CELLULAR
IMMUNOLOGY
139,208-2 17 (1992)
Human Cytotoxic T Lymphocytes and Activated Peripheral Blood Lymphocytes Enhance Cytochalasin B-Induced DNA Fragmentation of Indicator Bystander Cells MICHAEL
A.
KOLBER
Departments of Medicine, and Microbiology and Immunology, The University of Miami School of Medicine, Miami, Florida 33101 Received June 16, 1991; accepted August 14, 1991 The cytochalasins are known to have multiple effects on cellular function. Not only do they induce secretionfrom granule compartments but they can induce DNA fragmentation in numerous cells. Evidence is presented which shows that treatment of human cytotoxic T lymphocytes and activated peripheral blood lymphocytes with cytochalasin B induces release of a factor capable of enhancing DNA fragmentation in cytochalasin-susceptible target cells. This activity can be transferred in the supematants of cytochalasin B-activated CTL. o 1992 Academic PWS, h.
INTRODUCTION Cytochalasin B is a fungal metabolite. As with other cytochalasins, cytochalasin B affects dissolution of the microfilaments (1). The possibility that this agent has both chemotherapeutic potential and immune enhancing properties can be raised. Recently it has been shown that the cytochalasins are capable of inducing DNA fragmentation in susceptiblecell lines (2). Whether it is by this mechanism that cytochalasin B prolongs survival in animals hosting various subcutaneously injected tumors (3) is not known. Cytochalasin B can enhance lysis of target cells by cytotoxic T lymphocytes (CTL) when bridging polystyrene beads coated with antibodies directed against the T cell receptor and a target cell surface moiety are used to redirect the effector cell and stabilize the cellbeadzell complex (4). Since redirected lysis is thought to present an immunotherapeutic option (5, 6) against various cancers the possibility that cytochalasin B could enhance this processand provide an added chemotherapeutic thrust warrants further evaluation of its immunologic actions. Earlier work has shown that the cytochalasins can enhance secretion from various cell lines (7,8). In neutrophils, pretreatment with cytochalasin B can delineate various granule compartments by their distinct secretory response to rises in intracellular calcium (7). Since cytochalasins can differentially affect the ability of some cells to secretefactors from different compartments we postulated that such a discriminating effect on factors releasedby cytotoxic T lymphocytes (CTL) might be possible. Since CTLinduced target cell DNA fragmentation and lysis are potentially uncoupled events we evaluated whether cytochalasin B treatment would reflect differences in CTL-mediated target cell DNA fragmentation and lysis. 208 0008~8749192$3.00 Copy-i& Q I992 by Academic Fres, Inc. All rights of repmduction in any form reserved
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We demonstrate that human CTL and activated peripheral blood lymphocytes releasea factor capable of enhancing cytochalasin B-induced DNA fragmentation. This factor is not in the same compartment as BLT-esterase. The activity can be transferred in the supernatants from cytochalasin B-treated CTL. We discuss the role that such a soluble factor could play in light of the possible chemotherapeutic potential of cytochalasin B. MATERIALS AND METHODS Materials. Cytochalasin B was obtained from Sigma Chemical Co. (St. Louis, MO) and a stock solution prepared of 2 mg/ml in ethanol. Polystyrene beadswere obtained from Polysciences (Warrington, PA). Anti-CD3 (OKT3) was generated from hybridomas obtained from the American Type Culture Collection (Rockville, MD). Rabbit anti-DNP was obtained as a gift from Dr. David Segal(National Institutes of Health, Bethesda, MD). Hepes and trinitrobenzene sulfonic acid (TNBS) were obtained from Sigma Chemical Co. (St. Louis, MO). Secretion assaysand DNA fragmentation assayswere performed in medium consisting of Hanks’ balanced salt solution containing 0.02 M Hepes and 3% fetal calf serum (FCS) adjusted to a final pH of 7.4 with NaOH (referred to as BSSH). All assays were performed at 37°C in room air. All media were adjusted to pH 7.4 with NaOH unless otherwise specified. Adsorption of antibody to polystyrene beads. Antibody was adsorbed to 3-pm diameter beads as previously described (9). Briefly, lo8 beadswere incubated with 0.025 mg/ml of each antibody in 0.1 M NaHC03 at room temperature overnight, followed by washing and incubation in 1.Omg/ml BSA (pH 7.4) at 37°C for 0.5 hr. Cells. The human CTL lines (MC 1 and Q302) were generatedaspreviously described (9). Both lines use as stimulators and recognize specifically the allogeneic human EBV transformed line Z2B. The human CTL line, Q302, was a generous gift of Dr. R. Gress and P. Lucas (N.I.H.). The human CDCpositive CTL clone (10) was a generous gift from Dr. S. Shaw (N.I.H., Bethesda,MD). The type II CD4-positive murine helper cell clone (1 l), 8-5-5, was a generous gift of Dr. M. Taplits (F.D.A., Bethesda, MD). The murine CTL line, called DC 1, is a C57B l/6 anti-DBA/2 line generated by establishing a l-week primary in vivo and used after three in vitro passageswith weekly stimulations of the effector cells with irradiated splenic cells from DBA/2 mice in medium consisting of RPM1 (GIBCO, Grand Island, NY) containing 10% FCS and 6 X 10m5M 2-mercaptoethanol (Bio-Rad, Richmond, CA) plus L-glutamine, penicillin, and streptomycin. The cell lines Z2B (human EBV transformed line), BW5 147, EL4, WEHI(ATCC), and RBL (rat basophilic leukemia) were propagated by in vitro passage.Target cells were TNP (trinitrophenol) modified by incubation with 1 mg/ ml TNBS in PBS, for 10 min at 37°C. Peripheral blood was obtained from healthy volunteers by antecubital venipuncture. Lymphocytes were harvested by Ficoll separation followed by nylon wool column passageand collection. Lymphocytes were activated by a modification of a previously described procedure (12). Nickel (16 mM) wells were first coated with 0.5 ml 0.1 M NaHC03 (pH 8.5) of 10 pg/ml of OKT3 at room temperature overnight, followed by washing and incubation in 1.0 mg/ml BSA (pH 7.4) at 37°C for 0.5 hr. Lymphocytes were plated at 2 X IO6cells/well in human CTL medium (RPM1 1640 (GIBCO, Grand Island, NY) plus 10% FCS (Sigma Chemical Co., St. Louis, MO), 3% T cell growth
210
MICHAEL
A. KOLBER
factor (Cellular Products Inc., Buffalo, NY), 30 U/ml rIL-2 (Cetus Corp., Emeryville, CA), penicillin, and streptomycin) for 3 days in a 37°C 5% COZ incubator. After the initial activation period the cells were harvested, washed, and replated in human CTL medium in wells not coated with antibody. After 3 days in a 37°C 5% CO* incubator the cells were harvested and used in the enhancement experiments. DNA fragmentation assayed by labeled thymidine release. Target cell DNA fragmentation was assayed by first labeling target cells in their growth phase with [3H]thymidine ( 100 PCi) for approximately 18 hr in standard tissue culture medium. After the labeling period the target cells were washed and resuspended in unlabeled tissue culture medium for l-2 hr. After the resting period the target cells (50 ~1)were washed and added to the microtiter plate wells. Labeled target cells were incubated with effector cells and cytochalasin B (final well volume was 200 ~1) as determined appropriate for each experiment. Plates containing targets, effecters, and cytochalasin B were centrifuged at 200g for 3 min and then incubated for 4 hr at 37°C in a room air oven. After an appropriate incubation time, as dictated by the experiment, the extent of thymidine releasedfrom the experimental and spontaneous releasesamples was determined by adding Triton X-100 to each sample (final concentration 0.04%) mixing, centrifuging the plate for 5 min at 7008, and recovering 100 ~1of supernatant to count in a &scintillation counter. The total [3H]thymidine releasewas determined by adding 150 ~1of 0.5% SDS aqueous solution with 3 mM EDTA, 10 mJ4 Tris, pH adjusted to 7.4, to 50 ~1 of target cells, mixed, and 200 ~1 harvested and counted. It was found that the total determined by this technique was identical to the total radioactivity obtained when the triton releasable radioactivity in the supernatant was added to the radioactivity of the remnant pellet and supernatant solubilized with the SDS solution. The corrected percentage DNA fragmentation was determined by the equation: Corrected % DNA Fragmentation = 100 X (Cexp- Cmed)/(Cdet - Cm&, where C representsthe amount of [H3]thymidine released(in cpm) in the triton soluble supernatants for the samples specified by the subscripts, exp (experimental), med (targets incubated in medium alone), and det (targets solubilized with detergent). Spontaneous releasevalues for all labeled thymidine experiments were lessthan 10%unless otherwise indicated. Supernatant experiment. MC1 cells were suspended in BSSH (0.75 X 106/ml) at different concentrations of cytochalasin B and aliquoted (200 ~1) into wells of a Ubottom microtiter plate (six replicates per cytochalasin concentration). Control wells that contained only BSSH and cytochalasin B were prepared on the same plate. The plate was incubated for 3 hr at 37°C in a room air oven prior to centrifuging at 700g for 5 min. Supernatants from wells on the effector plate (150 ~1) were transferred to a target cell plate that was prepared previously. The target cell plate was set up in a configuration identical to that of the effector cell plate. Half the wells on the target cell plate (triplicates for each cytochalasin B concentration) contained TNBS-modified EL4 cells (50 ~1 containing 15K cells), and the remaining wells contained TNBSmodified EL4 cells and polystyrene beads coated with both OKT3 and anti-DNP (50 ~1containing 15K cells and IO6beads). The supernatants from the effector plate were transferred to the target plate and the target plate was centrifuged at 200g for 3 min and incubated for 3 hr at 37°C in a room air oven. After the final incubation period the releasable [3H]thymidine was determined as described above. The cytochalasin
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concentration given for each experiment is the cytochalasin concentration in the target plate wells. Statistics. Standard deviations are given for all points except when they are smaller than the symbol. Significance was calculated using the unpaired t test (Student’s). RESULTS Cytotoxic T lymphocytes enhance cytochalasin B-induced DNA fragmentation. Previously we showed that cytochalasin B had an effect on the secretion of lytic factors from CTL (4). In that work we demonstrated that CTL release both BLT-esterase, and lytic factors when the T cell receptor is cross-linked by beads coated with antiCD3. The secretion is biphasic in its concentration dependence on cytochalasin. We wished to evaluate whether DNA fragmentation induced by CTL had a similar biphasic dependence on cytochalasin concentration. In Fig. 1 is shown the corrected percent DNA fragmentation as measured by the releaseof tritated thymidine from TNP:EL4 cells as a function of cytochalasin B concentration. In this experiment radiolabeled TNP:EL4 were incubated with either human CTL (MC 1 cells) and polystyrene beads coated with OKT3 (anti-CD3) and anti-DNP (top curve), MCI cells and no beads (middle curve), or media alone (lower curve). The top curve demonstrates that when polystyrene beads, coated with the antibodies OKT3 (anti-CD3) and anti-DNP, are incubated with effector and target cells the DNA fragmentation is enhanced above that found for target cells alone. When the human CTL, MC 1, are incubated with the TNP:EL4 cells without beads the target cell DNA fragmentation induced at low cytochalasin B concentrations is the sameasif the effector cells were not present. However at higher cytochalasin B concentrations the target cell DNA fragmentation in the presence of MC1 cells approaches the DNA fragmentation found for effector cells
25
15
5
0
1
2
CYTOCHALASIN
3
4
5
6
B (pglml)
FIG. 1. Effect of cytochalasin B on release [3H]thymidine from TNP:EL4. The corrected percent DNA fragmentation is plotted as a function of cytochalasin B concentration. [‘Hlthymidine-labeled TNP:EL4 cells (1SK/well) were used as targets in the absence (m) or presence of MC1 cells (lOOK/well) with (0) or without (0) antibody-coated beads. The incubation time, at 37°C in room air, was 4 hr.
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MICHAEL A. KOLBER
incubated with target cells, and beads at higher cytochalasin B concentrations. The increase in DNA fragmentation as a function of cytochalasin B concentration for TNPEL4 cells alone has been described previously (2). The data from Fig. 1 show that the enhancement in DNA fragmentation in the presence of MC 1 CTL is not dependent on the presenceof beads at high cytochalasin B concentrations. A number of experiments were performed to demonstrate that CTL are enhancing target cell DNA fragmentation in the presence of cytochalasin. In Fig. 2 the corrected percent DNA fragmentation is shown as a function of effector to target cell ratio. When Z2B (cells with no effector potential) are incubated with radiolabeled EL4 cells there is no enhancement of DNA fragmentation above that found for fragmentation produced by cytochalasin B alone. Whereas, the DNA fragmentation is enhanced monotonically in the presenceof increasing numbers of MC 1 CTL. Further confirmation that MC 1 cells are releasinga factor capable of enhancing the cytochalasin B-induced DNA fragmentation is shown in Fig. 3. In that figure the corrected percent DNA fragmentation of EL4 cells is plotted, at a fixed cytochalasin B concentration, as a function of time. After a 3-hr incubation period MC1 cells induce a significant enhancement in target cell DNA fragmentation compared to the enhancement seen when target cells are incubated with RBL cells, cells with no effector potential. This enhancement is increased after 4 hr. Fluorescent single cell analysis of the MCI cell line revealed that they were approximately 96% CD8-positive and contained approximately 4% CD4-positive cells (data not shown). In order to evaluate which population of cells was responsible for the enhancement in target cell DNA fragmentation we evaluated a number of different CTL as shown in Table 1. In Table 1 the corrected percent DNA fragmentation is
5o’
0
0
2
4
6
8
10
EFFECTOFUARGET FIG. 2. DNA fragmentation as a function of the effecter/target cell ratio. The corrected percent DNA fragmentation, as indicated by the releaseof [3H]thymidine, from EL4 cells ( 15K/well) is plotted as a function of the effecter/target cell ratio. The effector cells were MC1 cells (0) and Z2B (m). Cytochalasin B at 2 Irg/ ml was usedin the experiment, and the values for the corrected percent DNA fragmentation were determined using the spontaneousthymidine releasefrom target cells without cytochalasin. The incubation time was 4 hr at 37°C in room air.
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5 loo2 3
80-
100 TIME (min)
200
FIG. 3. DNA fragmentation oftarget cells asa function of time. The corrected percent DNA fragmentation is plotted as a function of incubation time. [‘H]Thymidine labeled EL4 cells (I SK/well) were incubated in 2 pg/mI with either MCI cells (100 K/well; W)or RBL cells (lOOK/well; q ) at 37°C for the times shown.
shown for an effector cell population (column 1) under various conditions: effector cells, target cells, and cytochalasin B are present (column 2), only effector cells are present with target cells (column 3), or only cytochalasin B is present with target cells (column 4). In all casescytochalasin B alone produces approximately 15%DNA fragmentation of target cells (column 4), whereas effector cells with target cells alone produce no or little DNA fragmentation (column 3). When MC 1 cells are treated with anti-CD4 and complement the enhancement of target cell DNA fragmentation is unchanged from that produced by MC1 cells treated with complement alone. Another human CTL line, 4302, is also capable of enhancing cytochalasin B-induced DNA fragmentation. 8.9 is a human CD4-positive CTL clone that does not enhance DNA
TABLE I Human CTL Enhance Cytochalasin B-Induced DNA Fragmentation Corrected % DNA fragmentationb ElktoP
EFF + CYT@
EFF - CYTO
-EFF + CYTO
MC1 +C MC1 + C’ + anti-CD4 Q302 8.9 8-S-5 DC1
31.7 zk 2.2 33.3 iz 2.1 36.2 + 0.4 8.6 It 0.8 1I .4 + 2.2 13.5 f 0.3
0.0 * 0.4 0.7 It 0.2 0.5 -+ 1.1 -1.6 T 0.1 -2.8 +- 1.7 5.1 * 0.4
16.2 + 0.8 16.2 _’ 0.8 17.0 -t 2.2 13.1 2 0.6 14.3 t 2.1 13.6 + 0.7
a Effector cells were plated at 150 K/well with labeled EL4 cells (lOK/well). 6 Cells were incubated for 4 hr at 37°C. ’ Cytochalasin B, 5 FgjmI.
214
MICHAEL A. KOLBER TABLE 2 Activated Peripheral Blood Lymphocytes Releasea Factor Capable of Enhancing Cytochalasin B-Induced DNA Fragmentation Corrected % DNA fmgmentationb @EM) PT No.”
+PBL + CYTO’
+PBL - CYTO
-PBL + CYTO
1 2 3 4 5
31.8 (2.4) 15.5 (1.3) 33.4 (2.6) 38.8 (3.4) 36.8 (3.0)
-1.4 (1.4) -1.1 (1.6) -3.3 (0.6) 1.1 (0.4) -0.9 (6.4)
22.9 (2.5) 22.9 (2.5) 21.8 (2.0) 21.8 (2.0) 13.8 (2.3)
’ PBL were plated at 250K/well with labeled EL4 cells (lSK/well). bCells were incubated for 4 hr at 37°C. ’ Cytochalasin B, 5 &ml.
fragmentation of EL4 cells. A type II CDCpositive murine helper cell clone, S-5-5, and a murine CTL line, DC1 (C57B1/6 anti-DBA/Z), are not able to enhance cytochalasin B-induced DNA fragmentation. With the increasing interest in using activated lymphocytes in various immunologic therapies (e.g., 5, 12) we wanted to evaluate whether human peripheral blood T lymphocytes (PBL) activated via the cross-linked CD3 receptor could enhance cytochalasin B-induced DNA fragmentation. Blood obtained from five healthy volunteers was activated as described under Materials and Methods. Table 2 demonstrates that the activated PBL were capable of enhancing cytochalasin B induced DNA fragmentation in all but one case.PBL did not induce DNA fragmentation of the target cells in the absenceof cytochalasin (Table 2). When PBL were cultured in the human CTL medium (containing TCGF and IL2), without receptor cross-linking, they did not enhance the cytochalasin B-induced DNA fragmentation (data not shown). Human CTL enhance cytochalasin B-induced DNA fragmentation in EL4 cells. Whether this effect occurs with any target cell is not clear. In Table 3 we examined four different target cells for cytochalasin B-induced DNA fragmentation as well as
TABLE 3 Cytochalasin B-Induced DNA Fragmentation is Dependent on the Target Cell Corrected % DNA Fragmentationb Target” I. 2. 3. 4.
EL4 BW5147 RBL WEHI
+EFF+CYT’ 27.6 k 4.2 32.1 k 1.1 3.0 + 3.5 -0.1 zk 0.6
a 150K MC 1 cells/well and 15K target cells/well. bAfter 4-hr incubation at 37”. ’ Cytochalasin B, 1.7 &ml.
+EFF/-CYT 7.2 + -2.5 f 1.5 f 0.3 +
3.3 2.3 1.0 0.5
-EFF/+CYT 10.7 f 2.6 22.5 f 1.5 2.3 rfr 3.4 -0.2 + 0.8
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enhancement of DNA fragmentation with MC1 effector cells. As seen in Table 3 cytochalasin B can fragment DNA of EL4 and BW5 147, whereas cytochalasin B does not fragment DNA of the RBL and WEHI cells. In the limited number of targets used in this study whenever cytochalasin B fragmented DNA the MC1 cells enhanced the fragmentation. MC I cells did not enhance or induce DNA fragmentation in the RBL cells or the WEHI cells. Supernatantsfrom CTL treated with cytochalasin B can induce DNA fragmentation. The data presented so far suggestthat MC 1 cells releasea factor capable of enhancing cytochalasin B-induced target cell DNA fragmentation. Since there is no specific recognition between the MC 1 cells and the EL4 target cells this enhancement is a bystander effect. Such an effect indicates that a factor sufficiently stable to enhance DNA fragmentation in a bystander fashion might be found in the supematants from effector cells stimulated with cytochalasin B. To evaluate this possibility MC1 cells were incubated in different concentrations of cytochalasin B according to the protocol schematically illustrated in Fig. 4A. After a 3-hr incubation period at 37°C the supematants were harvested from the wells f human CTL and added to TNBS-modified EL4 cells with or without beads. After another 3-hr incubation period the DNA fragmentation induced in the target cells was assayed.If MC 1 cells are being transferred inadvertently then the beads will redirect lysis and an enhancement in DNA fragmentation will occur compared to the no bead control (seeFig. 1). In Fig. 4B is shown the corrected percent DNA fragmentation for three concentrations of cytochalasin B. Target cells incubated in the supematants from the MC1 cells showed a significant and dosedependent enhancement in DNA fragmentation above the DNA fragmentation induced by the cytochalasin alone. Polystyrene beads, coated with antibodies (OKT3 and anti-DNP), in the final incubation period did not enhance DNA fragmentation at any concentration when compared to the companion no bead controls. DISCUSSION In this paper we have shown that human cytotoxic T lymphocytes releasea substance capable of enhancing cytochalasin B-induced DNA fragmentation in a bystander fashion. Previously we have demonstrated that upon T cell receptor activation cytochalasin B will also enhance secretion of the granule enzyme BLT-esteraseand antibody-coated bead redirected lysis of target cells (4). However, as shown in that work CD3 receptor activation was necessary, and lysis and secretion were inhibited at high (5 pg/ml) cytochalasin concentrations. Figure 1 demonstrates that the DNA fragmentation of EL4 target cells in the presence of cytochalasin B and MC1 cells is not biphasic. The enhancement in DNA fragmentation occurring at high concentrations of cytochalasin B is the same whether beads that redirect lysis are present or not. The enhancement in cytochalasin B-induced DNA fragmentation by MCI cells is dependent on the number of effector cells present (Fig. 2) as well as the incubation period (Fig. 3). Receptor activation by cytochalasin B is unlikely since the secretion of BLT-esterase is diminished at concentrations of cytochalasin that enhance a DNA fragmentation (4). Support that the MC1 cells are releasing a factor contributing to the cytochalasin B-induced DNA fragmentation comes from the supematant experiment (Fig. 4). In that experiment supematants from MC1 cells treated with cytochalasin B enhance DNA fragmentation of target cells above that found when target cells are treated with cytochalasin alone.
216
MICHAEL A. KOLBER
A EFFECTOR CELL INCUBATION
TRANSFER OF SUPERNATANTS
TARGET CELL INCUBATION
TNP:EL4 - BEADS
B MC1
BEADS 5
5
-5 ’
3.75
0.0
1.9
CYTOCHALASIN
B (ugltnl)
FIG.4. The DNA fragmentation enhancing factor is stable.(A) A schematicrepresentationof the experiment for transferring supematants from cytochalasin treated effkctorsto TNPzEL4 targetswith or without antibodycoated beads. (B) The corrected percent DNA fragmentation for different cytochalasin B concentrations for the experiment shown in (A). Experimental conditions are described in the text.
Evaluation of numerous effector cell populations indicate that the CD8-positive human CTL are responsible for releasing the DNA fragmentation enhancing factor (Table 1). A murine CTL line and type II helper clone were unable to enhance the cytochalasin B DNA fragmentation, nor was a human CDCpositive CTL clone able to enhance the cytochalasin effect. Perhapsmore interesting is the finding that activated peripheral blood lymphocytes are capable of enhancing the cytochalasin B-induced DNA fragmentation (Table 2). These lymphocytes are generally of a high buoyant density and are enriched in both CD8- and CDCpositive cells (12). Table 1 supports the idea that the CD8 cells are responsible for the enhancement. Why human CTL should have a factor not found in the murine system is unclear. However such a difference could be secondary to culture conditions, or differences in cell types. It is
RELEASE OF A DNA DEGRADATION
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217
not likely that this factor is TNF since there is no DNA fragmentation induced in the TNF-sensitive target WEHI- 164 (Table 3). One hypothesis consistent with the data presented here is that a secretion pathway exists in CTL different from the one responsible for BLT-esterase secretion. This work indicates that human CD8-positive CTL can release a factor when stimulated with cytochalasin B. This factor is stable and can be transferred to a target cell system where it will enhance the cytochalasin B-induced DNA fragmentation. Whether this factor is secreted from CTL, and is a protein has yet to be determined. Numerous endogenous agents have been found that have a mechanism of action similar to the cytochalasins ( 13). We have demonstrated that villin, an endogenous capping protein, is capable of inducing DNA fragmentation in EL4 cells (2). Since proteins similar in action to the cytochalasins exist it is possible that the factor released from CTL may act in concert with these endogenous factors. Releaseof an enhancing factor could be potentially important in systems in which activated PBL are used to ablate tumor. Such speculation needs to be evaluated. ACKNOWLEDGMENTS The author thanks Patricia Hill and B. Landa-Gonzalez for technical assistance,Drs. M. Taplits and S. Shaw for cell lines, and Dr. D. Segal for the anti-DNP antibody.
REFERENCES 1. MacLean-Fletcher, S., and Pollard, T. D., Cell 20, 329, 1980. 2. Kolber, M. A., Brochat, K. O., and Landa-Gonzalez, B., FASEB J. 4,302 1, 1990. 3. Bousquet, P. F., Paulsen, L. A., Fondy, C., Lipski, K. M., Loucy, K. J., and Fondy, T. P., Cancer Res. 50,1431, 1990. 4. Kolber, M. A., Cell. Immunol. 133, 84, 1991. 5. Segal, D. M., Garrido, M. A., Perez, P., Titus, J. A., Winkler, D. A., Ring, D. B., Kaubisch, A., and Wunderlich, J. R., Mol. Immunol. 25, 1099, 1988. 6. Nitta, T., Sato, K., Yagita, H., Okumura, K., and Ishii, S., Lancet 335, 368, 1990. 7. Lew, P. D., Monod, A., Waldvogel, F. A., Dewald, B., Baggiolini, M., and Pozzan, T., J. Cell Biol. 102, 2197, 1986. 8. Michelakakis, H., and Danpure, C. J., Biochem. Pharm. 35, 933, 1986. 9. Kolber, M. A., Quinones, R. R., and Henkart, P. A., J. Immunol. 143, 1461, 1989. 10. Perez, M. S., Orejas, R. D., Petersen,J. W., DeMars, R., and Shaw, S., Eur. J. fmmunol. 20,673, 1990. 11. Asano, Y., and Hodes, R., J. Exp. Med. 158, 1178, 1983. 12. Ganido, M. A., Perez, P., Titus, J. A., Valdayo, M. J., Winkler, D. F., Barbieri, S. A., Wunderlich, J. R., and Segal, D. M., J. Immunol. 144,2891, 1990. 13. Pollard, T. D., and Cooper, J. A., Annu. Rev. Biochem. 55, 987, 1986.
IMMUNOLOGY
139,208-2 17 (1992)
Human Cytotoxic T Lymphocytes and Activated Peripheral Blood Lymphocytes Enhance Cytochalasin B-Induced DNA Fragmentation of Indicator Bystander Cells MICHAEL
A.
KOLBER
Departments of Medicine, and Microbiology and Immunology, The University of Miami School of Medicine, Miami, Florida 33101 Received June 16, 1991; accepted August 14, 1991 The cytochalasins are known to have multiple effects on cellular function. Not only do they induce secretionfrom granule compartments but they can induce DNA fragmentation in numerous cells. Evidence is presented which shows that treatment of human cytotoxic T lymphocytes and activated peripheral blood lymphocytes with cytochalasin B induces release of a factor capable of enhancing DNA fragmentation in cytochalasin-susceptible target cells. This activity can be transferred in the supematants of cytochalasin B-activated CTL. o 1992 Academic PWS, h.
INTRODUCTION Cytochalasin B is a fungal metabolite. As with other cytochalasins, cytochalasin B affects dissolution of the microfilaments (1). The possibility that this agent has both chemotherapeutic potential and immune enhancing properties can be raised. Recently it has been shown that the cytochalasins are capable of inducing DNA fragmentation in susceptiblecell lines (2). Whether it is by this mechanism that cytochalasin B prolongs survival in animals hosting various subcutaneously injected tumors (3) is not known. Cytochalasin B can enhance lysis of target cells by cytotoxic T lymphocytes (CTL) when bridging polystyrene beads coated with antibodies directed against the T cell receptor and a target cell surface moiety are used to redirect the effector cell and stabilize the cellbeadzell complex (4). Since redirected lysis is thought to present an immunotherapeutic option (5, 6) against various cancers the possibility that cytochalasin B could enhance this processand provide an added chemotherapeutic thrust warrants further evaluation of its immunologic actions. Earlier work has shown that the cytochalasins can enhance secretion from various cell lines (7,8). In neutrophils, pretreatment with cytochalasin B can delineate various granule compartments by their distinct secretory response to rises in intracellular calcium (7). Since cytochalasins can differentially affect the ability of some cells to secretefactors from different compartments we postulated that such a discriminating effect on factors releasedby cytotoxic T lymphocytes (CTL) might be possible. Since CTLinduced target cell DNA fragmentation and lysis are potentially uncoupled events we evaluated whether cytochalasin B treatment would reflect differences in CTL-mediated target cell DNA fragmentation and lysis. 208 0008~8749192$3.00 Copy-i& Q I992 by Academic Fres, Inc. All rights of repmduction in any form reserved
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209
We demonstrate that human CTL and activated peripheral blood lymphocytes releasea factor capable of enhancing cytochalasin B-induced DNA fragmentation. This factor is not in the same compartment as BLT-esterase. The activity can be transferred in the supernatants from cytochalasin B-treated CTL. We discuss the role that such a soluble factor could play in light of the possible chemotherapeutic potential of cytochalasin B. MATERIALS AND METHODS Materials. Cytochalasin B was obtained from Sigma Chemical Co. (St. Louis, MO) and a stock solution prepared of 2 mg/ml in ethanol. Polystyrene beadswere obtained from Polysciences (Warrington, PA). Anti-CD3 (OKT3) was generated from hybridomas obtained from the American Type Culture Collection (Rockville, MD). Rabbit anti-DNP was obtained as a gift from Dr. David Segal(National Institutes of Health, Bethesda, MD). Hepes and trinitrobenzene sulfonic acid (TNBS) were obtained from Sigma Chemical Co. (St. Louis, MO). Secretion assaysand DNA fragmentation assayswere performed in medium consisting of Hanks’ balanced salt solution containing 0.02 M Hepes and 3% fetal calf serum (FCS) adjusted to a final pH of 7.4 with NaOH (referred to as BSSH). All assays were performed at 37°C in room air. All media were adjusted to pH 7.4 with NaOH unless otherwise specified. Adsorption of antibody to polystyrene beads. Antibody was adsorbed to 3-pm diameter beads as previously described (9). Briefly, lo8 beadswere incubated with 0.025 mg/ml of each antibody in 0.1 M NaHC03 at room temperature overnight, followed by washing and incubation in 1.Omg/ml BSA (pH 7.4) at 37°C for 0.5 hr. Cells. The human CTL lines (MC 1 and Q302) were generatedaspreviously described (9). Both lines use as stimulators and recognize specifically the allogeneic human EBV transformed line Z2B. The human CTL line, Q302, was a generous gift of Dr. R. Gress and P. Lucas (N.I.H.). The human CDCpositive CTL clone (10) was a generous gift from Dr. S. Shaw (N.I.H., Bethesda,MD). The type II CD4-positive murine helper cell clone (1 l), 8-5-5, was a generous gift of Dr. M. Taplits (F.D.A., Bethesda, MD). The murine CTL line, called DC 1, is a C57B l/6 anti-DBA/2 line generated by establishing a l-week primary in vivo and used after three in vitro passageswith weekly stimulations of the effector cells with irradiated splenic cells from DBA/2 mice in medium consisting of RPM1 (GIBCO, Grand Island, NY) containing 10% FCS and 6 X 10m5M 2-mercaptoethanol (Bio-Rad, Richmond, CA) plus L-glutamine, penicillin, and streptomycin. The cell lines Z2B (human EBV transformed line), BW5 147, EL4, WEHI(ATCC), and RBL (rat basophilic leukemia) were propagated by in vitro passage.Target cells were TNP (trinitrophenol) modified by incubation with 1 mg/ ml TNBS in PBS, for 10 min at 37°C. Peripheral blood was obtained from healthy volunteers by antecubital venipuncture. Lymphocytes were harvested by Ficoll separation followed by nylon wool column passageand collection. Lymphocytes were activated by a modification of a previously described procedure (12). Nickel (16 mM) wells were first coated with 0.5 ml 0.1 M NaHC03 (pH 8.5) of 10 pg/ml of OKT3 at room temperature overnight, followed by washing and incubation in 1.0 mg/ml BSA (pH 7.4) at 37°C for 0.5 hr. Lymphocytes were plated at 2 X IO6cells/well in human CTL medium (RPM1 1640 (GIBCO, Grand Island, NY) plus 10% FCS (Sigma Chemical Co., St. Louis, MO), 3% T cell growth
210
MICHAEL
A. KOLBER
factor (Cellular Products Inc., Buffalo, NY), 30 U/ml rIL-2 (Cetus Corp., Emeryville, CA), penicillin, and streptomycin) for 3 days in a 37°C 5% COZ incubator. After the initial activation period the cells were harvested, washed, and replated in human CTL medium in wells not coated with antibody. After 3 days in a 37°C 5% CO* incubator the cells were harvested and used in the enhancement experiments. DNA fragmentation assayed by labeled thymidine release. Target cell DNA fragmentation was assayed by first labeling target cells in their growth phase with [3H]thymidine ( 100 PCi) for approximately 18 hr in standard tissue culture medium. After the labeling period the target cells were washed and resuspended in unlabeled tissue culture medium for l-2 hr. After the resting period the target cells (50 ~1)were washed and added to the microtiter plate wells. Labeled target cells were incubated with effector cells and cytochalasin B (final well volume was 200 ~1) as determined appropriate for each experiment. Plates containing targets, effecters, and cytochalasin B were centrifuged at 200g for 3 min and then incubated for 4 hr at 37°C in a room air oven. After an appropriate incubation time, as dictated by the experiment, the extent of thymidine releasedfrom the experimental and spontaneous releasesamples was determined by adding Triton X-100 to each sample (final concentration 0.04%) mixing, centrifuging the plate for 5 min at 7008, and recovering 100 ~1of supernatant to count in a &scintillation counter. The total [3H]thymidine releasewas determined by adding 150 ~1of 0.5% SDS aqueous solution with 3 mM EDTA, 10 mJ4 Tris, pH adjusted to 7.4, to 50 ~1 of target cells, mixed, and 200 ~1 harvested and counted. It was found that the total determined by this technique was identical to the total radioactivity obtained when the triton releasable radioactivity in the supernatant was added to the radioactivity of the remnant pellet and supernatant solubilized with the SDS solution. The corrected percentage DNA fragmentation was determined by the equation: Corrected % DNA Fragmentation = 100 X (Cexp- Cmed)/(Cdet - Cm&, where C representsthe amount of [H3]thymidine released(in cpm) in the triton soluble supernatants for the samples specified by the subscripts, exp (experimental), med (targets incubated in medium alone), and det (targets solubilized with detergent). Spontaneous releasevalues for all labeled thymidine experiments were lessthan 10%unless otherwise indicated. Supernatant experiment. MC1 cells were suspended in BSSH (0.75 X 106/ml) at different concentrations of cytochalasin B and aliquoted (200 ~1) into wells of a Ubottom microtiter plate (six replicates per cytochalasin concentration). Control wells that contained only BSSH and cytochalasin B were prepared on the same plate. The plate was incubated for 3 hr at 37°C in a room air oven prior to centrifuging at 700g for 5 min. Supernatants from wells on the effector plate (150 ~1) were transferred to a target cell plate that was prepared previously. The target cell plate was set up in a configuration identical to that of the effector cell plate. Half the wells on the target cell plate (triplicates for each cytochalasin B concentration) contained TNBS-modified EL4 cells (50 ~1 containing 15K cells), and the remaining wells contained TNBSmodified EL4 cells and polystyrene beads coated with both OKT3 and anti-DNP (50 ~1containing 15K cells and IO6beads). The supernatants from the effector plate were transferred to the target plate and the target plate was centrifuged at 200g for 3 min and incubated for 3 hr at 37°C in a room air oven. After the final incubation period the releasable [3H]thymidine was determined as described above. The cytochalasin
RELEASE OF A DNA DEGRADATION
FACTOR FROM CTL AND PBL
211
concentration given for each experiment is the cytochalasin concentration in the target plate wells. Statistics. Standard deviations are given for all points except when they are smaller than the symbol. Significance was calculated using the unpaired t test (Student’s). RESULTS Cytotoxic T lymphocytes enhance cytochalasin B-induced DNA fragmentation. Previously we showed that cytochalasin B had an effect on the secretion of lytic factors from CTL (4). In that work we demonstrated that CTL release both BLT-esterase, and lytic factors when the T cell receptor is cross-linked by beads coated with antiCD3. The secretion is biphasic in its concentration dependence on cytochalasin. We wished to evaluate whether DNA fragmentation induced by CTL had a similar biphasic dependence on cytochalasin concentration. In Fig. 1 is shown the corrected percent DNA fragmentation as measured by the releaseof tritated thymidine from TNP:EL4 cells as a function of cytochalasin B concentration. In this experiment radiolabeled TNP:EL4 were incubated with either human CTL (MC 1 cells) and polystyrene beads coated with OKT3 (anti-CD3) and anti-DNP (top curve), MCI cells and no beads (middle curve), or media alone (lower curve). The top curve demonstrates that when polystyrene beads, coated with the antibodies OKT3 (anti-CD3) and anti-DNP, are incubated with effector and target cells the DNA fragmentation is enhanced above that found for target cells alone. When the human CTL, MC 1, are incubated with the TNP:EL4 cells without beads the target cell DNA fragmentation induced at low cytochalasin B concentrations is the sameasif the effector cells were not present. However at higher cytochalasin B concentrations the target cell DNA fragmentation in the presence of MC1 cells approaches the DNA fragmentation found for effector cells
25
15
5
0
1
2
CYTOCHALASIN
3
4
5
6
B (pglml)
FIG. 1. Effect of cytochalasin B on release [3H]thymidine from TNP:EL4. The corrected percent DNA fragmentation is plotted as a function of cytochalasin B concentration. [‘Hlthymidine-labeled TNP:EL4 cells (1SK/well) were used as targets in the absence (m) or presence of MC1 cells (lOOK/well) with (0) or without (0) antibody-coated beads. The incubation time, at 37°C in room air, was 4 hr.
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MICHAEL A. KOLBER
incubated with target cells, and beads at higher cytochalasin B concentrations. The increase in DNA fragmentation as a function of cytochalasin B concentration for TNPEL4 cells alone has been described previously (2). The data from Fig. 1 show that the enhancement in DNA fragmentation in the presence of MC 1 CTL is not dependent on the presenceof beads at high cytochalasin B concentrations. A number of experiments were performed to demonstrate that CTL are enhancing target cell DNA fragmentation in the presence of cytochalasin. In Fig. 2 the corrected percent DNA fragmentation is shown as a function of effector to target cell ratio. When Z2B (cells with no effector potential) are incubated with radiolabeled EL4 cells there is no enhancement of DNA fragmentation above that found for fragmentation produced by cytochalasin B alone. Whereas, the DNA fragmentation is enhanced monotonically in the presenceof increasing numbers of MC 1 CTL. Further confirmation that MC 1 cells are releasinga factor capable of enhancing the cytochalasin B-induced DNA fragmentation is shown in Fig. 3. In that figure the corrected percent DNA fragmentation of EL4 cells is plotted, at a fixed cytochalasin B concentration, as a function of time. After a 3-hr incubation period MC1 cells induce a significant enhancement in target cell DNA fragmentation compared to the enhancement seen when target cells are incubated with RBL cells, cells with no effector potential. This enhancement is increased after 4 hr. Fluorescent single cell analysis of the MCI cell line revealed that they were approximately 96% CD8-positive and contained approximately 4% CD4-positive cells (data not shown). In order to evaluate which population of cells was responsible for the enhancement in target cell DNA fragmentation we evaluated a number of different CTL as shown in Table 1. In Table 1 the corrected percent DNA fragmentation is
5o’
0
0
2
4
6
8
10
EFFECTOFUARGET FIG. 2. DNA fragmentation as a function of the effecter/target cell ratio. The corrected percent DNA fragmentation, as indicated by the releaseof [3H]thymidine, from EL4 cells ( 15K/well) is plotted as a function of the effecter/target cell ratio. The effector cells were MC1 cells (0) and Z2B (m). Cytochalasin B at 2 Irg/ ml was usedin the experiment, and the values for the corrected percent DNA fragmentation were determined using the spontaneousthymidine releasefrom target cells without cytochalasin. The incubation time was 4 hr at 37°C in room air.
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FACTOR FROM CTL AND PBL
213
5 loo2 3
80-
100 TIME (min)
200
FIG. 3. DNA fragmentation oftarget cells asa function of time. The corrected percent DNA fragmentation is plotted as a function of incubation time. [‘H]Thymidine labeled EL4 cells (I SK/well) were incubated in 2 pg/mI with either MCI cells (100 K/well; W)or RBL cells (lOOK/well; q ) at 37°C for the times shown.
shown for an effector cell population (column 1) under various conditions: effector cells, target cells, and cytochalasin B are present (column 2), only effector cells are present with target cells (column 3), or only cytochalasin B is present with target cells (column 4). In all casescytochalasin B alone produces approximately 15%DNA fragmentation of target cells (column 4), whereas effector cells with target cells alone produce no or little DNA fragmentation (column 3). When MC 1 cells are treated with anti-CD4 and complement the enhancement of target cell DNA fragmentation is unchanged from that produced by MC1 cells treated with complement alone. Another human CTL line, 4302, is also capable of enhancing cytochalasin B-induced DNA fragmentation. 8.9 is a human CD4-positive CTL clone that does not enhance DNA
TABLE I Human CTL Enhance Cytochalasin B-Induced DNA Fragmentation Corrected % DNA fragmentationb ElktoP
EFF + CYT@
EFF - CYTO
-EFF + CYTO
MC1 +C MC1 + C’ + anti-CD4 Q302 8.9 8-S-5 DC1
31.7 zk 2.2 33.3 iz 2.1 36.2 + 0.4 8.6 It 0.8 1I .4 + 2.2 13.5 f 0.3
0.0 * 0.4 0.7 It 0.2 0.5 -+ 1.1 -1.6 T 0.1 -2.8 +- 1.7 5.1 * 0.4
16.2 + 0.8 16.2 _’ 0.8 17.0 -t 2.2 13.1 2 0.6 14.3 t 2.1 13.6 + 0.7
a Effector cells were plated at 150 K/well with labeled EL4 cells (lOK/well). 6 Cells were incubated for 4 hr at 37°C. ’ Cytochalasin B, 5 FgjmI.
214
MICHAEL A. KOLBER TABLE 2 Activated Peripheral Blood Lymphocytes Releasea Factor Capable of Enhancing Cytochalasin B-Induced DNA Fragmentation Corrected % DNA fmgmentationb @EM) PT No.”
+PBL + CYTO’
+PBL - CYTO
-PBL + CYTO
1 2 3 4 5
31.8 (2.4) 15.5 (1.3) 33.4 (2.6) 38.8 (3.4) 36.8 (3.0)
-1.4 (1.4) -1.1 (1.6) -3.3 (0.6) 1.1 (0.4) -0.9 (6.4)
22.9 (2.5) 22.9 (2.5) 21.8 (2.0) 21.8 (2.0) 13.8 (2.3)
’ PBL were plated at 250K/well with labeled EL4 cells (lSK/well). bCells were incubated for 4 hr at 37°C. ’ Cytochalasin B, 5 &ml.
fragmentation of EL4 cells. A type II CDCpositive murine helper cell clone, S-5-5, and a murine CTL line, DC1 (C57B1/6 anti-DBA/Z), are not able to enhance cytochalasin B-induced DNA fragmentation. With the increasing interest in using activated lymphocytes in various immunologic therapies (e.g., 5, 12) we wanted to evaluate whether human peripheral blood T lymphocytes (PBL) activated via the cross-linked CD3 receptor could enhance cytochalasin B-induced DNA fragmentation. Blood obtained from five healthy volunteers was activated as described under Materials and Methods. Table 2 demonstrates that the activated PBL were capable of enhancing cytochalasin B induced DNA fragmentation in all but one case.PBL did not induce DNA fragmentation of the target cells in the absenceof cytochalasin (Table 2). When PBL were cultured in the human CTL medium (containing TCGF and IL2), without receptor cross-linking, they did not enhance the cytochalasin B-induced DNA fragmentation (data not shown). Human CTL enhance cytochalasin B-induced DNA fragmentation in EL4 cells. Whether this effect occurs with any target cell is not clear. In Table 3 we examined four different target cells for cytochalasin B-induced DNA fragmentation as well as
TABLE 3 Cytochalasin B-Induced DNA Fragmentation is Dependent on the Target Cell Corrected % DNA Fragmentationb Target” I. 2. 3. 4.
EL4 BW5147 RBL WEHI
+EFF+CYT’ 27.6 k 4.2 32.1 k 1.1 3.0 + 3.5 -0.1 zk 0.6
a 150K MC 1 cells/well and 15K target cells/well. bAfter 4-hr incubation at 37”. ’ Cytochalasin B, 1.7 &ml.
+EFF/-CYT 7.2 + -2.5 f 1.5 f 0.3 +
3.3 2.3 1.0 0.5
-EFF/+CYT 10.7 f 2.6 22.5 f 1.5 2.3 rfr 3.4 -0.2 + 0.8
RELEASE
OF A DNA
DEGRADATION
FACTOR
FROM
CTL AND
PBL
215
enhancement of DNA fragmentation with MC1 effector cells. As seen in Table 3 cytochalasin B can fragment DNA of EL4 and BW5 147, whereas cytochalasin B does not fragment DNA of the RBL and WEHI cells. In the limited number of targets used in this study whenever cytochalasin B fragmented DNA the MC1 cells enhanced the fragmentation. MC I cells did not enhance or induce DNA fragmentation in the RBL cells or the WEHI cells. Supernatantsfrom CTL treated with cytochalasin B can induce DNA fragmentation. The data presented so far suggestthat MC 1 cells releasea factor capable of enhancing cytochalasin B-induced target cell DNA fragmentation. Since there is no specific recognition between the MC 1 cells and the EL4 target cells this enhancement is a bystander effect. Such an effect indicates that a factor sufficiently stable to enhance DNA fragmentation in a bystander fashion might be found in the supematants from effector cells stimulated with cytochalasin B. To evaluate this possibility MC1 cells were incubated in different concentrations of cytochalasin B according to the protocol schematically illustrated in Fig. 4A. After a 3-hr incubation period at 37°C the supematants were harvested from the wells f human CTL and added to TNBS-modified EL4 cells with or without beads. After another 3-hr incubation period the DNA fragmentation induced in the target cells was assayed.If MC 1 cells are being transferred inadvertently then the beads will redirect lysis and an enhancement in DNA fragmentation will occur compared to the no bead control (seeFig. 1). In Fig. 4B is shown the corrected percent DNA fragmentation for three concentrations of cytochalasin B. Target cells incubated in the supematants from the MC1 cells showed a significant and dosedependent enhancement in DNA fragmentation above the DNA fragmentation induced by the cytochalasin alone. Polystyrene beads, coated with antibodies (OKT3 and anti-DNP), in the final incubation period did not enhance DNA fragmentation at any concentration when compared to the companion no bead controls. DISCUSSION In this paper we have shown that human cytotoxic T lymphocytes releasea substance capable of enhancing cytochalasin B-induced DNA fragmentation in a bystander fashion. Previously we have demonstrated that upon T cell receptor activation cytochalasin B will also enhance secretion of the granule enzyme BLT-esteraseand antibody-coated bead redirected lysis of target cells (4). However, as shown in that work CD3 receptor activation was necessary, and lysis and secretion were inhibited at high (5 pg/ml) cytochalasin concentrations. Figure 1 demonstrates that the DNA fragmentation of EL4 target cells in the presence of cytochalasin B and MC1 cells is not biphasic. The enhancement in DNA fragmentation occurring at high concentrations of cytochalasin B is the same whether beads that redirect lysis are present or not. The enhancement in cytochalasin B-induced DNA fragmentation by MCI cells is dependent on the number of effector cells present (Fig. 2) as well as the incubation period (Fig. 3). Receptor activation by cytochalasin B is unlikely since the secretion of BLT-esterase is diminished at concentrations of cytochalasin that enhance a DNA fragmentation (4). Support that the MC1 cells are releasing a factor contributing to the cytochalasin B-induced DNA fragmentation comes from the supematant experiment (Fig. 4). In that experiment supematants from MC1 cells treated with cytochalasin B enhance DNA fragmentation of target cells above that found when target cells are treated with cytochalasin alone.
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MICHAEL A. KOLBER
A EFFECTOR CELL INCUBATION
TRANSFER OF SUPERNATANTS
TARGET CELL INCUBATION
TNP:EL4 - BEADS
B MC1
BEADS 5
5
-5 ’
3.75
0.0
1.9
CYTOCHALASIN
B (ugltnl)
FIG.4. The DNA fragmentation enhancing factor is stable.(A) A schematicrepresentationof the experiment for transferring supematants from cytochalasin treated effkctorsto TNPzEL4 targetswith or without antibodycoated beads. (B) The corrected percent DNA fragmentation for different cytochalasin B concentrations for the experiment shown in (A). Experimental conditions are described in the text.
Evaluation of numerous effector cell populations indicate that the CD8-positive human CTL are responsible for releasing the DNA fragmentation enhancing factor (Table 1). A murine CTL line and type II helper clone were unable to enhance the cytochalasin B DNA fragmentation, nor was a human CDCpositive CTL clone able to enhance the cytochalasin effect. Perhapsmore interesting is the finding that activated peripheral blood lymphocytes are capable of enhancing the cytochalasin B-induced DNA fragmentation (Table 2). These lymphocytes are generally of a high buoyant density and are enriched in both CD8- and CDCpositive cells (12). Table 1 supports the idea that the CD8 cells are responsible for the enhancement. Why human CTL should have a factor not found in the murine system is unclear. However such a difference could be secondary to culture conditions, or differences in cell types. It is
RELEASE OF A DNA DEGRADATION
FACTOR FROM CTL AND PBL
217
not likely that this factor is TNF since there is no DNA fragmentation induced in the TNF-sensitive target WEHI- 164 (Table 3). One hypothesis consistent with the data presented here is that a secretion pathway exists in CTL different from the one responsible for BLT-esterase secretion. This work indicates that human CD8-positive CTL can release a factor when stimulated with cytochalasin B. This factor is stable and can be transferred to a target cell system where it will enhance the cytochalasin B-induced DNA fragmentation. Whether this factor is secreted from CTL, and is a protein has yet to be determined. Numerous endogenous agents have been found that have a mechanism of action similar to the cytochalasins ( 13). We have demonstrated that villin, an endogenous capping protein, is capable of inducing DNA fragmentation in EL4 cells (2). Since proteins similar in action to the cytochalasins exist it is possible that the factor released from CTL may act in concert with these endogenous factors. Releaseof an enhancing factor could be potentially important in systems in which activated PBL are used to ablate tumor. Such speculation needs to be evaluated. ACKNOWLEDGMENTS The author thanks Patricia Hill and B. Landa-Gonzalez for technical assistance,Drs. M. Taplits and S. Shaw for cell lines, and Dr. D. Segal for the anti-DNP antibody.
REFERENCES 1. MacLean-Fletcher, S., and Pollard, T. D., Cell 20, 329, 1980. 2. Kolber, M. A., Brochat, K. O., and Landa-Gonzalez, B., FASEB J. 4,302 1, 1990. 3. Bousquet, P. F., Paulsen, L. A., Fondy, C., Lipski, K. M., Loucy, K. J., and Fondy, T. P., Cancer Res. 50,1431, 1990. 4. Kolber, M. A., Cell. Immunol. 133, 84, 1991. 5. Segal, D. M., Garrido, M. A., Perez, P., Titus, J. A., Winkler, D. A., Ring, D. B., Kaubisch, A., and Wunderlich, J. R., Mol. Immunol. 25, 1099, 1988. 6. Nitta, T., Sato, K., Yagita, H., Okumura, K., and Ishii, S., Lancet 335, 368, 1990. 7. Lew, P. D., Monod, A., Waldvogel, F. A., Dewald, B., Baggiolini, M., and Pozzan, T., J. Cell Biol. 102, 2197, 1986. 8. Michelakakis, H., and Danpure, C. J., Biochem. Pharm. 35, 933, 1986. 9. Kolber, M. A., Quinones, R. R., and Henkart, P. A., J. Immunol. 143, 1461, 1989. 10. Perez, M. S., Orejas, R. D., Petersen,J. W., DeMars, R., and Shaw, S., Eur. J. fmmunol. 20,673, 1990. 11. Asano, Y., and Hodes, R., J. Exp. Med. 158, 1178, 1983. 12. Ganido, M. A., Perez, P., Titus, J. A., Valdayo, M. J., Winkler, D. F., Barbieri, S. A., Wunderlich, J. R., and Segal, D. M., J. Immunol. 144,2891, 1990. 13. Pollard, T. D., and Cooper, J. A., Annu. Rev. Biochem. 55, 987, 1986.
