Developmentaland Comparative Immunology,Vol. 14, pp. 295-304, 1990 Printed in the USA.All rights reserved.

0145-305X/90 $3.00 + .00 Copyright © 1990 PergamonPressplc

PATHWAYS OF SIGNAL TRANSDUCTION IN TELEOST NONSPECIFIC CYTOTOXIC CELLS D o n a l d U Evans,* D a v i d T. H a r r i s , t D o n n a L. S t a t o n , * a n d Liliana Jaso-Friedmann* *Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602 and tDepartment of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85721 (Submitted March 1989;Accepted November 1989)

RAbstractlIn the present study evidence is presented that both a putative "receptor" binding monoclonal antibody (mAb) and the calcium ionophore A23187, either singly or together, increase receptor expression and lysis of IM-9 target cells by catfish NCC. NCC activity against IM-9 target cells was increased 55% after 1 h mAb treatment. Receptor expression determined by flow cytometry increased 95% following18-h treatment. A23187

treatment of NCC produced greater than 200% increases in receptor expression. Combined treatments of NCC with 10-4 M A23187 and 10-7 M PMA however augmented receptor expression only 22% above that produced by A23187 alone. MAb and A23187 comodulated cytotoxicityby a 65% increase over ionophore treatment alone. MAb and 10-1o M PMA comodulation produced only 10.6% increases in cytotoxicity compared to mAb. These data demonstrate that the moiety on NCC recognized by 5C6 may provide an activation signal for increased cytotoxicityand receptor expression. Calcium ionophore, either singly or together with mAb, provided an even stronger activation signal for increased lysis and receptor expression. :~,

DKeywords--Signal transduction; NCC; monoclonal antibody; receptor modulation;

A23187 modulation.

Research supported by National Cancer Institute grants Ca47338 and Ca48085 and by a USDA Science and Education Administration (Section 1433) grant. Address correspondence to Dr. Donald L. Evans, Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

Introduction We demonstrated previously that the calcium ionophore A23187 stimulated channel catfish nonspecific cytotoxic cell (NCC) activity. Treatment of NCC with p h o r b o l e s t e r s (PMA, PDD, Phorbol) produced either no activation or suppressed cytotoxicity (I). A synergism between PMA and A23187 for activation of NCC killing was demonstrated by cotreatment with nanogram amounts of PMA and m i c r o g r a m (optimum) a m o u n t s o f i o n o p h o r e . T h u s , the pathways of signal transduction (as determined by functional activity) in catfish NCC may differ from those in mammals wherein increases in free cytosol Ca + ÷ levels appear to provide a minimal signal for phosphatidyl inositol hydrolysis and protein kinase C (PKC) activation. The role of diacylglycerol (DAG) in the activation of PKC, however, appears less pivotal as a second messenger for enhancement of cytotoxicity as in mammalian systems. Analogous to the pathway of NCC activation by A23187, mAb binding may also produce increases in free cytosolic Ca ÷ +. Previous work with mammalian cells has shown that a second process by which increased cytosolic Ca ÷ ÷ levels are produced may be mediated by antibody binding to the TCR/CD3 complex or to surface immunoglobulin (2-5). Generally, cross-linkage of surface membrane ligands produces increased intracellular calcium release which may

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transduce a signal for increased proliferative activity. The requirements for mAb activation of human alloreactive CTL's (3) and also for mouse L A K cells (6,7) have been reported. In these studies anti-CD3 mAb activated cytotoxicity in the absence of A23187 (3) or phorbol ester (6,7). In the present experiments, channel catfish NCC were treated with a monoclonal antibody which reacts with a putative antigen receptor complex involved in target cell binding (8,9). The mAb enhances cytotoxicity both in the presence and absence of A23187. In addition, enhancement of cytotoxicity was accompanied by an increase in membrane antigen receptor expression.

Reagents

Materials and Methods

Monoclonal Antibody Modulation

Fish Ictalurus punctatus (catfish) of both sexes weighing 20-60 g and approximately 6 months to 11/2years of age were obtained from local commercial farms. These outbred catfish were maintained and fed as described previously (10).

Me~um All cell culture was performed in RPMI-1640 adjusted with water to 250 mlOsmol/Kg H20 (Cellgro TM Mediatech, Washington, DC).

Target Cell IM-9 (ATCC CCL 159) is a human lymphoblastoid cell line originally obtained from a multiple myeloma patient. This line was maintained at 37°C in RPMI-1640 plus 10% fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, NY).

PMA (phorbol 12 myristate 13-acetate) and calcium ionophore were obtained from Sigma Chemical Corporation (St. Louis, MO). Stock solutions were made and stored as previously described (I).

Monoclonal Antibodies Monoclonal antibodies 5C6 and 603.4 were originally derived and characterized as reported previously (8). Briefly, these mAbs recognize a m e m b r a n e dimer which is associated with the recognition/binding phase of catfish NCC and rat and human NK Cells (12,13).

Monoclonal antibody (5C6) was produced, c o n c e n t r a t e d by a Millipore Minitan S TM system (Millipore Corp., Bedford, ME) and purified by Con-A sepharose chromatography (Biorad Laboratories, Richmond, CA) as previously d e s c r i b e d (8). To d e m o n s t r a t e mAb modulation, 350 microliters of NCC at a concentration of 4 x 106 cells/mL were a d d e d to 12 x 75 mm glass r o u n d bottom tubes. Cells were centrifuged at 40 x g, the supernatants decanted, and 350 microliters of the mAb were added. Undiluted mAb 5C6 contained 205.8 micrograms/mL. Four million NCC were resuspended in 205.8 micrograms of mAb 5C6 (51.45 ~gm/106 NCC) and incubated for 1 h, after which appropriate E:T ratios were tested for cytotoxicity responses.

Calcium lonophore and Phorbol Ester Treatment Anterior kidney cells from channel catfish were prepared as a source of

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NCC as previously described (1). For mAb inhibition of A23187 activated cells, NCC were first treated with either 10 -4 M or 10 -5 M A23187 for 2 h; washed, and monoclonal antibody was added 1 h prior to addition of the target cells. NCC were not subjected to a preincubation activation period of 4 h.

mAb cotreatment, NCC were treated with 10 -s, 10 -1°, o r 10 -12 M PMA followed by 2 h incubation in the presence of mAb 5C6. NCC were then washed and added to labeled IM-9 target cells (using liP IM-9 cells at each E:T ratio). Cytotoxicity was determined after 4 - 6 h incubation at 25°C/5% CO2.

Monoclonal Antibody Modulation of Receptor Expression

Cytotoxicity

One million NCC were incubated for 18 h with 1 mL of fresh hybridoma tissue culture supernatants. After overnight incubation, cells were washed 2 times and incubated an additional 2 h in medium without mAb. Cytotoxicity and membrane binding with mAb 5C6 were then examined as previously described (8).

Calcium lonophore and mAb Comodulation NCC were first preincubated for 4 h at 25°C/5% CO2 prior to each coincubation experiment. Without this preincubation step comodulation effects could not be produced (data not shown). For c o m o d u l a t i o n , 1 x 106 N C C w e r e treated with 10 -4 M A23187 and Con-A purified 5C6 (undiluted 5C6 contained 73.5 micrograms/mL). This mixture was i n c u b a t e d for 3 h at 25°C/5% CO2. Treated cells were washed, mixed with chromium-51 labeled IM-9 target cells at various E:T ratios, and incubated for 4 - 5 h at 25°C/5% CO 2. The supernatants were harvested and counted for 51Cr as previously described (8).

PMA and mAb Comodulation To determine if modulation of cytotoxicity could be produced by PMA and

One to five × 106 IM-9 target cells were labeled for I h at 37°C with I00 vLCi of 5~Cr-Na2CrO4 (Amersham Corporation, Chicago, IL). Cells were washed three times in medium and diluted to 1 x 105 cells/mL; liP cells in I00 IxL were delivered to 96-well round-bottom tissue culture plates (Linbro Plastics, McLean, VA). Effector cells were then added in 100 v,L v o l u m e s at different effector:target cell ratios. The Linbro plates were centrifuged (50 x g) for 1 min and incubated at 25°C for 4 h. Supernatants were harvested and radioactivity was determined in a Beckman Biogamma II.

Cell Cycle Analysis To determine the effects of phorbol esters and/or mAb on DNA synthesis, cell cyclic analysis was done. Phorbol ester, mAb, or ionophore-treated cells were analyzed following propidium iodide (PI) staining. NCC were lysed by nuclear isolation medium (NIM) containing NP-40 (.75%) and R N A s e (10 vLg/mL) in phosphate-buffered saline (pH 7.4). P r o p i d i u m iodide ( F l u k a Chemical Corp., Ronkonkoma, NY) was resuspended in PBS (1 mg/mL) and diluted 1:20 for staining. D N A staining was accomplished by adding 200 microliters of NIM to 1 drop of NCC. Cells were stained for 15 min with 2 ml of a 5% solution of PI.

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Table 1. Modulation of catfish NCC cytotoxlcity by mAb 5C6. Monoclonal Antibody Concentration =

Percent Specific Release b

Percent Increase in Cytotoxicityc

Control 51.45 25.7 12.85 6.43 3.22 1.61 0.80 0.40

22.5 17.1 18.0 24.7 25.1 22.9 24.5 35.0 34.5

-( - ) 24.0 ( - ) 20.0 9.7 11.5 1.7 8.8 55.5 53.3

• MAb concentration (p,g)/10e cells/mE Cytotoxicity was measured following 5 h cocultivation with IM-9 target cells at a 40:1 E:T ratio. Values are means of triplicate samples. c Percent of Increase in Cytotoxicity = (Test - Control/Control) 100.

Flow Cytometry Flow cytometry was accomplished using an EPICS ® 541 system (Coulter Electronics, EPICS Division, Hialeah, FL). The instrument was standardized using 9.75 p.m diameter Fullbright GR-II fluorospheres (Coulter Electronics), and was operated at constant laser power and PMT settings. A 5W argon-ion laser (Coherent, Palo Alto, CA) was tuned to 488 nm light. Fluorescence (green) was detected as orthogonal light using (in sequence) a 488 dichroic mirror, a 488 LP laser blocker, a 550 SP dichroic mirror, and PMT-1 contained a 525 BP interference filter. Data storage and analysis used an E.A.S.Y. II system (Coulter Electronics, Hialeah, FL). Analysis of propidium iodide staining was accomplished using PMT-2 with the same dichroic and an additional 590 LP absorbance filter.

Results

Ability of Monoclonal Antibody (5C6) to Modulate Cytotoxicity The binding specificities, cytotoxicity i n h i b i t o r y c h a r a c t e r i s t i c s , and the species distribution of 5C6 activity have been described previously elsewhere (1,8,11-13). In the present study, Con A

purified 5C6 was added to NCC to determine the requirements for modulation of cytotoxic activity. Anterior kidney cells were h a r v e s t e d , c o u n t e d , and added directly without preincubation to different concentrations of 5C6. In Table 1, a mAb titration experiment demonstrated that 25.7 and 51.45 micrograms/ mL of 5C6 added to 106 NCC inhibited cytotoxicity. However enhancement of cytotoxicity was produced by 0.40 and 0.80 micrograms. This relationship was consistent, that is, higher concentrations of mAb routinely produced a net inhibitory effect, whereas nanogram concentrations increased cytotoxicity.

Monoclonal Antibody Inhibition of A23187 Activated Cytotoxicity We have previously shown that the calcium ionophore A23187 produces increased cytotoxicity of NCC (1). To determine if this activity was mediated by increased receptor expression, A23187 treated cells were incubated with two different mAbs, both of which previously have been shown to bind NCC (8). NCC were first treated with 10 -4 M and 10 -5 M A23187/106 cells for 2 h, cells were washed, and either 5C6 or 6D3.4 was added for 1 h prior to addition of the IM-9 target cells (Fig. 1). Each

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Monoclonal Antibody Dilution Figure 1. Effects of mAb treatment following activation by A23187 on catfish NCC. NCC were first incubated with 10 -4 M (A) or 10 -5 M (B) A23187 for 2 h at 25°C/5% CO 2 followed by treatment with different concentrations of mAb for 1 h. IM-9 target cells were added and supernatants harvested after 5 h cocuitivation. Data points are mean values of triplicate samples.

mAb was added at 50 micrograms/106 cells for the undiluted concentration. NCC were not subjected to a preincubation step. Both mAbs significantly inhibited the increased cytotoxicity seen with A23187-treated cells (Fig. 1).

Effects of Cotreatment of NCC with A23187 and mAbs Using similar concentrations of mAb 5C6 as those shown in Fig. 1, a different treatment regimen was used to determine if differences in cytotoxicity could be o b t a i n e d by c o t r e a t m e n t w i t h A23187. When 10 -4 M A23187 was coincubated with different concentrations of 5C6 for 3 h (Fig. 2), significantly increased cytotoxicity of IM-9 target cells was seen. This comodulatory effect was

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Figure 2. Modulation of NCC activity by cotreatment with A23187 and mAb 5C6. NCC were preincubated for 4 h followed by addition of either mAb 5C6 (0) only, 10 -4 M A23187 plus 5C6 (X), or 10 -a M A23187 plus 5C6 (©). Medium points (Y-axis) do not contain mAb and percent increases were determined by comparisons of treatments with these values. Cotreatment was for 3 h followed by addition of IM-9 target cells at a 40:1 E:T ratio and are representative of two experiments. Data points are means of triplicate samples.

produced only after first preincubating the NCC for 4 h. The percent increase values were obtained by comparisons with medium (Y-axis) control values, that is, the medium controls did not contain mAb.

Effects of Cotreatment of NCC with PMA and rnAb Similar effects, but with lower differences between control and cotreatment cytotoxicity responses, were observed following PMA and 5C6 modulation (Table 2). NCC were first treated with PMA for I h, followed by 2 h 5C6 treatment. Following a 4-h NCC preincubation (and sequential treatments), only a combined treatment with 18.37 micro-

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Table 2. Effects o f c o m b i n e d t r e a t m e n t s with PMA and monoclonal a n t i b o d y (5C6) on NCC activity. Monoclonal Antibody Concentration =

Percent Specific Releaseb

10 -= M ~

none 147.00 73.50 36.75 18.37 9.19 4.59 2.30 1.15 NMS =

31.9 35.2 34.0 37.3 36.2 38.2 40.8 38.0 35.4 26.2

-30.9 16.4 22.2 22.1 23.0 32.3 32.6 27.6 --

Percent Inhibition = 1 0 - lo M

10-12 M

-4.2 6.3 5.1 10.6 0.5 1.7 4.2 29.7 --

(-) (-) (-) (-) (-)

6.5 ( - ) 7.1 9.1 ( - ) 4.9 ( - ) 2.5 6.9 4.2 ( - ) 5.3

• MAb 5C6 concentration/10a cells/mL; NMS = normal mouse serum. Cytotoxicity of NCC in the presence of mAIDs only. = Compared to percent specific release values of mAb treatments. d Expressed as Molar concentrations of PMA (e.g., 10 -a M contains 1.54 x 10 -s micrograms of PMA/10e cells). • 1:1000 dilution.

grams 5C6 and 10-lo M PMA produced a significant (10.6%) increase in cytotoxicity (Table 2).

Modulation of Receptor Expression by mAb Increases in both cytotoxicity and membrane 5C6 + receptor expression were produced by 18-h pretreatment with mAb (Table 3). Increases were produced at two different concentrations of mAb, that is, undiluted and 1:16. Two Table 3. C o r r e l a t i o n o f NCC c y t o t o x l c i t y with receptor e x p r e s s i o n f o l l o w i n g 18 h pretreatment with m A b 5C6. Treatment b (Dilution of 5C6) 0 1:2 1:4 1:8

1:16 1:32

Percent Increase = Cytotoxicity = Binding d 145.4 8.4 0 0 42.8 0

67.3 94.7 42.9 21.9 49.8 0

• Values expressed are minus normal mouse serum controls. Percent increase is calculated as given in Table 1 footnote. b Medium control had 6.09 percent specific release; mAb was fresh tissue culture supernatant. ©40:1 E:T ratio; 4 h cytotoxicity assay; IM-9 target cells. = Medium control had 26.4% specific membrane binding of 5C6.

populations of mAb binding cells were further indicated by flow cytometric analysis of the activated NCC. In Fig. 3, two mAb binding cell populations were found in cells stained with 5C6 and conjugate. These activated cells had approximately a 50% increase in 5C6 ÷ expression compared to nonactivated cells.

Modulation of Receptor Expression by A23187 NCC were preincubated for 4 h and then treated for 1 h with media (control) or PMA, and then sequentially treated for 3 h with A23187. Cells were analyzed for expression of membrane receptor by staining with mAb 5C6 (Table 4). A23187 produced a greater than 235% increase in 5C6 ÷ expression. PDD had no effect either alone or in combination with A23187. Combined treatment with PMA and A23187 produced a slight comodulation effect.

Cell Cycle Analysis Cells prepared and analyzed in Table 4 were examined by flow cytometry for

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301

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Relative Fluorescence (Log 1o) Figurs 3. Flow cytometric detection of 5C6 + membrane receptor on catfish NCC. NCC were incubated for 18 h at 25°C in the presence of a 1:16 dilution of mAb 5C6. Cells were washed two times. incubated for 2 additional h without antibody and either the FITC conjugate (A) or 5C6 plus the conjugate was added (B).

PI staining as indicated in Table 5. Fifteen hours following treatment, compared to nontreated controls, A23187 caused a shift into the S-phase from

5.86% (control) to 46.08%. Phorbol esters PDD and PMA individually had no effects on DNA synthesis, however when NCC were cotreated with A23187,

Table 4. Effects of phorbol esters and/or calcium Ionophore on monoclonal antibody binding to catfish NCC. NCC were treated with PMA, PDD, or A23167 either singly or in combinations end 5C6 binding was determined by flow cytometry 15 h after treatment. Treatment

Percent Specific Binding

Percent Increase in Binding ©

Medium Control PDD (10 -a M) PMA (10 -8 M) A23187 (10 -4 M) PMA + A23187 PDD + A23187

14.67 14.42 19.32 49.19 60.21 50.63

-0 31.7= 235.3= 310.4 (22.4)b 245.1 (2.9) b

• Compared to medium control. b Compared to A23187 (10-4 M) treatment. Test - Medium c 100 Medium

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Table 5. Effects of phorbol eaters and/or calcium Ionophore on DNA synthesis by catfish NCC. NCC were treated and stained with PI after 15 h Incubation as Indicated In Table 4. Analysis was by flow cytomatry. Treatment

Go/G1

Medium control PDD (10 -a M) PMA (10 -a M) A23187 (10 -4 M) PMA + A23187 PDD + A,?.3187

93.11 94.85 88.75 50.41 67.56 58.05

a decrease in the S-phase shift was observed. A similar experiment was conducted to examine the effects of mAb modulation on DNA synthesis. In Table 6, cells were treated for 18 h with 5C6 (same experiment as shown in Table 3) and cell cycle analysis was conducted by PI staining. An S-phase shift was observed in the cell population treated with undiluted 5C6.

Discussion In the present study an attempt was made to determine select requirements for the activation of teleost NCC. These data indicate that (putative) receptor cross-linkage by mAbs and increased intracellular calcium levels (either combined with mAb treatment or singly) significantly affect signal transduction for cytotoxicity. Previously a monoclonal antibody with specificity against the epsilon chain of the murine CD3 complex has been shown to modulate mouse L A K cell lysis of P815 and H F L / b targets (14). Kinetics for activation re-

Percent in Interval S 5.86 3.98 10.18 46.08 26.37 36.39

G2M 1.03 1.16 1.07 3.52 6.08 5.56

quired 1-day p r e t r e a t m e n t and, depending on the target cells used, augmented cytotoxicity was either dependent (HFL/b) or not (P815) on continued presence of mAb during the killing assay (14). However, mAb modulation of catfish NCC cytotoxicity, as reported here, required as little as 1 h incubation with 5C6 (Table 1) with maximum modulation occurring following 18 h-treatment (Table 3). This is a much higher activation signal compared to that produced by anti-CD3 treatment of HFL/b cells (14). The murine CD3 complex transducing signal has also been shown previously to be capable of initiating cellular DNA synthesis (15) as shown by the induction of a proliferative response. A similar function for the 5C6 + receptor with catfish cells has not yet been demonstrated. Others working with human cells have shown that monoclonal binding of membrane receptors mimic receptormediated cell activation by causing an increase in cytoplasmic free Ca + + (15). Addition of A23187 in the absence of mAb (15) produced an increased pattern of phosphoprotein synthesis, suggesting

Table 6. Effects of 5C6 binding on DNA synthesis by catfish NCC. Treatment"

Go/G1

Percent in Interval S

G2/M

Medium 5C6 (0) 5C6 (1:2) 5C6 (1:4)

81.37 56.33 78.48 78.13

10.28 41.23 15.80 18.63

8.34 2.44 5.72 3.24

• Cells obtained from the experiment as described in Table 3.

Signal transduction in NCC

similar pathways of PKC mobilization. The present work suggests that mAb (5C6) binding to NCC.likely induces increased levels of c y t o s o l free Ca + + which in turn could rapidly (1 h) provide a cell activation or second messenger pathway leading to increased cytolysis. In this same regard, we have previously shown that teleost cytotoxic cells (NCC) may utilize Ca + + (independent of a cosignal provided by diacyglyceride) to activate protein kinases (1). To determine if changes in extracellular Ca + + affected mAb-associated NCC activation, experiments were next conducted to determine if an A23187mediated influx in Ca + ÷ combined with mAb binding could activate cytotoxicity. Previously studies have shown that for murine B cells, cross-linkage of surface immunoglobulin causes both a Ca + ÷ influx and increased Ca + ÷ release from endoplasmic reticulum stores (4). A similar association between mAb binding by anti-CD2 or anti-CD3 and A23187 treatment was observed for killing of nonM H C related targets by alloreactive human T cells (3). In the present study using catfish cells mAb 5C6 may recognize a receptor which provides an activation signal, which in turn may depend on levels of extracellular calcium and calcium influx to fully activate the lytic apparatus. When NCC were cotreated with an o p t i m u m c o n c e n t r a t i o n of A23187 (for activation) and otherwise inhibiting concentrations of 5C6 (Fig. 2), significant increases in cytotoxicity (compared to A23187 alone) were observed. These results can be interpreted as indicating that A23187 facilitated transport of Ca ÷+ (influx) apparently reversed the mAb-induced inhibitory signal which would have otherwise produced a net inhibition of cytotoxicity. It thus appears that the initial 5C6 receptor signal, produced by subsaturating mAb concentrations, may either up or down regulate cytotoxicity depending on concentrations of Ca + +. Evidence for this

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can also be d e d u c e d from the data shown in Fig. 2, where a suboptimal A23187 concentration had no comodulating effect on cytotoxicity. We (1) and others (16) have previously shown that certain phorbol esters (PMA) inhibit NCC and CTL activities, respectively. However, phorbol ester plus calcium ionophore treatment (1,17) significantly augments selected cell activation processes. In the present study, in an effort to identify signals required or a s s o c i a t e d with r e c e p t o r b i n d i n g b y N C C , we d e t e r m i n e d whether PMA (at nonsuppressive levels) and mAb 5C6 could activate cytotoxicity. Previously, it was shown (2) that pretreatment of mammalian NK-like cells with anti-CD3 and PMA either had no effect or was inhibitory. In Table 2, sequential treatment of catfish NCC with 10 -l° M PMA followed by treatment with a p p r o x i m a t e l y 18 micrograms/106 cells of 5C6 produced only a slight augmentation of cytotoxicity. Additional evidence that the 5C6 + binding structure on NCC membranes is a signal t r a n s d u c i n g m o l e c u l e was strongly suggested by e x p e r i m e n t s which correlated mAb binding with increased cytotoxicity (Table 3) and increased 5C6 + membrane expression (Table 3 and Fig. 3). However, compared to the A23187-induced 5C6 ÷ expression (Table 4), it appears that calcium may have more direct, longer lasting, and higher amplification capabilities than the perhaps more singular activation pathway provided by mAb binding. It is not clear if receptor activation in fish NCC produces a transient increase in cytosolic calcium. Perhaps alternate or non-calcium-associated activation pathways exist in fish cells. Furthermore, the kinetics of calcium transport or its release from intracellular stores may be different in teleost cells following receptor activation. These questions have not yet been answered. Further support that the 5C6 ÷ recog-

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n i z e d s t r u c t u r e is a s s o c i a t e d d i r e c t l y with N C C activation was provided by a n a l y s i s o f PI s t a i n i n g o f t r e a t e d cells. B o t h A23187 a n d m A b 5C6 i n d u c e sign i f i c a n t S - p h a s e shifts (Tables 5 a n d 6, r e s p e c t i v e l y ) . C o t r e a t m e n t o f N C C with A 2 3 1 8 7 a n d p h o r b o l e s t e r s ( T a b l e 5) p r o d u c e d a n e t i n h i b i t o r y effect o n this shift. T h e s e e x p e r i m e n t s h a v e s h o w n that

the 5C6 r e c e p t o r b i n d i n g m A b 5C6 provides a n a c t i v a t i o n signal for N C C . This a c t i v a t i o n is m a n i f e s t b y (i) significant i n c r e a s e s in c y t o t o x i c i t y , (ii) b y the exp r e s s i o n o f i n c r e a s e d 5C6 ÷ r e c e p t o r s , a n d (iii) b y i n c r e a s e s in D N A s y n t h e s i s . A d e q u a t e t r a n s d u c t i o n o f this signal is l i k e l y to b e c l o s e l y a s s o c i a t e d w i t h levels of increased intracellular free C a + +.

References 1. Evans, D. L.; Harris, D. T.; Jaso-Friedmann, L. Effects of phorbol esters and calcium ionophore on nonspecific cytotoxic cells. Dev. Comp. Immun. (in press). 2. Dobbs, J. E; Katz, D. R. Human T-cell activation: comparative studies on the role of different phorboi esters. Immunology 63:133137; 1988. 3. Inverardi, L.; Geller, R. L.; Gleason, J. T.; Gromo, G. Anti-CD2 monoclonal antibodies and calcium ionophore A23187 modulate lytic activity in CD4 ÷ and CD8 ÷ alloreactive clones. J. Immunol. 140:2876-2879; 1988. 4. Ransom, J. T.; Chen, M.; Sandoval, V. M.; Pasternak, J. A.; Diguisto, D.; Cambier, J. C. Increased plasma membrane permeability to Ca + + in anti-Ig stimulated B-lymphocytes is dependent on activation of phosphoinositide hydrolysis. J. Immunol. 140:3150-3155; 1988. 5. Thiele, D. L.; Patei, S. S.; Lipsky, E E. AntiCD3 and phorboi myristate acetate regulation of MHC unrestricted T cell cytotoxicity. Lack of a requirement for CD3/T cell receptor complex expression during tumor cell lysis. J. Immunol. 140:3253-3260; 1988. 6. Ting, C-C.; Hargrove, M. E.; Yun, Y. S. Augmentation by anti-T3 antibody of the lymphokine-activated killer cell-mediated cytotoxicity. J. Immunol. 141:741-748; 1988. 7. Yun, Y-S.; Hargrove, M. E.; Ting, C-C. Heterogeneity of long-term cultured activated killer cells induced by anti-T3 antibody. J. Immunol. 141:1390-1397; 1988. 8. Evans, D. L.; Smith, E. E.; Jaso-Friedmann, L.; St. John, A.; Koren, H. S.; Harris, D. T. Identification of a putative antigen receptor on fish nonspecific cytotoxic cells with monoclonal antibodies. J. Immunol. 141:324-332; 1988. 9. Jaso-Friedmann, L.; Evans, D. L.; Grant, C. C.; St. John, A.; Harris, D. T.; Koren, H. S. Characterization by monoclonal antibodies of a target cell antigen complex recog-

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