate and use Fas-L and TRAIL to kill tumors.



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  		Figure 2. TALL-104 and T-LAK cells (1x106) were incubated in the presence or absence of 10 nM PMA and 1 mM ionomycin for 2 h at 37°C, stained for the presence of membrane-bound TRAIL or Fas-L, and analyzed by flow cytometry. Results are shown for one representative experiment out of three with similar results.

Supernatant of CD3-stimulated TALL-104 cells is cytostatic
We next tested whether activated TALL-104 cells release TNF-{alpha} to mediate target cell cytotoxicity. Tumor targets were incubated overnight in the presence of supernatants from unstimulated and anti-CD3-stimulated TALL-104 cells. The latter supernatants decreased the proliferation of Jurkat, U937, and Daudi cells by two- to fivefold, as measured in a 3H-thymidine incorporation assay, but did not affect RPMI-8866 cells (Fig. 3 ). Control supernatants derived from CD3-stimulated cultures of Jurkat or the noncytotoxic leukemic T cell line TALL-106 [13 ] had no antiproliferative effect on any target (results not shown). Inclusion of 1 µg/ml anti-TNF-{alpha} mAb partially antagonized the cytostatic effect of the anti-CD3-stimulated TALL-104 cell supernatants on Jurkat and U937 targets and completely abolished the effect on Daudi (Fig. 3) .



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  		Figure 3. Tumor target cells were seeded at 5 x 104/well in a 96-well round-bottom microplate in the presence of medium or serial supernatant dilutions from TALL-104 cells stimulated with 2.5 µg/ml-immobilized anti-CD3 mAb. Antibodies against Fas, TNF-{alpha}, or TRAIL were added to the wells at the concentration of 1 µg/ml. After 24 h incubation, 10 µCi 3H-thymidine was added for 10 h. Cells were harvested, and radioactivity was measured in a ß-counter. Results, expressed in counts per minute (cpm), are the mean ± SD of three determinations. SDs <5 x 103 for Jurkat, U937, and RPMI-8866 cells and <2 x 103 for Daudi cells are not shown.

These data indicate that TNF-{alpha}, released by anti-CD3-stimulated TALL-104 cells, is mainly responsible for inhibition of proliferation of Daudi cells and partially inhibits proliferation of Jurkat and U937 targets. It is not surprising that mAb against Fas or TRAIL (both at 1 µg/ml) were found to be ineffective (Fig. 3) , in line with the finding that neither FasL nor TRAIL could be detected in these supernatants by ELISA (not shown). The presence of TNF-{alpha} in the supernatant of anti-CD3-stimulated TALL-104 cells was confirmed by ELISA (Fig. 4B ): Anti-CD3-stimulated TALL-104 cells (2.5x106) were found to produce 85 ng/ml TNF-{alpha} after 24 h versus the 25 ng/ml released at the basal level by the unstimulated cultures. Together, these data indicated that TNF-{alpha} is the effector of cytostatic activity. However, although supernatants from anti-CD3-stimulated LAK cells contained ~50 ng/ml TNF-{alpha} (Fig. 4B) , they did not show cytostatic effect on tumor targets (results not shown).



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  		Figure 4. (A) Jurkat, Daudi, and U937 cells were incubated for 24 h in 1 ml medium [control (C)] or supernatant (SPN) from TALL-104 cells stimulated with 2.5 µg/ml-immobilized anti-CD3. Cells were collected, DNA was extracted, and 1 µg DNA per sample was processed for ladder formation. Results are shown from one experiment out of three performed with similar results. (B) TALL-104 and T-LAK cells (2.5x106/ml) were incubated in the presence or absence of 2.5 µg/ml-immobilized anti-CD3 antibody for 24 h. The supernatant was collected and TNF-{alpha} measured by ELISA. Results are the mean ± SD of three determinations for TALL-104 cells and of two determinations made on LAK cells from two different donors.

To investigate whether the antiproliferative effects of the TALL-104 supernatants were associated with apoptosis, DNA fragmentation was measured in Jurkat, U937, and Daudi cells. Figure 4A shows that DNA extracted from Jurkat or U937 cells previously incubated with the supernatant of anti-CD3-stimulated TALL-104 cells was fragmented, and the DNA from Jurkat or U937 cells cultured in regular medium was not. Conversely, Daudi cells incubated in the TALL-104 supernatant did not show DNA fragmentation (Fig. 4A) . In total, these results indicate that the antiproliferative effect of the TALL-104 supernatant on Daudi was solely a result of TNF-{alpha}, possibly through cytostatic or alternative apoptotic mechanisms. This is consistent with a previous report that TNF-{alpha} induces apoptosis by Daudi cells without DNA fragmentation [39 ]. Alternatively, the apoptotic death observed in Jurkat and U937 cells exposed to TALL-104 supernatants is likely a result of factors other than TNF-{alpha}.

Potential role of CD1 molecules in tumor cell recognition
We next wanted to identify the receptors mediating target cell killing by TALL-104 cells, which express high levels of TCR/CD3 and respond to TCR stimulation by producing lymphokines and killing resistant tumor targets [14 15 16 ], thus suggesting the possibility for a direct role of TCR in antigen recognition by TALL-104 cells. However, recognition of tumor antigens in the context of classical MHC-I molecules was ruled out in another study using anti-HLA blocking antibodies (ref. [40 ] and unpublished observations). We therefore investigated the possibility that TALL-104 cells use TCR-CD3 mechanisms typical of NKT cells, which recognize glycolipids presented by CD1 molecules; to do so, cytotoxic assays were performed using Hela cell transfectants carrying different CD1 epitopes as targets. In those experiments, TALL-104 cells killed mock-transfected CD1– parental Hela cells to the same extent as Hela cell transfectants bearing CD1a, -b, -c, or -d molecules (data not shown), arguing against a role for CD1 in tumor antigen presentation to these effectors.

Role of NK cell receptors on effector function
Initially discovered on NK cells, KIR and NCR have been shown to regulate tumor killing by some cytotoxic T cells [41 42 43 ]. We investigated the role of NKp44 [44 ] and NKp46 [45 , 46 ], KIR3DL2, KIR3DL1, NKG2A, 2B4, and NKG2D on TALL-104 cell-killing responses. Immunofluorescence analysis showed that TALL-104 cells express low levels of NKp46, while being negative for NKp44 (Fig. 5A ). T-LAK cells from two donors also stained negative for NKp44 and were marginally (8–14%) reactive for NKp46. Conversely, the NK-92 cell line, used as positive control, stained moderately for NKp44 and NKp46 (Fig. 5A) . We further found that TALL-104 expresses NKG2D and 2B4 but not NKG2A (Fig. 5B) . In addition, the antibody 5.133, which recognizes KIR3DL1, KIR3DL2, and KIR2DS4, binds to TALL-104 cells, as does the antibody DX31 reacting with KIR3DL2 (Fig. 5C) . To the contrary, antibody DX9, which recognizes KIR3DL1 and an antibody specific for KIR2DS4 (116.1), failed to stain TALL-104 cells (Fig. 5C and results not shown). These results indicate that TALL-104 cells are positive for KIR3DL2.



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  		Figure 5. TALL-104 cells, T-LAK cells from two donors, and NK-92 cells were incubated with IgG control, anti-NKp46, or anti-NKp44 antibodies (A), with 5.133 (recognizing KIR3DL2, KIR2DS4, and KIR3DL1), DX31 (recognizing KIR3DL2), or DX9 (recognizing KIR3DL1) mAb (C) or with NKG2A, NKG2D, and 2B4 (C1.7) mAb (B) and were analyzed by flow cytometry.

To further characterize the role of NKp46, NKp44, and KIR3DL2 in TALL-104 killing of target cells, 51Cr-release assays against several tumor targets were performed in the presence and absence of these antibodies to block the influence of individual receptors on cytotoxicity. The anti-NKp46 antibody 9E2 protected RPMI-8866 (a B cell lymphoma) from TALL-104 cell lysis and slightly protected MDA-MB231 (a breast carcinoma) but had no effect on the killing of Daudi, K562 (Fig. 6 ), Jurkat, or U937 cells (not shown). IFN-{gamma}, TNF-{alpha}, and GM-CSF were measured in the supernatant of TALL-104 cells stimulated in the presence or absence of immobilized anti-NKp46 antibody for 18 h (Fig. 7A ). Indeed, cross-linking NKp46 increased the production of IFN-{gamma}, GM-CSF, and TNF-{alpha} by TALL-104 cells but not by T-LAK cells (Fig. 7A) . Conversely, anti-CD3 antibody stimulated the production of all three cytokines by both cells (Fig. 7A) . Anti-NKp46 antibody also stimulated granule exocytosis by TALL-104 cells as measured by ß-hexosaminidase release (Fig. 7B) . Taken together, these results indicate that NKp46 functions as an activating receptor on TALL-104 cells.



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  		Figure 6. TALL-104 cells were incubated in the absence or presence of antibodies against NKp46 or NKp44. 51Cr-labeled target cells were added, and isotope release was measured 18 h later.



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  		Figure 7. (A) TALL-104 or T-LAK cells from a donor (1x106/ml) were incubated overnight in medium (control; IgG) or with immobilized anti-CD3 (2.5 µg/ml) or anti-NKp46 antibodies. Supernatants were collected, and the production of IFN-{gamma}, TNF-{alpha}, and GM-CSF was measured by ELISA. The results are representative of at least four assays performed in duplicate, and individual duplicate values did not vary more than 10% from the mean. (B) ß-Hexosaminidase release assay of supernatants from TALL-104 cells stimulated for 3 h with immobilized antibodies toward CD56, NKp46, NKG2D, or 2B4 (2.5 µg/ml). Results are representative of two experiments with similar results. Standard deviation from triplicate determinations was less than 20% of the mean.

Redirected lysis of P815 was not affected by inclusion of anti-NKG2A mAb (Fig. 8A ). Conversely, anti-2B4 or anti-NKG2D stimulated redirected cytotoxicity (Fig. 8A) and granule exocytosis (Fig. 7B) responses by TALL-104. These results confirm that NKG2A is not expressed on TALL-104, and 2B4 and NKG2D function as activating receptors on the cells. Similarly, NKG2D and 2B4 can stimulate robust cytotoxicity by NK-92 cells (Fig. 8A) , and antibody to NKG2A does not inhibit the NK-92 killing response.



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  		Figure 8. (A) TALL-104 cells or NK-92 cells were tested for redirected lysis of the 51Cr-labeled P815 target pretreated with no mAb (No Ab), anti-NKG2A (2.5 µg/ml), anti-2B4 (C1.7: diluted 1:25), anti-CD56 (5 µg/ml), or anti-NKG2D (5 µg/ml) antibodies. Isotope release was measured 3 h later. (B) TALL-104 cells were tested for cytolytic capacity toward the MHC-I-deficient target cell, 721.221 (Phebo: empty vector transfectant lacking MHC-I expression), or transfectants expressing HLA-A0301, HLA-Cw3, or HLA-B*2702 in an 18-h 51Cr-release assay. In the right panel, cytotoxicity by TALL-104 was tested against HLA-A0301-transfected target cells in the presence or absence of anti-CD56 (B159), KIR (5.133), or MHC-I (W6/32) mAb (all at 1 µg/ml) in an 18-h 51Cr-release assay. E:T, Effector:target.

Finally, we tested whether the KIR3DL2 on TALL-104 can transduce a negative signal by engagement with its MHC-I ligand, HLA-A3 [47 ]. Cytotoxicity assays were performed against MHC-I-deficient 721.221 cells and transfectants bearing different HLA haplotypes. Figure 8B shows that TALL-104 cells display low levels of killing of the HLA-A0301-expressing 721.221 cells, and cytotoxicity of the HLA– parent (Phebo) as well as HLA-B+ (HLA-B*2702) and HLA-C+ (HLA-Cw3) transfectants was not suppressed (Fig. 8B , left panel). Moreover, antibodies toward KIR3DL2 or MHC-I effectively blocked inhibition toward the HLA-A3-expressing targets, whereas anti-CD56 did not disrupt the inhibition (Fig. 8B , right panel). Thus, TALL-104 cell killing is decreased by interaction of KIR3DL2 with its ligand HLA-A3 on target cells.


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DISCUSSION
 
Adoptive transfer of irradiated TALL-104 cells represents a promising, new form of allogeneic T cell therapy for cancer because of the demonstrated anti-tumor efficacy of these effectors in a preclinical setting and their safety in humans [17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ]. Particularly fascinating is the ability of TALL-104 cells to recognize tumors of various histological types and across species, along with their unique tumor-seeking and homing properties [23 , 26 ]. The reason for the highly potent and broad tumoricidal function of the TALL-104 cell line, as compared with other types of cytokine-activated MHC-nonrestricted effectors, has remained unknown and is of great interest. A precise understanding of the mechanisms by which TALL-104 cells execute tumoricidal action and the receptors that allow TALL-104 cells to spare healthy tissues is essential for the use of these cells in anti-tumor therapy. TALL-104 cells were previously shown to use the perforin/granzyme intracytoplasmic lytic machinery, as well as apoptotic pathways to kill different tumors [14 , 16 , 17 ]. However, the role played by TNF family components in their tumoricidal activity was never investigated. In this study, it was found that Fas-L, TRAIL, and TNF-{alpha} are present in TALL-104 cells and their normal T-LAK counterparts. However, at odds with T-LAK cells, which in this study, were found to kill tumors, preferentially through a perforin-dependent pathway, TALL-104 cells appeared highly versatile in their use of perforin and TNF family mediators. In particular, TALL-104 cells can kill tumors using released TNF-{alpha} or membrane-bound Fas-L and TRAIL, depending on the nature of the tumor target: Tumors such as RPMI-8866 and Daudi required combined perforin-dependent and TRAIL-dependent mechanisms to be lysed by TALL-104 cells, and Jurkat and U937 targets were killed efficiently by perforin or Fas-dependent mechanisms. U937 targets also exhibited some TRAIL-dependent killing. Indeed, TRAIL is expressed at low levels on activated TALL-104 and T-LAK cells, and this low amount of TRAIL might be neutralized by TRAIL decoy receptors present on Jurkat cells [48 ].

Although CMA treatment resulted in almost complete suppression of lysis of RPMI-8866 and Daudi, TALL-104 cells could also use a TRAIL-mediated mechanism to kill these targets, as blocking of TRAIL/TRAIL receptor interaction resulted in protection. That a Fas/Fas-L interaction failed to contribute to the death of RPMI-8866 or Daudi cells likely reflects the fact that Fas is expressed on a lower percentage of RPMI-8866 (40%) and Daudi (10%), as compared with Jurkat (98%; data not shown). TALL-104 and T-LAK cells can produce LT-{alpha} (data not shown). However, in our hands, inclusion of a neutralizing LT-{alpha} mAb failed to prevent killing of tumors by either effector population (unpublished results).

Supernatants from anti-CD3-stimulated TALL-104 cells decreased the proliferation of Jurkat and U937 cells and caused their DNA fragmentation. Daudi cells, which were also sensitive to the antiproliferative effect of the anti-CD3-stimulated TALL-104 supernatants, did not show DNA fragmentation. Moreover, a blocking antibody against TNF-{alpha} partially decreased the cytostatic action of TALL-104 cell supernatants on Jurkat and U937 cells and totally abolished the effect on Daudi cells. By contrast, mAb against Fas and TRAIL had no effect on the cytotoxicity of the TALL-104 supernatants on Jurkat, Daudi, or U937 cells. The levels of TNF-{alpha} produced by TALL-104 cells may be sufficient to block Daudi cell growth but not to induce DNA fragmentation-associated apoptotic death, as suggested in other studies [39 , 49 ]. As the TNF-{alpha} mAb only partially protected Jurkat and U937 cells from the antiproliferative effects of the TALL-104 supernatants, it is conceivable that TALL-104 cells release other, as yet unknown, cytostatic factors, which may synergize with TNF-{alpha} to inhibit cell growth. By contrast, T-LAK cell supernatants had no cytostatic effect on any tumor target tested, despite the presence of 50 ng/ml TNF-{alpha}, suggesting that a TNF-{alpha}-antagonizing factor may coexist.

TALL-104 cells express high levels of TCR/CD3 and respond to TCR stimulation by producing lymphokines and killing resistant tumor targets [14 15 16 ], thus suggesting the possibility of a direct role in antigen recognition by TALL-104 cells. However, recognition of tumor antigens in the context of classical MHC-I molecules was ruled out in another study using anti-HLA blocking antibodies (ref. [40 ] and unpublished observations). As described in the present study, we also tested whether recognition of nonproteic antigens presented by CD1 molecules may be involved, despite the fact that TALL-104 lacks the distinguishing invariant V{alpha}24 TCR of classical NKT cells. The finding that TALL-104 cell killing was independent of CD1 epitope expression on Hela target cells argues against a role for CD1 in tumor antigen presentation to these effectors (results not shown).

NKp46, described as the main triggering receptor on NK cells [43 , 45 , 46 ], was found to be expressed on a proportion of TALL-104 cells. Inclusion of an anti-NKp46 antibody in 51Cr release assays blocked their ability to lyse the RPMI-8866 targets, previously shown to express NKp46 ligand [50 ], and partially blocked killing of MDA-MD231. Failure of NKp46 to block the killing of the other targets tested is likely a result of their lack of NKp46 ligand and their expression of ligands for other TALL-104-activating receptors. Triggering of NKp46 had effects similar to CD3 stimulation, resulting in increased production of TNF-{alpha}, IFN-{gamma}, and GM-CSF. Thus, NKp46 acts as an important stimulating receptor on TALL-104 cells. Anti-NKG2D and anti-2B4 mAb enhanced the redirected lysis of the Fc{gamma}R+ P815 target by TALL-104 cells, and antibodies toward NKG2A or CD56 did not affect cytotoxicity. Thus, we have defined NKp46, NKG2D, and 2B4 as functional activating receptors on TALL-104. Activating and inhibitory roles for 2B4 have previously been reported in NK cells [51 , 52 ]. It is still conceivable that other activating receptors (e.g., NKp30) may play roles in TALL-104 cell triggering.

In addition to the activating receptors, TALL-104 cells were found to express the KIR3DL2-inhibitory receptor. Furthermore, reduced killing was observed toward 721.221 target cells transfected with HLA-A0301, which has been shown to be recognized as a ligand by KIR3DL2 in only three previous reports [47 , 53 , 54 ]. Alternatively, efficient killing was seen toward 721.221 cells transfected with HLA-B*2702 or -Cw3, which is consistent with the lack of detectable, inhibitory KIR, recognizing those HLA ligands. Thus, HLA-A3 was shown to serve as an inhibitory ligand for TALL-104 cells. This is important for potential tolerance of TALL-104 cells toward normal cells or certain targets that may express appropriate HLA-A ligands for KIR3DL2.

In summary, TALL-104 cells have advantages over LAK cells as a result of their ability to use a variety of mechanisms to kill tumor cells, such as TRAIL and Fas-L, in addition to perforin-granzyme release. The expression of NK cell-activating receptors, such as NKp46, 2B4, and NKG2D, also makes TALL-104 cells more effective in killing tumors bearing ligands for these receptors. Alternatively, the presence of KIR3DL2 prevents TALL-104 cells from killing HLA-A3+ cells. TALL-104 cells lack NKp44 and do not appear to express inhibitory NKG2A, recognizing HLA-E or KIR, recognizing HLA-Cw3 or -B*2702. The present study significantly improves our understanding of available receptor cells and lytic mediators that may possibly offer some predictive capacity to aid in patient selection for TALL-104 therapy. Tumors with more ligands for available activating receptors and lacking inhibitory class I HLA haplotypes (such as A0301) might be more amenable to this type of therapy. The results also offer insight to further manipulate TALL-104 functional capacity. This includes pretreatment with polyclonal activators and/or lymphokines to induce higher expression of killer mediators, as well as transfection with receptors or other molecules (such as NKp44 or activating KIR), which are lacking in the parent cell to improve anti-tumor activities according to specific needs.


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ACKNOWLEDGEMENTS
 
This work was supported by the National Cancer Institute Grants 5RO1 CA20833 (to D. S.) and CA83859 (to K. S. C.). The authors thank Dr. Steven Porcelli (Albert Einstein College of Medicine, Bronx, NY) for providing Hela cell transfectants and helpful advice, Dr. Marco Colonna for antibodies, Drs. H. Ertl (The Wistar Institute, Philadelphia, PA) and D. Kappes (Fox Chase Cancer Center) for critical review of the manuscript, Mr. J. Faust for assistance in flow cytometry analysis, Mr. A. Lucente and Ms. Christine DeLaurentis for help with graphics, and Ms. M. Sacks for preparing the manuscript. This paper is dedicated to Dr. Daniela Santoli; death took her away from us too soon, but the memory of her example and inspiration will always remain with us.


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FOOTNOTES
 
1 Current address: The Walter Reed Institute for Research, Division of Communicable Diseases and Immunology, Department of Immunology, Silver Spring, MD 20910. Back

2 Current address: Corning Inc., Corning, NY 14831. Back

3 Current address: AppTec, Inc., Philadelphia, PA 19112. Back

4 Current address: Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107. Back

Received June 25, 2004; revised April 28, 2005; accepted May 6, 2005.


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