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,§
*
Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan;
Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo, Japan; and
Department of Immunology and Medical Zoology and
§
Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, which
may protect the cells from death domain-containing TRAIL-R-mediated
apoptosis (8, 9). Although mRNAs for these TRAIL-Rs have
been detected in a wide variety of tissues and cells, physiological
roles of these multiple TRAIL-Rs remain largely unknown. CTL rapidly kill target cells in vitro via two major effector pathways, the perforin-mediated and FasL-mediated pathways (10, 11, 12). Perforin is stored in the cytoplasmic granules of CTL and secreted into intercellular junction between a CTL and a target cell after granule exocytosis, which makes pores in the target plasma membrane and leads to osmotic lysis and influx of granule-derived granzyme B. Granzyme B also activates the caspase cascade which leads to apoptotic cell death of target cells (13, 14). In the FasL-mediated pathway, the intercellular interaction between FasL on a CTL and Fas on a target cell induces Fas-mediated apoptosis of target cells via Fas-associated death domain-mediated activation of the caspase cascade. In contrast to the perforin-mediated pathway that can potentially lyse any target cell, FasL-mediated pathway is dependent on the susceptibility of target cells to FasL which is determined primarily by the expression of Fas on the target cells and secondarily by intracellular antiapoptotic molecules such as FLICE-like inhibitory protein (15). In addition to these two effector pathways, we recently demonstrated that TRAIL was constitutively expressed on human CD4+ T cell clones and was involved in their cytotoxicity against TRAIL-sensitive target cells (16). Thomas et al. also reported that TRAIL was partly responsible for mediating the cytotoxicity of human CD4+ T cell clones against melanoma cells (17). These findings supplemented TRAIL as an additional component of T cell cytotoxicity. However, the expression and function of TRAIL on the other types of effector cells such as NK cells remain largely unknown.
NK cells play important roles in immune surveillance against
transformed cells and virus-infected cells. In response to infection by
certain viruses and pathogens, NK cells produce proinflammatory and
antiviral cytokines such as TNF-
and IFN-
(18, 19).
Moreover, NK cells directly kill transformed cells and virus-infected
cells in a MHC-unrestricted and Ag-independent manner. Previous studies
with perforin- or FasL-deficient mice showed that these effector
molecules constitute predominant pathways of NK cell cytotoxicity
(20, 21). It has been also reported that the FasL- and
perforin-mediated NK cell cytotoxicity can be augmented by some
cytokines including IL-2, IL-15, and IL-18 (22, 23, 24, 25).
In the present study, by using newly generated anti-murine TRAIL mAbs, we examined the expression and function of TRAIL on murine lymphocytes. Cell surface TRAIL expression was not detectable on freshly isolated splenic lymphocytes as assessed by flow cytometry. However, a remarkable level of TRAIL was induced preferentially on CD3- NK1.1+ NK cells after stimulation with IL-2 or IL-15, which was involved in their cytotoxicity against TRAIL-sensitive target cells along with perforin and FasL. In contrast to IL-2 and IL-15, IL-18 did not induce TRAIL expression but augmented NK cell cytotoxicity in a perforin- and FasL-dependent manner. These results indicated that IL-2 and IL-15 are potent inducers of TRAIL-mediated NK cell cytotoxicity against tumor target cells. The physiological and clinical relevancies of this finding are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six-wk-old male C57BL/6 (B6) mice and F344/DuCrj rats were purchased from SLC (Shizuoka, Japan).
Cell lines
Mouse B lymphoma 2PK-3, myeloma P3U1 (P3 x 63Ag8U.1), mastocytoma P815 (H-2d), T lymphoma YAC-1 (H-2a), melanoma B16 (H-2b), macrophage RAW 264, and hamster fibroblast BHK21 were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 containing 10% FCS, 100 µg/ml streptomycin and penicillin, and 2 mM glutamine (culture medium). A mouse fibrosarcoma L929 (H-2k) was obtained from Japanese Cancer Research Resource Bank (Tokyo, Japan) and maintained in the culture medium. 2PK-3 transfectants, mTRAIL/2PK-3 and mFasL/2PK-3, that stably express mTRAIL and mFasL, respectively, were also maintained in the culture medium. For preparation of IL-2, IL-15, or IL-18 blasts, B6 splenocytes were cultured at 2 x 106 cells/ml in the culture medium supplemented with IL-2 (500 U/ml), IL-15 (150 ng/ml), or IL-18 (100 ng/ml) for 6 days.
Reagents
Mouse IL-15 was purchased from PharMingen (San Diego, CA). Human
IL-2 was provided by Shionogi (Kamakura, Japan). Mouse IL-18 was
prepared as described before (26). An anti-mFasL mAb
(MFL2) and soluble DR5-Ig fusion protein were prepared as described
previously (16, 27). An anti-murine TNF- mAb (MP-6
XT22) and mouse TNF- were purchased from PharMingen.
Preparation of mTRAIL or mFasL transfectants
mTRAIL cDNA was prepared by RT-PCR amplification of total RNA from Con A-activated B6 splenocytes with an oligonucleotide primer corresponding to the first six codons as the 5'-primer and that corresponding to the last six codons as the 3'-primer, according to the published sequence (1). The 5'- and 3'-primers were tagged with a XhoI and a NotI site, respectively. After XhoI and NotI digestion, the PCR product of 850 bp was subcloned into pBluescript II SK(+) and the nucleotide sequence was confirmed by using an automated sequencer (Applied Biosystems, Foster City, CA) and a fluoresceinated dye terminator cycle sequencing method. The 850-bp cDNA was then transferred into the XhoI and NotI sites of the pMKITNeo expression vector (kindly provided by Dr. K. Maruyama, Tokyo Medical and Dental University, Tokyo, Japan). For generating mTRAIL transfectants, mTRAIL/pMKITNeo vector was transfected into 2PK-3 cells by electroporation (290 V, 960 µF) with a Gene Pulser (Bio-Rad, Hercules, CA). After selection with 1 mg/ml G418 and cloning by limiting dilution, a stable transfectant, designated mTRAIL/2PK-3, was selected by the staining with DR5-Ig. In a similar way, BHK21 cells stably expressing mTRAIL (mTRAIL/BHK) were prepared. Murine FasL-expressing 2PK-3 cells (mFasL/2PK-3) were generated as described before (27).
Flow cytometric analysis
2PK-3, mTRAIL/2PK-3, and mFasL/2PK-3 cells (1 x 106) were incubated with l µg of biotinylated mAb or DR5-Ig for 1 h at 4°C followed by PE-labeled avidin or PE-labeled anti-human IgG (PharMingen), respectively. After washing with PBS, the cells were analyzed on a FACScan (Becton Dickinson, San Jose, CA), and data were processed by using the CELLQuest program (Becton Dickinson). In some experiments, lymphocytes were stained with 1 µg biotinylated mAb followed by PE-labeled avidin, FITC-labeled anti-NK1.1 mAb (PharMingen), and peridinin chlorophyll protein-labeled anti-CD3 mAb (PharMingen).
Generation of anti-mTRAIL mAbs
A F344/DuCrj rat was immunized with mTRAIL/2PK-3 (2 x
107 cells) several times at 10-day intervals.
Three days after final immunization, the splenocytes were fused with
P3U1 mouse myeloma cells as described previously (28).
After hypoxanthine-aminopterin-thymidine selection, the Abs that can
inhibit cytotoxic activity of mTRAIL/2PK-3 against L929 were screened.
Two hybridomas producing mAbs (N2B1 and N2B2) were identified by their
strong inhibitory effects and cloned by limiting dilution. N2B1 and
N2B2 (both rat IgG2a,) were purified from culture supernatants by
protein G affinity chromatography.
Cytotoxic assay
A standard 51Cr release assay was performed as described previously (29). Briefly, 51Cr-labeled target cells (1 x 104) and effector cells were mixed in U-bottom wells of a 96-well microtiter plate at the indicated E:T ratios. After 8 h of incubation, cell-free supernatants were collected, and radioactivity was measured in a gamma counter. The percentage of specific 51Cr release was calculated as described before (30). In some experiments, the effector cells were pretreated with 20 nM concanamycin A (CMA) (Wako Pure Chemicals, Osaka, Japan) for 2 h to inactivate perforin (31). Anti-mFasL mAb (MFL2) and/or N2B2 were added to a final concentration of 10 µg/ml at the start of the cytotoxic assay.
In some experiments, IL-2- or IL-15 blasts were stained with PE-labeled anti-CD3 mAb (PharMingen) or PE-labeled anti-NK1.1 mAb (PharMingen). Then, CD3- (NK) and NK1.1- (T) populations, respectively, were isolated by sorting on a FACStar (Becton Dickinson) and used as effector cells. The purities were >95% CD3-NK1.1+ and >90% CD3+NK1.1-, respectively.
A [3H]TdR release assay was performed as described previously (16).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although we and others have recently showed that TRAIL is involved
in cytotoxic activity of human CD4+ T cell clones
against certain target cells, physiological functions of TRAIL remain
largely unknown. Further studies in the murine system will be helpful
for elucidating the physiological and pathological roles of TRAIL. To
characterize the expression and function of TRAIL in the murine system,
we first generated stable cDNA transfectants expressing mTRAIL. The
mTRAIL cDNA was isolated by RT-PCR from Con A-activated B6 splenocytes
and subsequently subcloned into expression vector pMKITNeo. Mouse B
lymphoma 2PK-3 cells, which were totally resistant to recombinant
TRAIL-induced cytotoxicity (our unpublished data), were transfected
with mTRAIL/pMKITNeo. Cell surface expression of TRAIL was first
verified by cell surface staining with DR5-Ig. As represented in Fig. 1
A, DR5-Ig specifically
stained mTRAIL/2PK-3 but not 2PK-3. An anti-mFasL mAb (MFL2), one
we previously generated (27), stained mFasL/2PK-3 but not
mTRAIL/2PK-3 (not shown), indicating that MFL2 does not cross-react
with mTRAIL.
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B, both mTRAIL/2PK-3 and mFasL/2PK-3 cells efficiently
lysed L929 cells in the 8-h 51Cr release assay,
whereas no significant cytotoxicity was observed with the parental
2PK-3 cells. Because L929 cells are highly sensitive to TNF-mediated
cytotoxicity, we tested mTRAIL/2PK-3 or mFasL/2PK-3 cytotoxicity
against L929 in the presence of anti-TNF mAb. However, no
inhibition was observed even with 50 µg/ml anti-TNF mAb (not
shown). In addition, neither soluble TNF activity in their supernatants
nor membrane-bound TNF on these cells was detected (not shown). These
results ruled out the possible involvement of TNF in the cytotoxicity
of these transfectants against L929. A mastocytoma cell line P815 was
sensitive to FasL but not to TRAIL. A melanoma cell line B16 and a T
lymphoma cell line YAC-1 were resistant to both TRAIL and FasL. Similar
levels of mTRAIL/2PK-3 or mFasL/2PK-3 cytotoxicity against these
targets were also observed as estimated by
[3H]TdR release assay (not shown), suggesting
apoptotic death of these target cells. Characterization of anti-mTRAIL mAbs
To further characterize the expression and function of mTRAIL, we
generated two mAbs that specifically bind to mTRAIL and block its
cytotoxic activity. Hybridomas were prepared from splenocytes from a
F344/DuCrj rat immunized with the mTRAIL/2PK-3 cells. Two hybridomas,
producing mAbs designated N2B1 and N2B2 (both rat IgG2a), were
selected by their strong ability to block the mTRAIL/2PK-3 cytotoxicity
against L929 cells. As represented in Fig. 2A, both N2B1 and N2B2 bound
to mTRAIL/2PK-3, but not to 2PK-3, cells as estimated by cell surface
staining. These mAbs also reacted with mTRAIL/BHK, but not BHK, cells
(not shown). Furthermore, as represented in Fig. 2B, both
N2B1 and N2B2 mAbs neutralized mTRAIL/2PK-3 cytotoxicity against L929
cells in a dose-dependent manner. Neither N2B1 nor N2B2 could inhibit
the cytotoxic activities of recombinant mTNF or mFasL/2PK-3 against
L929 cells (not shown). In addition, these mAbs could not stain
mFasL/2PK-3 cells or LPS-stimulated RAW 264 cells expressing membrane
TNF (not shown). These results indicated that these mAbs do not
cross-react with mFasL or mTNF.
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We recently showed that some human CD4+ T
cell clones constitutively expressed TRAIL on their surface as
estimated by staining with anti-human TRAIL mAbs (16).
However, it remains to be determined whether TRAIL can be expressed not
only on particular T cell clones but also on primary lymphocytes.
Especially in the murine system, expression of TRAIL at the protein
level remains largely unknown. Thus, we first examined the expression
of TRAIL on freshly isolated splenic lymphocytes by 3-color flow
cytometric analysis with N2B2. Freshly isolated B6 splenocytes can be
classified by their expression of CD3 and NK1.1 into four populations,
CD3+ NK1.1- (T cells),
CD3-NK1.1+ (NK cells),
CD3-NK1.1- (mostly B
cells), and CD3+NK1.1+ (NKT
cells). No detectable level of TRAIL expression was found on all of
these cells (Fig. 3A).
Likewise, neither freshly isolated thymocytes nor lymph node cells
expressed TRAIL on their surface (not shown). We also examined the
surface expression of FasL, which constitutes another pathway of T and
NK cell cytotoxicity (10, 11, 12, 20). As represented in the
bottom panels of Fig. 3A, a marginal but significant level
of FasL expression was observed on NK cells but not on the other cells,
as reported by others (20).
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B). When stimulated with IL-2, the resulting
lymphoblasts were composed of two populations,
CD3+NK1.1- (T) and
CD3-NK1.1+ (NK) cells. A
remarkable level of TRAIL expression was found on the NK cells but not
on the T cells. IL-15, which shares ß and subunits of the
receptor complex with IL-2 (33, 34, 35), exhibited an effect
similar to that of IL-2, which produced both T and NK populations and
induced TRAIL expression preferentially on the latter. In contrast to
these cytokines, IL-18, which stimulates a distinct receptor
(IL-18R/IL-1Rrp) (36, 37), induced only lymphoblasts of
the NK phenotype (Fig. 3B). No detectable level of TRAIL
expression was found on these IL-18 blasts. Taken together, these
results indicated that IL-2 and IL-15 but not IL-18 are potent inducers
of TRAIL expression on murine NK cells. We further examined the
kinetics of TRAIL expression on NK cells after IL-2 stimulation. The
TRAIL expression appeared on NK cells after 2 days after the IL-2
stimulation and reached peaks at 5 days (Fig. 3C). A similar
kinetics of TRAIL expression on NK cells was found after the IL-15
stimulation (not shown). We also examined whether IL-2, IL-15, and
IL-18 can up-regulate the surface expression of FasL and found that all
these cytokines substantially up-regulated the FasL expression on both
T and NK cells (Fig. 3B bottom). These results suggested a
differential regulation of TRAIL and FasL expression by these
cytokines. Involvement of TRAIL in IL-2- or IL-15-activated NK cell cytotoxicity
It has been well known that IL-2-activated lymphocytes exhibit
MHC-unrestricted, Ag-independent cytotoxicity against various tumor
cells, including NK-resistant targets such as P815, i.e., so-called
lymphokine-activated killer (LAK) activity (32, 38). We
compared the LAK activity of the IL-2, IL-15, or IL-18 blasts against
those of several target cells. The B6-derived IL-2 blasts spontaneously
killed not only the NK-susceptible YAC-1 but also NK-resistant B16,
P815, and L929 target cells in an MHC-unrestricted manner (Fig. 4). A similar level of LAK activity
against these target cells was observed with the IL-15 blasts. The
IL-18 blasts exhibited much higher cytotoxic activities against all
these target cells than those of the IL-2 and IL-15 blasts (Fig. 4).
Similar results were also obtained as estimated by
[3H]TdR release assay (not shown).
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A), indicating a dominant
contribution of perforin. Although cytotoxic activity of the IL-18
blasts against B16 was almost completely abrogated by the CMA
treatment, that against YAC-1 was not completely inhibited even by the
combination of CMA, MFL2, and N2B2. This suggests a possible existence
of another cytotoxic pathway that does not depend on perforin, FasL, or
TRAIL. When the FasL-sensitive but TRAIL-resistant P815 was used as the
target, CMA mostly inhibited and the combination with MFL2 completely
abrogated cytotoxic activities of the IL-2, IL-15, and IL-18 blasts
(Fig. 5A), indicating major and minor contribution of
perforin and FasL, respectively. When the TRAIL- and FasL-sensitive
L929 was used as the target, CMA alone only partially inhibited
cytotoxic activities of the IL-2 blasts and the IL-15 blasts (Fig. 5A). Further inhibition was observed by the combination with
either MFL2 or N2B2, and almost complete inhibition was achieved by the
combination with both. These results indicated that TRAIL, in addition
to perforin and FasL, is involved in the cytotoxic activities of the
IL-2 and IL-15 blasts against L929. In contrast, the IL-18 blasts
appeared to exert perforin- and FasL-dependent but TRAIL-independent
cytotoxicity, because the combination of CMA and MFL2 completely
inhibited the cytotoxic activity of the IL-18 blasts against L929 in
the absence of N2B2.
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B, the IL-2 or IL-15 blasts contain two
populations of T and NK cells. To investigate the effector mechanisms
for these cells separately, we isolated each population by flow
cytometry. Then, the cytotoxic activities of these cells against L929
were tested in the presence or absence of CMA, MFL2, and/or N2B2. As
shown in Fig. 5B, IL-2-stimulated NK cells exhibited
substantially higher cytotoxicity than T cells. The combination of CMA,
N2B2, and MFL2 was required to completely inhibit the cytotoxic
activity of IL-2-activated NK cells against L929. In contrast, N2B2 was
not required for complete inhibition of the IL-2-activated T cell
cytotoxicity. Similar results were obtained with the IL-15 blasts (Fig. 5B). These results indicated that functional TRAIL was
expressed on IL-2- or IL-15-activated NK cells but not on T cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we observed that IL-2 and IL-15 exhibited similar
effects; i.e., both induced the surface expression of TRAIL
preferentially on NK cells and induced LAK activities to a similar
level (
Figs. 3–5). These results are consistent with previous reports
indicating that IL-2 and IL-15 share two receptor components
(IL-2Rß/IL-15Rß and IL-2R/IL-15R chains) and exert mostly
overlapped biological activities including the up-regulation of NK
activity (33, 34, 35). It has been reported that exogenously
administered or genetically expressed IL-2 or IL-15 exhibited antitumor
effects in mouse models by activating NK cells (38, 39, 40).
The TRAIL-mediated NK cell cytotoxicity might be partly responsible for
the antitumor effects of IL-2 and IL-15 in vivo. It has also been shown
that IL-15 plays an important role in the development and maturation of
NK cells in vivo (41, 42). Indeed, IL-15R-chain
knockout mice have a defect in the development of NK cells and almost
completely lack mature NK cells (41). Thus, IL-15 might
up-regulate NK activity in vivo not only by inducing the development of
NK cells but also by augmenting the perforin-, FasL-, and
TRAIL-dependent cytotoxicity.
As demonstrated in Fig. 3, no detectable level of TRAIL expression was
found on freshly isolated lymphocytes. Furthermore, in our ongoing
studies with N2B2, we could not find a detectable level of mTRAIL
expression on T and B cells, even when these cells were activated by
various stimuli such as anti-CD3 mAb, PHA, CD40 ligand, LPS, and
PMA + ionomycin (our unpublished data). Thus, the cell surface
expression of mTRAIL presently appeared to be largely confined to
activated NK cells in vitro. This is inconsistent with a previous
report (43), which demonstrated the cell surface
expression of TRAIL on freshly isolated murine B cells and T cell
blasts by utilizing a rabbit polyclonal Ab (pAb) to a peptide sequence
in the extracellular region of human TRAIL. However, they did not
address functional activity of TRAIL on these cells. It has been
noticed that some commonly used pAbs to FasL exhibited aberrant
reactivities (44). In this respect, their staining might
result from some nonspecific reactivity of their pAb.
Molecular mechanisms for the restricted induction of TRAIL expression
on NK cells by IL-2 or IL-15 remain to be determined. This could not be
explained by a differential expression of IL-2R and IL-15R among
lymphocytes, because FasL expression was inducible on both NK and T
cells after IL-2 or IL-15 stimulation (Fig. 3B, bottom).
Further studies are needed to elucidate the mechanisms for restricted
expression of TRAIL on NK cells.
IL-18 is a recently identified cytokine that strongly induces IFN-
production by NK cells (26, 37) and augments T and NK cell
cytotoxicity (24, 25, 26, 37). In the present study, we found
that the stimulation of splenocytes with IL-18 selectively produced
lymphoblasts of NK cell phenotype. This might be the result of the
constitutive expression of IL-18R on NK cells but not on the other
types of splenic lymphocytes (25, 45, 46). IL-18 augments
NK cell cytotoxicity in a FasL- and perforin-dependent manner
(24, 25). Consistently, we also found that IL-18-activated
NK cells up-regulated the cell surface expression of FasL and that
these cells killed Fas-sensitive targets such as P815 and L929 via
FasL- and perforin-mediated pathways (Fig. 5A). However, in
contrast to IL-2 and IL-15, IL-18 did not induce the TRAIL expression
(Fig. 3). The apparently distinct regulation of TRAIL-, FasL-, and
perforin-mediated cytotoxicity by these cytokines might be relevant to
differential roles of these effector molecules in the immune
system.
We observed that the IL-18-activated NK cells could lyse YAC-1 target
cells even in the presence of CMA, anti-FasL, and anti-TRAIL
mAbs (Fig. 5A). It is has been known that membrane-bound or
soluble form of TNF- can participate in CTL or IL-2-induced LAK
cytotoxicity (32, 47). However, the addition of a
neutralizing anti-TNF- mAb did not abolish the IL-18-induced LAK
cytotoxicity against YAC-1 even in the presence of CMA + anti-FasL
+ anti-TRAIL (data not shown). These results suggest a possible
existence of another effector mechanism for NK cytotoxicity, which can
be specifically induced by IL-18. Further study is now under way to
characterize this perforin-, FasL-, TRAIL-, and TNF-–independent
cytotoxic pathway.
It has been suggested that perforin and FasL constitute two predominant pathways of NK cell cytotoxicity (20, 21). In the present study, we demonstrated an additional TRAIL-mediated pathway in NK cell cytotoxicity. A very recent study by Zamai et al. also showed that human CD161+CD56- immature NK cell lines, which were established from CD34+Lin- progenitor cells in cord blood by using stem cell factor + IL-2, exhibited TRAIL-dependent but FasL- and perforin-independent cytotoxicity (48). Their results together with our present study supplement TRAIL as an additional component of NK cell cytotoxicity.
Although our present study demonstrated the potential involvement of TRAIL in NK cell cytotoxicity, its physiological roles remain to be determined. It has been well known that NK cells play important roles in immune surveillance against virus-infected or transformed cells (18, 19). In this respect, the TRAIL-mediated NK cytotoxicity may at least partly participate in the elimination of virus- or intracellular pathogen-infected cells. In fact, IL-15 production was inducible following infection with certain viruses such as herpesvirus (49). The neutralizing anti-mTRAIL mAb generated in the present study will be useful for further investigation of the physiological functions of TRAIL in the murine system.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hideo Yagita, Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail address:
3 Abbreviations used in this paper: TRAIL, TNF-related apoptosis-inducing ligand; mTRAIL, murine TRAIL; FasL, Fas ligand; mFasL, murine FasL; pAb, polyclonal Ab; CMA, concanamycin A; LAK, lymphokine-activated killer.
Received for publication March 16, 1999. Accepted for publication June 9, 1999.
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References |
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), mFasL/2PK-3 (
), or 2PK-3 (
) was
tested against the indicated target cells in an 8-h 51Cr
release assay at an E:T ratio of 10. Data represent mean ± SD of
triplicate samples. Similar results were obtained in two independent
experiments.
) or presence of serially diluted N2B1 (
) or N2B2 (•) in an
8-h 51Cr release assay at an E:T ratio of 10. Data
represent mean ± SD of triplicate samples. Similar results were
obtained in two independent experiments.
) for 6 days was tested against YAC-1, B16, P815, or L929
target cells by an 8-h 51Cr release assay at the indicated
E:T ratios. Data represent mean ± SD of triplicate samples.
Similar results were obtained in three independent experiments.