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Originally published online as doi:10.1189/jlb.0506305 on September 22, 2006

Published online before print September 22, 2006
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(Journal of Leukocyte Biology. 2006;80:1345-1353.)
© 2006 by Society for Leukocyte Biology

Cytokine-induced killer T cells kill immature dendritic cells by TCR-independent and perforin-dependent mechanisms

Pramod S. Joshi*, Jin-Qing Liu*, Yin Wang*, Xing Chang*, John Richards*, Erika Assarsson{dagger}, Fu-Dong Shi{ddagger}, Hans-Gustaf Ljunggren{dagger} and Xue-Feng Bai*,1

* Department of Pathology and Comprehensive Cancer Center, The Ohio State University Medical Center, Columbus, Ohio, USA;
{dagger} Center for Infectious Medicine, Department of Medicine, Karolinska Institute, F59, Karolinska University Hospital Huddinge, Stockholm, Sweden; and
{ddagger} Barrow Neurological Institute, NRC-428, St. Joseph’s Hospital and Medical Center, Phoenix, Arizona, USA

1 Correspondence: Department of Pathology and Comprehensive Cancer Center, The Ohio State University Medical Center, 129 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, USA. E-mail: xue-feng.bai{at}osumc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytokine-induced killer (CIK) cells are ex vivo, expanded T cells with proven anticancer activity in vitro and in vivo. However, their functional properties with the exception of their cancer cell-killing activity are largely unclear. Here, we show that CIK T cells recognize dendritic cells (DC), and although mature DC (mDC) induce CIK T cells to produce IFN-{gamma}, immature DC (iDC) are killed selectively by them. Moreover, CIK T cell activation by mDC and their destruction of iDC are independent of the TCR. The cytotoxicity of CIK T cells to iDC is perforin-dependent. Our data have revealed an important regulatory role of CIK cells.

Key Words: cytotoxicity • IFN-{gamma} • dendritic cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytokine-induced killer (CIK) T cells are ex vivo, expanded T cells with NK activity [1 ]. In humans, CIK T cells are mainly CD3+CD56+ cells and are derived from T cells, which may express CD4 or CD8 [2 3 4 ]. The majority of the CD3+CD56+ cells is TCR-{alpha}/β+ [5 ]. In mice, CIK cells can be easily expanded from the spleen, similar to humans. CIK cells from mice express NK markers such as NK1.1 and NKG2D; however, the cells are mainly CD8+ cells, which have similar TCR useage as normal CD8+ T cells [5 , 6 ]. CIK cells can be generated from CD1d–/– mice, suggesting that these cells differ from the traditional invariant chain NKT cells (iNKT cells) [5 ]. CIK T cells produce IFN-{gamma} and exhibit potent cytotoxicity against a variety of malignant cells [5 ]. The cytotoxicity to tumor cells is non-MHC-restricted, relies on cell-cell contact, is perforin-dependent, and is Fas-independent [7 ]. Recently, it was demonstrated that NKG2D-NKG2D ligand interactions mediated the antitumor activity of CIK cells [8 ]. In in vivo models, CIK cells can traffic efficiently to tumor sites and exhibit antitumor activity [2 , 3 , 9 ]. These cells can also be used as carriers for a virus to lyse tumor cells [10 ]. In addition, adoptive transfer of CIK cells may serve as an alternative approach for donor lymphocyte infusions, a method being used for treating relapse after hematopoietic cell transplantation for a variety of malignancies, because of their low graft-versus-host effects [6 , 7 ]. Because of the important potentials of this type of cell in cancer immunotherapy, it is important to learn if these cells have other functional properties. It is also important to understand molecular interactions that mediate or regulate the effector functions of CIK T cells.

Dendritic cells (DC) have emerged as the major regulator in T cell response and NK cell activation [11 12 13 ]. Unlike other cell types, DC have vastly different functions depending on their stage of maturation. Thus, DC precursors may mediate innate immunity such as production of type I IFN and phagocytosis [14 ]. As the precursor differentiates into immature DC (iDC), they play a critical role in the induction and maintenance of immune tolerance [15 ]. Upon activation, DC initiate and sustain T, B, NK, and NKT responses [13 , 16 17 18 ]. Thus, the relative frequency of DC at different stages may be a deciding factor for immunity versus tolerance. DC commitment and maturation are known to be regulated by other factors, including TLR ligands, cytokines, and immunological help [19 , 20 ]. Theoretically, their frequency can also be modulated by selective elimination of given subsets.

NK cells have been shown to interact with DC [11 , 21 ]. Given the similarities of the patterns of cellular cytotoxicity of CIK cells to NK cells, we asked whether CIK cells could interact with DC. Here, we show that CIK cells recognize DC by a TCR-independent mechanism. It is interesting that CIK cells selectively eliminate iDC by direct cytotoxicity via a perforin-dependent mechanism. Our data thus reveal an important function of CIK cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C57BL/6 mice and perforin-deficient mice (C57BL/6-Prf1tm1SdZ/J) were purchased from the Jackson Laboratory (Bar Harbor, ME) and were maintained in the animal facilities of the Ohio State University under specific pathogen-free conditions.

Cell lines
3B11 (H-2b) is a mouse DC-like line originally isolated from a CD8{alpha}+ DC lymphoma [22 , 23 ]. 3B11 cells were maintained in RPMI-1640 medium containing 5% FBS, L-glutamine (2 mM), penicillin (100 µg/ml)/streptomycin (100 µg/ml), and 2-ME (1 mM). Murine L cell fibroblast lines (L-control) and L cells transfected with cd1d1 cDNA (L-CD1) [24 ] were cultured in DMEM with the same supplements as above. The V{alpha}14+ CD1d-specific NKT cell hybridoma DN32.D3 [25 ] was cultured in IMDM supplemented with 5% FBS and 2 mM L-glutamine. L-control, L-CD1d, and DN32.D3 cells were kindly provided by Dr. Randy R. Brutkiewicz (Indiana University, Bloomington).

CIK cell culture and subsequent cloning
Cell suspensions from spleens or lymph nodes were depleted of erythrocytes and resuspended in Click’s Eagle’s Hanks’ amino acid (EHAA) medium containing 10% FCS, L-glutamine (2 mM), penicillin (100 µg/ml)/streptomycin (100 µg/ml), 2-ME (1 mM), and recombinant human (rh)IL-2 (PeproTech, Rocky Hill, NJ) at the concentration of 5 ng/ml. Cells were cultured at 37°C in a humidified 5% CO2 atmosphere in air for 10 days. The culture supernatants were replaced with IL-2-containing fresh medium every 3 days. For cells maintained for more than 10 days, irradiated, syngeneic splenocytes (2000 rad), anti-CD3 antibody (2C11, 100 ng/ml), and IL-2 were added to the culture medium. For cloning of IL-2-expanded cells, we seeded cells at a concentration of 0.5 cell/well into round-bottomed 96-well plates and 2 x 104/well-irradiated syngeneic splenocytes and IL-2. Supernatants of cell culture were replaced with fresh medium containing IL-2 at weekly intervals. A similar procedure was used for subcloning of clonal cells. By using this method, we obtained a cell clone CIKG12, which can be maintained in IL-2 alone.

Antibodies and flow cytometry
The following antibodies were used in the experiments according to the manufacturer’s recommendations. Purified and FITC-, PE-, Percp-, or biotin-labeled anti-CD3 (145-2C11), -CD4 (GK1.4), -CD8{alpha} (53-6.7), -CD11c (HL3), -CD25 (7D4), -CD28 (37.51), -CD30 (5F-2D), -CD40 (3/23), -CD44 (IM7), -CD62 ligand (CD62L; Mel-14), -CD69 (H1.2F3), -CD80 (16-10A), -CD86 (GL-1), -CD122 (TM-β1), -CTLA4 (4F10), -4-1BB (2A), -B220 (RA3-6B2), -Db (28-14-8) , -Kb (AF6-88.5), -I-Ab (AF6-120.1), -Qa-1b (6F10), -IFN-{gamma} (XMG1.2), -IL-4 (11B11), -Ly49A (A1), -Ly49C/I (5E6), -Ly49D (4E5), -NK1.1 (PK136), -CD49b (DX5), -CD94 (18d3), -NKG2A B6 (16a11), -NKG2D (C7), -TCRβ (H57-597), -Vβ2 (B20.6), -Vβ3 (KJ25), -Vβ4 (KT4), -Vβ5.1/5.2 (MR9-4), -Vβ6 (RR4-7), -Vβ7 (TR310), -Vβ8.1/8.2 (MR5-2), -Vβ9 (MR10-2), -Vβ10 (B21.5), -Vβ11 (RR3-15), -Vβ12 (MR11-1), -Vβ13 (MR12-3), -Vβ14 (14-2), and -VLA4 (R1-2) were obtained from BD PharMingen (San Diego, CA). Anti-TRAIL antibody (N2B2) was purchased from Biolegend (San Diego, CA). {alpha}-Galactosylceramide ({alpha}-GalCer)/tetramer was a gift from Dr. Yang Liu (University of Michigan, Ann Arbor). For flow cytometry analysis, cells were incubated with antibodies on ice for 30 min followed by extensive washing. Cells were analyzed on a FACSCalibur cytometer (Becton Dickinson, Mountain View, CA). Flowjo software (Tree Star, Inc., Ashland, OR) was used for further analysis of data.

Intracellular cytokine staining
CIKG12 cells or Day 13 polyclonal CIK cells were first incubated with different stimuli. GolgiStop (BD PharMingen) was added into the culture medium in the last 5 h of culture. Viable cells were first stained for cell surface markers (in the case of polyclonal CIK cells), then fixed with Cytofix/Cytoperm buffer (BD PharMingen), followed by washing with Perm/Wash buffer (BD PharMingen), and incubated with PE-labeled anti-IFN-{gamma} (XMG1.2), -IL-4 (11B11), or -rat IgG1 (A110-1) control antibody (BD PharMingen) for 30 min on ice. Cells were analyzed on a FACSCalibur cytometer.

RNase protection assay (RPA)
Total RNAs from CIKG12 cells were isolated with Trizol reagent (Life Technologies, Gaithersburg, MD). The concentration of RNA in each sample was assessed by spectrophotometry. Multiprobe RPA kits (RiboQuant, BD PharMingen) were used, and the assay was performed according to the manufacturer’s protocol. Briefly, a set of 32P-labeled RNA probes synthesized from DNA templates using T7 polymerase was hybridized with 20 µg total RNA, after which free probes and other single-strand RNA were digested with RNase. The remaining RNase-protected probes were purified and then resolved on denaturing polyacrylamide gels. For detection of cytokines, we used template set mCK1b, which detects mRNA of IL-4, IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-3, IFN-{gamma}, L32, and GAPDH. The template set mCK-5c detects chemokine mRNA of lymphotactin (Ltn), RANTES, MIP-1β, MIP-1{alpha}, MIP-2, IFN-inducible protein 10 (IP-10), MCP-1, TCA-3, eotaxin, L32, and GAPDH.

Cytokine ELISA
IFN-{gamma} and IL-2 ELISA were performed by following standard procedures (BD PharMingen). The antibody pairs used for IL-2 were: capture antibody (JES6-1A12) and biotinylated anticytokine detection antibody (JES6-5H4). The antibody pairs used for IFN-{gamma} were: capture antibody (R4-6A2) and biotinylated anticytokine detection antibody (XMG1.2). Avidin-HRP (BD PharMingen) and o-phenylenediamine substrate (Sigma Chemical Co., St. Louis, MO) were used to develop color. OD was read at 490 nm in a microplate reader.

51Cr release assay
CIKG12 cells or freshly generated polyclonal CIK cells were used as effector cells. As targets, we used 51Cr-labeled 3B11 cells and bone marrow-derived DC. The effector and target cells were incubated at indicated E:T ratios for 6 h, and the percentages of specific lysis were calculated based on the following formula: specific lysis % = 100 x (cpmsample–cpmmedium)/(cpmmax–cpmmedium).

Culture of DC from bone marrow
We followed an established procedure [26 ]. Briefly, bone marrow cells from C57BL/6 mice were cultured in Click’s EHAA medium containing 5% FCS, L-glutamine (2 mM), penicillin (100 U/ml)/streptomycin (100 µg/ml), 2-ME (1 mM), and murine rGM-CSF (PeproTech) at the concentration of 20 ng/ml. Fresh medium containing GM-CSF was added every 2 days. After 7 days of culture, cells were then left untreated or treated with LPS (Sigma Chemical Co.) at a concentration of 1 µg/ml for another 2 days to allow maturation of DC. CD11c+ cells were purified with CD11c-Dynabeads (Dynal Biotech LLC, Brown Deer, WI) by following the manufacturer’s manual.

Flow cytometry-based cytotoxicity assay (FloKA)
A flow-based killing assay was developed to measure in vitro cellular cytotoxicity of syngeneic targets as described [27 , 28 ]. Target cells (3B11 cells or DC) were washed with PBS, resuspended at 1 x 106 cell/ml, and then labeled at 37°C for 15 min with 10 nM CFSE (Molecular Probes, Junction City, OR). Labeling reactions were stopped with RPMI medium containing 10% FBS. Labeled target cells (1x105) were added to 96-well plates along with indicated effector cells in complete RPMI medium. The E:T ratio was 4:1 for all the experiments unless otherwise indicated. Immediately before analysis, 1 µg/mL (final concentration) of 7-amino-actinomycin D (7-AAD; Calbiochem, San Diego, CA) was added to each sample. 7-AAD incorporation was used as a surrogate marker for late cell death/apoptosis as it intercalates with DNA in cells that have lost membrane integrity. FloKA assays were compared with standard chromium release assays and gave similar results (data not shown). All cytotoxicity assays were performed in triplicate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of polyclonal and monoclonal CIK cells
We cultured total splenocytes from C57BL/6 mice with rhIL-2. As shown in Fig. 1a , before culture, 2.27% of total splenocytes coexpressed TCR-β and NK1.1 markers. After culture with IL-2 for 1 week, the NK1.1+ T cell population was up-regulated extensively to 27.3%. By Weeks 2 and 3, this population of cells was expanded further to 38.8% and 78%, respectively. However, the maintenance of those cells required the addition of APC and a low dose of anti-CD3 antibodies after 10 days of culture. As reported previously [5 , 6 ], the majority of the IL-2-expanded T cells was CD8± cells, and no CD4+ T cells were expanded (data not shown). Moreover, the CIK T cells expressed high levels of CD122, CD44, and CD69, with almost undetectable CD62L (data not shown). The IL-2-expanded T cells also expressed a variety of NK markers including 2B4, NKG2D, CD94, and low levels of NKG2A and LY49C/I/F/H. Other NK markers such as Ly49A, Ly49D, and pan-NK marker DX5 were negative (data not shown). TCR analysis by flow cytometry revealed a polyclonal population similar to naïve CD8+ T cells (data not shown). Intracellular cytokine staining revealed that the majority of the cells contained IFN-{gamma} protein, even in the resting state (only in the presence of IL-2), and upon activation with anti-CD3 antibody, drastic up-regulation of IFN-{gamma} was detected. In contrast, IL-4 was detected in neither the resting cells nor in the anti-CD3-activated cells (Fig. 1b) .


Figure 1
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  		Figure 1. Characterization of polyclonal and monoclonal CIK cells. (a) Splenocytes from C57BL/6 mice were cultured with rhIL-2 (5 ng/ml) in vitro, and cells were stained with anti-TCR-β and anti-NK1.1 antibodies and analyzed by flow cytometry. Day 13 and Day 21 cells were stimulated with IL-2 and anti-CD3 antibody (2C11, 100 ng/ml) for 3 days in the presence of irradiated (2000 Rad), syngeneic splenocytes (2x105/ml) before being analyzed. (b) Intracellular cytokine staining was performed on Day 13 cells, which were gated on TCR-β+ cells. (a, b) Data shown represent two to five repeated experiments with similar results. (c) TCR β-chain staining of a CIK cell clone. Antibody staining verified the β chain of the CIKG12 cells as Vβ12. Dotted lines represent isotype controls, and solid lines represent respective TCR antibodies. (d) Cytokine and chemokine mRNA expression by CIKG12 cells. RPA was used to measure cytokine mRNA expression (probe set mCK-1b for cytokines; probe set mCK-5c for chemokines; both probe sets from BD PharMingen). Cells were cultured in the presence of different stimuli, and 24 h later, total RNA was purified and analyzed by RPA. Total RNA from the spleen was used as a positive control. (c, d) Data shown represent two independent experiments with similar results. Ham, hamster.

 
To understand further the functional characteristics of the IL-2-expanded CIK cells, we cloned the IL-2-expanded CIK population and obtained a clonal cell CIKG12, which can be cultured permanently in vitro with IL-2 alone. By using the RACE technique, we cloned and sequenced the {alpha} chain and β chain of the cell clone, which used Vβ12.1Jβ2.1 and V{alpha}10.3J{alpha}18 as its TCR (data not shown). Flow cytometry analysis further confirmed this clonal cell bore the Vβ12 TCR (Fig. 1c) , a little CD8 but no CD4 (not shown). Like polyclonal CIK cells, the clonal CIK cells constitutively expressed varieties of NK markers such as NK1.1 and 2B4 but no Ly49A, D, C, I, CD94, NKG2A, and NKG2D (data not shown). RPA revealed that the cell clone expressed mRNA of IFN-{gamma} but not other cytokines such as IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-13, and IL-15 (Fig. 1d) . After activation with anti-CD3 mAb or PMA plus ionomycin, the IFN-{gamma} mRNA level was enhanced greatly. A number of chemokines such as Ltn, RANTES, MIP-1{alpha}, MIP-1β, MCP-1, and TCA-3 were detectable prior to activation and were elevated significantly upon activation with anti-CD3 antibody or PMA plus ionomycin (Fig. 1d) . Direct cytokine staining confirmed that the cell clone produced large amounts of IFN-{gamma} but not IL-4 protein after activation through the TCR (not shown). Thus, the CIKG12 clone resembles polyclonal CIK cells phenotypically and functionally.

Syngeneic mature DC (mDC) induce CIK cells to produce IFN-{gamma} by TCR-independent mechanisms
The clonal cell CIKG12 did not recognize the CD1d-{alpha}-GalCer tetramer (Fig. 2a ). Moreover, this clone also failed to produce IFN-{gamma} (Fig. 2c) when stimulated with L cells transfected with CD1d, which were capable of stimulating V{alpha}14+ iNKT cell clone DN32.D3 to produce IL-2 (Fig. 2b) . When stimulated with CD1d-transfected L cells, polyclonal CIK cells also did not produce IFN-{gamma} (Fig. 2d) . These results demonstrate that the IL-2-induced CIK cells are not CD1d-restricted. It is interesting that upon coculture with 3B11 cells, a DC line from syngeneic mice [22 ], CIKG12 cells produced moderate levels of IFN-{gamma} (Fig. 2c) . To test whether the ability to activate the CIK cell is a general feature of DC, we tested whether bone marrow-derived DC can induce IFN-{gamma} production by CIK cells. As shown in Fig. 3a , immature bone marrow-derived DC failed to stimulate CIK cells to produce IFN-{gamma}. However, mDC stimulated CIK cells to produce IFN-{gamma}. This stimulatory effect was specific to DC, as similarly stimulated macrophages failed to stimulate CIK cells to produce IFN-{gamma}. Moreover, our intracellular staining suggested that the detected IFN-{gamma} was derived from CIK cells but not DC (Fig. 3a , inset). When culturing in vitro, we frequently found a down-regulation of TCR by CIKG12 cells (Fig. 3b , inset). However, the cells that lost their TCR expression from the cell surface did have the same TCR gene rearrangement (Fig. 3b , inset). We therefore determined whether mDC could stimulate a TCR– variant of CIKG12 cells to produce IFN-{gamma}. As shown in Figure 3b , the TCR– CIKG12 cells produced the same amount of IFN-{gamma} as the TCR+ cells, and anti-CD3 antibody failed to stimulate TCR– CIKG12 cells to produce IFN-{gamma}. Thus, induction of IFN-{gamma} production in CIKG12 cells after stimulation with mDC was not via the TCR. Therefore, DC can activate CIK cells and trigger IFN-{gamma} production by CIK cells via a TCR-independent manner.


Figure 2
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  		Figure 2. IL-2- and anti-CD3-expanded CIK cells are non-CD1d-dependent NKT-like cells. (a) CD1d tetramer failed to stain CIKG12 cells. Thymocytes from a C57BL/6 mouse or CIKG12 cells were double-labeled with anti-NK1.1 antibody and CD1d-{alpha}-GalCer tetramer followed by flow cytometry analysis. (b) CD1d-transfected L cells stimulated a NKT hybridoma DN32.D3 to produce IL-2, and a DC line 3B11 failed to stimulate the NKT hybridoma to produce IL-2. CD1d-transfected L cells, control cells, or 3B11 cells were cocultured with NKT hybridoma DN32.D3 cells for 24 h, and IL-2 released to the supernatant was measured by IL-2 ELISA. (c) 3B11 cells stimulated CIKG12 cells to produce IFN-{gamma}, and CD1d- or control vector-transfected L cells failed to stimulate CIKG12 cells to produce IFN-{gamma}. The culture condition used was the same as in b. (d) L-CD1d cells failed to stimulate polyclonal CIK cells to produce IFN-{gamma}. The culture condition used was the same as in b (*, P>0.05, compared with L-CD1d culture alone; Student’s t-test was used). (a–d) Data shown were representative of two experiments with similar results.

 

Figure 3
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  		Figure 3. DC stimulate CIK cells to produce IFN-{gamma}. (a) Induction of IFN-{gamma} by bone marrow-derived DC. Spleen macrophages were used as control cells. CIK cells (2x104) and equal numbers of iDC, mDC, or control macrophages (purified with anti-CD11b beads from spleen) were cocultured for 48 h. IFN-{gamma} released in the supernatant was measured. Mature DC (mDC) or immature macrophage (mMac): iDC or spleen macrophages were stimulated with LPS (1 µg/ml) for 48 h before they were used to stimulate CIK cells. Data represent four independent experiments with similar results. (Inset) CIKG12 cells were cocultured with mDC or iDC for 48 h, and cells were then stained for IFN-{gamma} and NK1.1. Data shown were gated on NK1.1+ cells. (b) TCR-β+ and TCR-β– CIKG12 cells produced IFN-{gamma} after coculture with mDC. TCR+ or TCR– variant CIKG12 cells (2x104) were cocultured with the same numbers of mDC in each well of 96-well plates. Forty-eight hours later, IFN-{gamma} ELISA was performed to measure IFN-{gamma} release in the culture supernatants. Error bars represent SD of triplicate wells of culture. *, P < 0.01, compared with unstimulated CIK cells. (Inset) TCR-β+ and TCR-β– CIKG12 subclones had the same TCR gene rearrangement. Genomic DNA from TCR-β+ and TCR-β– CIKG12 subclones was amplified by PCR. The primers used for typing the Vβ12 gene rearrangement were Vβ12.F: 5'-ATG GGC ATC CAG ACC CTC TGT TGT G-3' and Jβ2.1.R: 5'-GGA AAT GCT GGC ACA AAC CTC CTC T-3'. Data shown represented two experiments with similar results.

 
CIK cells preferentially kill syngeneic iDC in vitro and in vivo
To test whether CIK cells recognize DC and had cytotoxicity against DC, we generated iDC from the bone marrow of C57BL/6 mice. For generation of mDC, we stimulated iDC with LPS at a concentration of 1 µg/ml for 48 h. We then purified CD11c+ cells by MACS and labeled purified iDC and mDC with 51Cr. As shown in Fig. 4a 4b 4c , CIKG12 cells and polyclonal CIK cells killed bone marrow-derived iDC. A better lytic activity of iDC was detected in fresh polyclonal CIK cells compared with that of monoclonal CIKG12 cells. After being stimulated with LPS, iDC became resistant to lysis by the CIK cells.


Figure 4
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  		Figure 4. CIK cells killed iDC. 51Cr-release assay was used to measure the lytic activity of CIK cells against DC. Target cells were labeled with 51Cr and incubated with CIK cells at the indicated E:T ratio. Bone marrow cells of C57BL/6 mice were cultured in medium containing GM-CSF (20 ng/ml) for 7 days. Cells were then treated with LPS (mDC) or without LPS (iDC) for 48 h (LPS was used at a concentration of 1 µg/ml). Cells were then purified by magnetic cell sorting using CD11c-Microbeads and labeled with 51Cr. Cells were then cocultured with CIK cells for 4 h. (a) Polyclonal CIK cells and monoclonal CIK cells (CIKG12) killed iDC. (b) iDC was more sensitive to lysis by CIKG12 cells compared with mDC. (c) iDC was more sensitive to lysis by Day 13 CIK cells compared with mDC. (a–c) Data shown represented three to five experiments with similar results.

 
As an alternative approach, we labeled mDC and iDC with CFSE and cocultured them with CIK cells for 4 h. After staining with 7-AAD, the percent of dead cells was quantitated by flow cytometry. As shown in Fig. 5a , monoclonal CIKG12 cells killed the iDC but not the mDC. Although the polyclonal CIK cells also killed mDC to some extent, the iDC were preferentially killed (Fig. 5c and 5d) . The flow cytometry assay allowed us to test susceptibility of other cell types that are difficult to label with 51Cr. As shown in Fig. 5b , although the iDC were killed efficiently, peritoneal cells expressing CD11b or F4/80 were resistant. Likewise, activated B cells and CD4 and CD8 T cells were also resistant to CIK cells. Therefore, the cytotoxicity of CIK cells is highly selective against iDC.


Figure 5
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  		Figure 5. Flow cytometry-based killing assay. Bone marrow cells of C57BL/6 mice were cultured in medium containing GM-CSF (20 ng/ml) for 7 days. Cells were then treated with LPS (mDC) or without LPS (iDC) for 48 h (LPS was used at a concentration of 1 µg/ml). Cells were then purified by magnetic cell sorting using CD11c-Microbeads and labeled with CFSE. Cells were then cocultured with CIK cells for 4 h followed by staining with 7-AAD and flow cytometry analysis. (a) Flow cytometry killing assay was used to test the lytic activity of CIKG12 to iDC or mDC. E:T, 4:1. (b) Flow cytometry killing assay was used to test the lytic activity of CIKG12 to other activated targets. E:T, 4:1. SD bars represent triplicate wells (*, P<0.01, Student’s t-test compared with mDC-targeted killing). Thioglycolate (3%; Sigma Chemical Co.) was injected into C57BL/6 mice (2 ml/mouse) i.p.; 72 h later, peritoneal cells were collected and positively selected with F4/80 or CD11b beads (Dynal Biotech LLC). For generation of activated B cells, splenocytes from C57BL/6 mice were stimulated with LPS (10 µg/ml) for 48 h. Cells were then positively selected with anti-B220 beads. For selection of activated CD4 or CD8 cells, splenocytes from C57BL/6 mice were stimulated with anti-CD3 antibody (Clone 2C11, concentration: 1 µg/ml) for 48 h. Activated CD4 or CD8 cells were then selected with anti-CD4 or anti-CD8 beads. (c) Flow cytometry killing assay was used to test the lytic activity of Day 13 CIK cells. E:T, 4:1. (d) Flow cytometry killing assay. Effector cells were polyclonal CIK cells. SD bars represent triplicate wells (*, P<0.05, Student’s t-test compared with mDC-targeted killing). Data are representative of three to five experiments with similar results.

 
We tested whether the injection of CIK cells into normal mice could deplete DC in vivo. C57BL/6 mice were injected with 4 x 106/mouse polyclonal CIK cells i.v.. Two days after treatment, splenocytes were stained for CD11c and CD45.2 markers. As shown in Fig. 6a and 6b , an ~50% reduction of CD11chigh cells was observed in spleens of CIK-treated mice.


Figure 6
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  		Figure 6. In vivo injection of CIK cells reduced the numbers of splenic DC. C57BL/6 mice (n=5) were treated with Day 13 CIK cells (derived from C57BL/6 mice, 4x106/mouse, i.v.). Forty-eight hours later, the treated mice and untreated control mice (n=5) were killed, and splenocytes from each mouse were stained for CD45.2 and CD11c markers. (a) Flow cytometry diagram shows a representative mouse in each group. (b) Number of CD11c+ cells reduced in CIK cell-treated mice compared with control mice (*, P<0.01, Student’s t-test). Bars represent SD of five mice per group. Sp, Spleen.

 
CIK cells kill iDC by contact-dependent, perforin-dependent but TCR-independent mechanisms
Polyclonal and monoclonal CIK cells killed the 3B11 cell line over a 4-h period; however, the polyclonal CIK cells were significantly more potent (Fig. 7a ). The lysis of 3B11 cells by CIKG12 cells needed cell-cell contact, as separation of effector cells (CIKG12) from target cells (3B11) in a transwell setting showed no killing (Fig. 7b) . The killing of 3B11 cells was diminished by incubation of CIKG12 cells with Con A (Fig. 7c) . As Con A is known to be a specific inhibitor of vacuolar-type H+-dependent adenosine triphosphatase, and it inhibits the activity of perforin [29 , 30 ], this result suggests that CIKG12-mediated lysis of 3B11 cells was perforin-dependent. We verified this point further by generating CIK cells from perforin-knockout mice and tested their cytotoxicity to iDC. As shown in Fig. 7d , Day 13 CIK cells generated from perforin-deficient mice had greatly diminished (approximately tenfold reduction) lytic activity to iDC compared with their wild-type counterparts. To determine whether the killing activity of CIKG12 cells was mediated by TCR, we obtained TCR+ and TCR– subclones from the CIKG12 cells. We then tested whether they differed in lysing the same 3B11 target. As shown in Fig. 7e , TCR-negative cells had vigorous lytic activity against 3B11 cells. In three independent experiments, we detected equal killing of 3B11 target cells by TCR-negative cells compared with TCR-positive cells. Moreover, by using anti-Vβ12 antibody to block their specific TCR, we failed to demonstrate any effect on the lytic activity of CIKG12 cells against 3B11 (data not shown). Thus, the lytic activity of CIK cells against target DC was not mediated by the TCR.


Figure 7
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  		Figure 7. Characterization of the lytic activity of CIK cells against DC. (a) Polyclonal and monoclonal (CIKG12) CIK cells could lyse 3B11 cells, a DC-like cell line. Higher lytic activity was detected in polyclonal CIK cells. Day 13 CIK cells and CIKG12 cells were used as effectors. (b) The lytic activity of CIKG12 cells needed cell-cell contact. 3B11 cells were labeled with 51Cr and were incubated with CIKG12 cells with or without (W/o) transwell, which allowed the effector and target cells to share medium and soluble factors. (c) Concanamycin A (CMA) activation of CIKG12 cells diminished their lytic activity against 3B11 targets. CIKG12 cells were incubated with or without CMA for 2 h, and then target cells were added into plates and cocultured for 5 h. (d) The DC-lytic activity of perforin-deficient CIK cells was diminished greatly. Day 13 CIK cells were generated from a perforin-knockout mouse and a wild-type (wt) mouse simultaneously and were used as effector cells. Day 7 iDC were used as target cells. Data shown represent two experiments with similar results. (e) The lytic activity of CIKG12 cells against targets was independent of the TCR. TCR+ and TCR– subclones of CIKG12 cells were used as effectors, and 3B11 cells were used as targets. Data shown represent the average of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CIK cells are ex vivo-expanded T cells with proven anticancer activity in vitro and in vivo [1 , 3 , 7 , 10 , 31 ]. One important feature of CIK cells is their cytotoxicity against transformed cells [32 ]. The cytotoxicity to tumor cells is non-MHC-restricted, relies on cell-cell contact, and is perforin-dependent and Fas-independent [7 ]. Another feature of CIK cells is the production of effector cytokines and chemokines. Previous gene chip studies using human CIK cells have revealed that CIK cells produce IFN-{gamma}, a number of other cytokines such as IL-4 and IL-10, and chemokines such as RANTES, MIP-1{alpha}, and MIP-1β [33 ]. In this study, we have characterized the pattern of cytokines and chemokines in clonal CIK cells. RNA and protein analysis have revealed that CIK cells produce IFN-{gamma} but not IL-4 or other Th2 cytokines after activation. RPA has also revealed constitutive and inducible production of chemokines including Ltn, RANTES, MIP-1{alpha}, and MIP-1β by CIK cells. The major role of these chemokines is to function together with IFN-{gamma} as Type 1 cytokines [34 ], which enables them to potentially tilt the immune response toward a Th1 direction.

By using polyclonal and monoclonal CIK cells, we have made two noteworthy observations. First, CIK cells recognize DC. Second, in addition to its defining feature as the lack of CD1d restriction, CIK cells resemble neither T cells nor NK cells in its target recognition.

It is of particular interest that the consequences of CIK cells and DC interaction differ based on the stage of the DC differentiation. iDC failed to trigger cytokine production by CIK cells but were killed efficiently by the CIK cells. In contrast, mDC were efficient inducers of IFN-{gamma} production by CIK cells but were poor targets for the CIK-mediated lysis. The selective cytotoxicity of CIK cells makes it likely that this type of cell can regulate the relative frequency of mDC versus iDC. As the iDC and mDC have distinct functions in tolerance versus immunity [15 , 16 ], and CIK cells exist in vivo in significant numbers under physiological conditions [5 ], it is plausible that CIK cells may play a significant role in maintaining the balance between T cell tolerance and immunity. Our in vivo data suggest that upon adoptive transfer, CIK cells do have the capacity to deplete CD11chigh cells. It remains to be determined what the consequences are of in vivo depletion of DC by CIK cells.

It is most important that the recognition of target by CIK cells is completely independent of the TCR, as TCR+ and TCR– subclones are equally competent in cytokine production and cytolysis. The involvement of known NK receptors including Ly49A, D, C, I, CD94, NKG2A, and NKG2D is unlikely, as the CIK clone CIKG12, which does not express these NK cell receptors, retains the function of the polyclonal CIK cells, which express most of the receptors. Blocking TRAIL or its receptor DR5 has been shown to block the lysis of iDC mediated by mouse NK cells [21 ]. As neither monoclonal nor polyclonal CIK cells express TRAIL (data not shown), TRAIL is not responsible for the recognition of DC by CIK cells. Blocking antibodies against conventional MHC Class I, nonconventional MHC Class I Qa-1b, and MHC Class II also failed to show their involvement (data not shown). Thus, at this stage, neither the ligand on DC nor the receptor on the CIK cell has been identified, although it is clear that the ligand is expressed on mDC and iDC, and the cytotoxicity of CIK cells to DC is dependent on the perforin pathway.

Taken together, we have shown several important functions of CIK cells. Our work provides compelling evidence that this type of cell may serve as an important immune-regulatory function through the cross-talk between CIK cells and DC. As CIK cells exist in vivo under physiological conditions and during immune response [35 ], the effects of CIK cells in shaping adaptive immune responses in vivo remain to be investigated further. Moreover, our clonal cells may provide useful tools for identifying novel ligands that are involved in CIK-DC interactions.


   ACKNOWLEDGEMENTS
 
This study was supported in part by NIH/NCI (IR210A1166T8 to X.F.B.) and Ohio Cancer Research Associates (X.F.B.).

Received May 5, 2006; revised July 1, 2006; accepted August 22, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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