Original Paper International Journal of Cell Cloning 9:65-77 (1991)
Gamma-Irradiated Peripheral Blood Mononuclear Cells Can Express LAK Activity Anita S.-E Chong".b,c,D. E. Bier! U? J. Grimes', E. M. Hershb.' aDepartment of General Surgery, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois, USA; bDepartments of Internal Medicine, 'Biochemistry, dRadiation-Oncology, and eImmunology/Microbiology, The University of Arizona, Tucson, Arizona, USA. Key Words. Feeder cells ' LAK
IL-2
*
y-Irradiation
*
Mitogen-PHA
*
Cytotoxicity
Abstract. Peripheral blood mononuclear cells (PBMC) irradiated with high dose y-radiation (1000-5000 rad) are commonly used as feeder cells during the cloning of T lymphocytes, natural killer (NK) and lymphokine activated killer (LAK) cells. We report here that such y-irradiated PBMC can be stimulated with interleukin 2 (IL-2) to express the ability to lyse a variety of tumor cell targets. The non-major histocompatibility complex (MHC) restricted cytotoxicity demonstrated by irradiated PBMC is, however, lower than that expressed by their non-irradiated counterparts. The numbers of viable, ?irradiated LAK cells are significantly increased by the addition of the mitogen, phytohernagglutinin(PHA). Purification of the y-irradiated cells expressing cytotoxic activity by flow cytometry determined that the effector cells were predominantly CD3- cells, although some CD3' cells also expressed moderate LAK activity. The ability of y-irradiated cells to proliferate in the presence of PHA alone, or with IL-2 PHA, was maximal at day 4-5; but proliferation, as detected by 3H-thymidmeuptake, was not detectable beyond 12-15 days of in vitro culture. Because many of the LAK, T cell and NK cell cloning procedures require the presence of feeder layers, growth factors (usually IL-2) and mitogens, the presence of residual feeder cells expressing cytotoxic activity may affect the specificity of such clones. Thus, efforts should be made to ensure that such y-radiation-resistant cells capable of expressing cytotoxic activity are completely eliminated before the cloned cells are used for further experiments.
+
Introduction Peripheral blood mononuclear cells (PBMC) are stimulated by interleukin 2 (IL-2) to exhibit the ability to kill a wide range of tumor cell types in vitro [l-31. Correspondence: Dr. Anita S.-F. Chong, Department of General Surgery, RushPresbyterian-St. Luke's Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612, USA. Received June 27,1990; provisionally accepted August 8,1990; accepted for publication October 5, 1990. 0737-14541911$2.00/0 QAlphaMed Press
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The non-major histocompatibilitycomplex (MHC) restricted cytolytic activity is termed lymphokine-activatedkiller (LAK) activity [4-lo]. It is now clear that a variety of cell types from peripheral blood can exhibit LAK activity. The cell type that expressesthe highest level of LAK activity is the IL-2-stimulatedNK cell expressing the Leu 19(CD56),Leu ll(CD16),CD3- phenotype [ll-141. However, T lymphocytes that express Leu 19’have also been reported to exhibit significant LAK activity [fi, 161. It is known that irradiated cells retain some of their functions for a short period of time. We report here the PBMC pretreated with 1000-5000rad y-radiation can still be stimulated by IL-2 to exhibit LAK activity. The y-irradiated cell population expressing LAK activity is similar to that reported for non-irradiated PBMC, where the predominant cytotoxic cell population is CD3-. Since irradiated (2000-5000rad) PBMC, IL-2, and PHA have all been used in the generation of T, NK, and LAK cell clones [17-221, our findings indicate that steps should be taken to ensure that these y-radiation-resistantcells capable of expressing LAK activity are completely eliminated from the populations before they are used in further experiments. Materials and Methods
Cells and Culture Conditions PBMC were isolated from normal donor blood by Ficoll-hypaque separation (Pharmacia, Piscataway, NJ). Cells collected from the interface were washed with Hanks buffered salt solution (HBSS; GIBCO, Grand Island, NY) and resuspended to required concentrations. Complete medium consists of RPMI-1640 (GIBCO), 10% heat-inactivated fetal calf serum (FCS; GIBCO) supplemented with 100 U/ml each of penicillin and streptomycin (GIBCO). PBMC were resuspended at 1 X lo6cells/ml in complete medium or in defined serum-free medium (AIM-V; GIBCO). To stimulate PBMC, 1000 U/ml recombinant human (rh)IL-2 (a gift from Cetus Corp., Emeryville, CA) and/or 0.01 pg/ml purified PHA from Phaseolus spp. (HA 16; Burroughs Wellcome, Research Triangle Park, NC) were used, unless otherwise indicated. Cells were incubated in a humidified, COz incubator at 37” C. Non-adherent cells were collected after 5 days, washed and adjusted to the required concentrations for further use. The tumor cell lines K562 (erythroleukemia)and Daudi (Burkitt lymphoma), were maintained as suspension cultures, and MCF-7 (breast carcinoma), A375M (melanoma), and primary cultures of skin fibroblast cells were maintained as adherent cultures. All cells were maintained in RPMI-1640 (GIBCO), 10% heatinactivated fetal calf serum (FCS) with 100 U/ml each of penicillin and streptomycin (complete medium), and were subcultured every 3-4 days. All cell lines except A375M (gift from J. Fidier, M.D. Anderson Cancer Center, Houston), and skin fibroblastswere obtained from the American Type Culture Collection. Skin fibroblasts were cell outgrowths from normal skin (gift from Dr. S . Leong, Anzona Cancer Center, Tucson, AZ), cultured in RPMI, supplemented with 10% FCS and penicillin, streptomycin and gentamycin (50 pg/ml; GIBCO).
?-Irradiated PBMC Express LAK Activity
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ylrradiation of PBMC PBMC were resuspended in complete medium or AIM-V, placed in a Petri dish (Falcon, Becton Dickinson, Lincoln Park, NJ), and irradiated with 1000-5000 rad, using a 4MV linear accelerator (Varian Associates, Pa10 Alto, CA). Calibration was performed weekly and technical assistance and dosimetry were provided by the Physics Section, Department of Radiation Oncology, University of Arizona. Irradiated cells were washed once and resuspended in the indicated culture medium at a concentration 1 X 10Vml with 1000 U/ml IL-2, or/and 0.01 pg/ml PHA, unless otherwise indicated. After 4-5 days of culture, the viable non-adherent cells were separated on Ficoll-hypaque, washed three times in HBSS, and resuspended in complete medium for further use. Assay for Cell-Mediated Cytotoxicity A modification of the standard Tr-release assay was used. Target cells were incubated overnight at 1 X 106/mlin complete medium containing 100 pCi/ml of W r . Target cells were washed, and 5 X lo3to 10 x lo3labeled target cells were incubated with the indicated ratios of effector cells for 4 h in a total volume of 200 pl in round-bottom microtiter plates (Costar, Boston, MA). After 4 h, 100 pl of supernatant were harvested and assayed for released T r with a y-counter. All assays were performed in triplicate. Percentage specific lysis (SL) was calculated as follows: % SL = cpm - SR x 100 MR - SR cpm being the mean counts-per-minute of the experimental sample, SR being the mean spontaneous release and MR being the mean maximum release of 1 X lo4 SICr-labeled cells incubated with 0.1 M HCI. Proliferation Assay Cells were plated in quintuplicate at a density of 1 x 10s cells/well, with 1000Ulml IL-2, or 0.1 pg/ml PHA, or both IL-2 and PHA. For long-term cultures, 100 pI/well of spent medium was removed and 100 &'well fresh medium, with the indicated additives, was added to each well every fourth day. Proliferation was assessed by measuring tritiated thymidine (3H-Tdr)uptake (2 mCi/mM; NEN/ Dupont, Boston, MA) during the last 8 h of incubation. Each well received 1 pCi of 3H-Tdr on the days indicated. Cells were harvested on a glass filter using a cell harvester (Cambridge Tech. Inc., Cambridge, MA), and the amount of 3H-Tdr incorporated by the cells determined by scintillation counter. Monoclonal Antibody Staining and Flow CytometricAnalysis of the Cloned
LA K Cell Population The surface phenotypes of the PBMC were determined by the binding of fluorescein isothiocyanate- and phycoerythrin-conjugatedmonoclonal antibodies (Becton Dickinson, Mountain View, CA).The test antibodies were Leu 19, Leu 7,
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Leu 11 (CD16), Leu 4 (CD3), Leu 2 (CD8), Leu 3 (CD4) and anti-IL-2 receptor (CD25). Controls using non-specific IgG were performed to determine non-specific binding. Cells were harvested and washed three times in PBS and made up to 2 x lo7cells/ml. Fifty microliters of the cell suspension were incubated with the conjugated antibodies, at concentrations recommended by the manufacturer, for 20-30 min at 4°C. Cells were washed twice in PBS then fixed in 1% formalin. Fixed cells were analyzed by flow cytometry on FACSCAN (Becton Dickinson). Purification of Subpopulations of Effector, ?Irradiated Cell Populations PBMC were irradiated at 3000 rad, and then stimulated with 1000 U/ml IL-2 and 0.01 pg/ml PHA for 5 days in complete medium. The cells were then centrifuged on Ficoll-hypaque to enrich for viable cells. Cells were then washed 2 x in HBSS, and resuspended at 1 X lo7celldm1 in HBSS. Viable cells were incubated with 100 ,ul/ml of antibody for 30 min on ice. Fluorescein isothiocyanateand phycoerythrin-conjugated monoclonal antibodies, Leu 4, Leu 2 and Leu 19 were purchased from Becton Dickinson. Excess antibodies were removed and cells washed 2 x in HBSS. Cells were then resuspended at 1 X lo7 cells/ml in complete medium before sorting under sterile conditions on FACSTAR (Becton Dickinson). Only strongly positive cells and strongly negative cells were collected on ice in 20% FBYRPMI. Cells were washed, counted and their cytolytic potential measured in a 4 h Wr-release assay. Cells pretreated with antibody, but not sorted, were also assayed as controls for the effects of antibody binding to the lymphoid cells. Statistics Paired, one-tailed, Student’s t test was performed on data to test their significance, using the Statsview 512+ program (Brainpower Inc., Calabasas, CA) for the Macintosh SE/30 computer.
Results y-Irradiated PBMC Are Stimulated by IL-2 to Exhibit LAK Activity PBMC, or y-irradiated PBMC, were cultured in complete medium, either alone or supplemented with IL-2 and/or PHA. After 5 days, the viable cells were assayed for their ability to lyse Daudi and K562 cells. Figure 1 demonstrates that both PBMC and y-irradiated PBMC were stimulated by IL-2 to exhibit LAK activity. Both non-irradiated PBMC and irradiated PBMC, co-stimulated with IL-2 + PHA, consistently expressed lower levels of cytotoxic activity for the tumor cells than when stimulated with IL-2 alone. The y-irradiated cells exhibited a lower cytolytic activity than their non-irradiated counterpart. Finally, K562 cells were more susceptible than Daudi cells to the cytolytic activities of the irradiated LAK cells ( p < 0.05). Data from one donor is presented in Figure 1, although similar results were observed with PBMC from 5 different donors.
69
y-Irradiated PBMC Express LAK Activity
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'
1A:mn-irradlatmdMecion. Daudl tarpels
1 C non-radiatedaffecton. K562 targets
. 100
20 40
'
0
201
101
5:l
231
2O:l
5 1
101
2.5:1
x
0 'O
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50
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30
18:inidlated affecton. Dud1lug&
80
1 D irradiated Mecton. K562 tarpats
- 60
.#
40
201
l0:l 5:l 251 EFFECTOR : TARGET
2O:l
101 5:l EFFECTOR : TARGET
2.5:1
Fig. 1. Irradiated PBMC partially retain their ability to be stimulated by IL-2 to express LAK activity. Irradiated @ and D) and non-irradiated (A and C) PBMC are incubated for 5 days in medium (0, 1OOO U/ml IL-2 (0, R),0.01 pg/ml PHA ( X , A ) or both IL-2 +PHA (El, A). The viable cells are harvested and assayed in triplicate for LAK activity using a standard 4 h SICr-releaseassay. The tumor targets used were Daudi (A, B) and K562 (C, D) cells. Data obtained from one representative experiment of five are presented as mean percent cytotoxicity f SE.
o),
To investigate the radiation sensitivity of the LAK cell precursors, PBMC were subjected to increasing doses of y-irradiation. The cell viabilities, as determined by their ability to exclude trypan blue, and cytotoxic activities of these cells stimulated for 5 days with IL-2, were examined and the results presented in Figure 2. Increasing doses of y-radiation decreased the number of viable cells as well as their cytotoxic activities against K562 and Daudi cells. Specificiry of the LAK Activity in y-Irradiated PBMC IL-2-activated PBMC are reported to kill NK cell-resistant tumor cells and cultured primary-fibroblasts, but not fresh, unstimulated PBMC. We examined the ability of y-irradiated PBMC, stimulated with IL-2 PHA, to kill two NK cell-resistant cell lines (MCF-7 and A375M), fibroblasts and PBMC. Figure 3 demonstrates that y-irradiated PBMC, stimulated with PHA IL-2, exhibit LAK activity similar to, albeit lower, than non-irradiated IL-2 activated PBMC, and were able to kill primary fibroblast cells, MCF-7 and A375M cells, but not fresh PBMC (Fig. 3).
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ChonglBierlGrimeslHersh
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3000
4000
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GAMMA-RADIATION (RADS)
0
1000
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GAMMA-RADIATION (RADS)
Fig. 2. The ability of PBMC to express LAK activity is radiosensitive. PBMC were irradiated at the indicated doses, then cultured in IL-2 (1000 U / d ) and PHA (0.01 pglml) for 5 days. The cells were centrifuged on Ficoll-hypaque and the number of viable cells determined by trypan blue exclusion on a hemocytometer (A). 100 % represents the nurnber of cells at the start of the experiment. The viable cells are then assayed in triplicate for LAK activity with a standard 4 h W3-release assay (B). The tumor targets used were Daudi (H)and K562 (0)cells. Data obtained from one representative experiment of two, are presented as mean percent cytotoxicity k SE.
LAK Activity by Irradiated PBMC is Dependent on IL-2 Concentration The effects of different concentrationsof IL-2 and PHA on y-irradiated PBMC were determined next. Irradiated cells were cultured with different concentrations of IL-2 and/or PHA for 5 days. The viable cells were counted and assayed for their ability to kill Daudi and K562 cells (Fig. 4). IL-2 only marginally increased the number of viable cells recovered from 8% to approximately 20%. However, the addition of increasing concentrationsof PHA, from 0 to 0.1 pg/ml, dramatically increased the number of viable cells recovered from 20% to approximately 70%; 100%represents the total number of PBMC at the start of the experiment. In contrast, the cytolytic activity of the surviving y-irradiated cells for K562 and Daudi targets was primarily dependent on the IL-2 concentration. Proliferation of y-Irradiated PBMC when Stimulated with IL-2 or PHA A time course to determine the survival of y-irradiated cells, measured by the uptake of 3H-Tdr,when stimulatedwith IL-2 andor PHA, was next performed. 3H-Tdr uptake by the irradiated cells was highest 4-5 days after y-radiation of the PBMC, beyond which the uptake of 3H-Tdr by the irradiated cells declined, and there was no detectable uptake after 15 days (Table I). These data indicate that the survival of the irradiated cells was limited. In contrast, the non-irradiated PBMC continued to proliferate beyond 20 days when cultured under similar culture conditions. Su$ace Phenotype of Surviving y-Irradiated Cells The phenotypes of surviving y-irradiated PBMC stimulated with IL-2 andlor
71
?-Irradiated PBMC Express LAK Activity
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60
3: PBMC
3: FIBROBLAST
401
50
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6
c
1O:l 60
0 40 30
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5:l
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1O:l
;& 3: MCF-7
101
5:l 251 1.25:l EFFECTOR :TARGET
%
5:l
2.5:l
1.25:l
\
101
60
3: A375M
t
50 40
5:1 2.5:l 1.253 EFFECTOR : TARGET
Fig, 3. Specificity of LAK activity expressed by lL-2-activated, yirradiated PBMC. Irradiated PBMC are incubated for 5 days in loo0 U/ml IL-2 and 0.01 pglml PHA (A). Nonirradiated PBMC were cultured in medium (U)or with loo0 Ulml (H) for 5 days. The viable cells are then assayed in triplicate for LAK activity with a standard 4 h 51Crrelease assay. The targets used were fresh PBMC, primary-cultured fibroblasts, MCF-7 and A375M cells. Data obtained from one representative experiment of three are presented as mean percent cytotoxicity f SE.
PHA were determined by flow cytometric analysis. As expected, both startingPBMC and IL-2-activated PBMC population were heterogeneous, consisting largely of T cells of either CD4 or CD8 phenotype, with approximately 10%NK cells (Leu U+, Leu 19.) (Table XI). -,-irradiation consistently (4out of 4 donors tested) reduced the percent of surviving CD8 and Leu 11 expressing cells compared to their nonirradiated counterpart ( p < 0.05). Stimulation of the y-irradiated PBMC with IL-2 and/or PHA increased the number of surviving CD8 cells ( p < 0.05), but not the Leu 11 cells ( p > 0.05) (Table II). Considerable variation in the actual percentages of cells expressing each phenotype was observed among different donors (A. Chong, data not shown), and based on such phenotypic analyses we could not determine the phenotype of cells expressing LAK activity. Determination of y-Irradiated-LymphoidPopulation Expressing LAK Activity The cell that expresses high levels of LAK activity in peripheral blood is reported to be the NK cell. To determine the phenotype of cells that express LAK activity, -,-irradiated lymphocytes were incubated for 5 days in IL-2 and PHA.
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Fig. 4. Phenotypeof the irradiated PBMC that express LAK activity. Irradiated PBMC are incubated for 5 days in the indicated concentrationsof E-2 and 0.1 (E3) ,0.01 (8),0.001 (H)pgglml PHA. The viable cells were determined by trypan blue dye exclusion, and then counted by hemocytometer (A). The cells were also assayed in triplicate for LAK activity with a standard 4 h 50-release assay. The targets used were Daudi (B) and K562 (C) cells. Data obtained from one representativeexperiment of four are presented as mean percent cytotoxicity & SE.
The viable cells were then sorted into populations that expressed, or did not express, CD3,CD8 and Leu 19. The cytolytic activity of each of these cells for K562 tumor cells is presented in Figure 5. Leu 19- cells exhibited virtually undetectable cytolytic activity, and there were insufficient Leu 19' cells to perform a cytotoxicity
y-Irradiated PBMC Express LAK Activity
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Table I. Proliferative index of y-irradiated cells stimulated with IL-2 and/or PHA Mean cpmb (stimulation index)' Medium Day 4, non-irradiated" Day 4, yirradiated Day 8, non-irradiated Day 8, yirradiated Day 12, non-irradiated Day 12, yirradiated Day 15, non-irradiated Day 15, y-irradiated Day 20, non-irradiated Day 20, yirradiated
IL-2
787 1,512 1,881 589 2,355 465 5,423 970 4,063 343
PHA
35,548 (45.2) 1,759 (1.2) 56,652 (30.1) 2,291 (3.9) 61,483 (26.1) 973 (2.1) 85,682 (15.8) 1,248 (1.3) 68,541 (16.9) 459 (1.3)
PHA
+ IL-2
92,466 (117.5) 249,033 (316.4) 14,895 (9.9) 13,419 (8.9) 25,558 (13.6) 20,336 (10.8) 5,244 (8.9) 1,399 (2.3) 10,397 (4.4) 92,979 (39.5) 3,503 (7.5) 654 (1.4) 83,894 (15.5) 108,678 (20.0) 2,639 (2.7) 1,890 (1.9) 23,656 (5.8) 46,017 (11.3) 1,138 (3.3) 824 (2.4)
"Control and 7-irradiated (3000 rad) PBMC were cultured with lo00 Ulml IL-2 and/or 0.01 pglml PHA for 5 days in AIM-Vmedium. 100 pl of fresh medium with the indicated stimulators were added every 4 days of culture. bCells are pulsed with 1 pCi/well "-Tdr on the indicated days for 8 h. The amount of 3H-Tdr incorporated in the cells were determined by scintillation counter. 'Stimulation index is the ratio of the cpm of the stimulated cultures to the cpm of the cultures in medium only.
Table II. Surface phenotype of PBMC and y-irradiated PBMC after stimulation with IL-2 and/or PHA" Cell treatment -~
non-irradiated PBMC only PBMC + IL-2 PBMC PHA PBMC IL-2 i PHA y-Irradiated PBMC only PBMC + IL-2 PBMC PHA PBMC + IL-2 -t PHA
+ +
+
Leu 11 Leu 19 CD2
CD3
CD4
CD8 IL-2Rb
10.20 0.56 12.02 8.87
21.86 10.57 19.24 14.13
52.46 72.90 73.85 74.09
66.02 84.61 69.05 80.77
34.44 28.27 27.38 29.50
22.21 3.46 27.60 8.23 26.91 20.00 39.02 35.34
2.27 7.28 2.17 6.8
22.73 13.67 8.29 9.84
48.12 82.19 56.95 54.86
73.14 94.63 86.64 90.82
26.22 8.66 2.80 33.75 33.16 13.76 46.66 21.25 3.59 29.34 30.10 2.80
~
aControl and y-irradiated (3000 rad) PBMC were cultured with loo0 Ulml IL-2 andlor 0.01 pglml PHA for 5 days in RPMI containing 10%autologous serum. The surface phenotype of the viable cells was determined by monoclonal antibodies and flow cytometric analyses as described in the Materials and Methods section. bIL-2R = IL-2 receptor
assay (Figs. 5A and 5B). The LAK activity was highest in the CD3- population, although CD3+cells could also kill K562 tumor cells (Fig. 5A). Finally, both CD8+
74
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untreated --t CD3 pretreat CD3+
+
U CD3-
-.-
Leu19 pretreat U CDlQ-
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g
1O:l
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25
---*----=-+ Leu19 pretreat
20 15
+ LeulQ-
10
0 d z 5 0
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101
5:l
231
EFFECTOR: TARGET RATIO
Fig. 5. Irradiated PBMC are incubated for 5 days in loo0 U/ml and 0.01 pglml PHA. The viable cells are then sorted into Leu 19, CD3 (A) and CD8 (B) expressing and nonexpressingpopulations by monoclonal antibodies and flow cytometry. The sorted cell subpopulationsare then assayed in triplicate for LAK activity with a standard 4 h Wr-release assay. The tumor targets used were K562 cells. Data obtained from one representativeexperiment of two are presented as mean percent cytotoxicity f SE.
and CD8- cells expressed comparable cytolytic activity for K562 cells (Fig. 5B), indicating that the increased numbers of CD8' cells, observed when IL-2 was added to the cell culture, were not responsible for the observed rise in cytotoxic activity. The y-irradiated cells expressingthe highest levels of LAK activity were the CD3cells. Similar results, but with considerably lower levels of cytotoxicity, were observed with Daudi targets.
Discussion The data presented in this paper demonstrate that PBMC, when irradiated with high dose (1000-5000 rad) y-radiation, partially retain their ability to be stimu-
y-Irradiated PBMC Express LAK Activity
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lated by IL-2 to express LAK activity ( p < 0.05). The addition of mitogen and PHA significantly increases the viability of these y-irradiated LAK cells ( p C 0.05). The phenotype of the effector cells mediating the radiation-resistantLAK activity cells were identified using monoclonal antibodies and flow cytometry. The CD3- cells expressed the highest, while the Leu 19' cells expressed the lowest levels of cytotoxic activity. Moderate levels of LAK activity were observed with the CD3+, and CDS' and CD8- subsets of T cells. These observations support the conclusion that the phenotype of irradiated cells can express LAK activity similar to those of the non-irradiated PBMC [3, 21, 221. The observationthat highdose (1000-5000 rad) irradiation did not completely eliminate LAK activity is somewhatunexpected. PBMC irradiated with 2000-3000 rad is a routine protocol for the generation of feeder cells [17-221. These feeder cells are used together in the presence IL-2 and/or mitogen, usually PHA, in the cloning of LAK, NK or T cells. Addition of feeder cells to the cloned population every 3-7 days is not uncommon. Under such cloning conditions and based on the data presented above, it is conceivable that some of the cytotoxic activity observed with cloned cells may be due to residual feeder cells. To eliminate such a contamination, cloned cells should be used in functional assays at least 12-15 days after the last addition of feeder layer, since most of the feeder cells should have disintegrated by then. The primary target of ionizing radiation in lymphocytes is believed to be DNA and the radiation results in interphase and/or mitotic cell death [23-251. Miyahoshi et al. have suggestedthat IL-2-stimulated lymphocyteshave some capacity to repair y-radiation-induced, sub-lethal damage [24]. Although the y-radiation doses used in our studies are greater than theirs, and were not split doses, IL-2 and PHA may act synergistically to generate some repair capacity in the irradiated lymphocytes. This repair mechanism may then act to prolong the survival of a larger percentage of lymphocytes, and to induce proliferativecell death, rather than mitotic cell death in the irradiated PBMC [25]. An alternativeexplanation for the ability of some irradiated PBMC to express cytolytic LAK activity is that induction of cytotoxicity is relatively radio resistant, and is not dependent on the ability of the replicative capacity of the LAK cell precursors. This is supported by reports that demonstrate that selected genetic repertoires are expressed, and certain cell functions are retained in heavily irradiated cells [26, 271. If this hypothesis was true, then it would imply that the IL-2 can directly induce activation and differentiation of LAK cell precursors to express non-MHC-mediated cytotoxicity.
Acknowledgments This work was supported by the following grants from the American Cancer Society, Illinois Division (#90-38),Arizona Disease Commission Grant ( 8 2 7 7 - ~ 1 4 - Y R - 9 3 0 1 ) and the National Institutes of Health (COREgrant #CA 23074 and MOPP grant #CA 17094).
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We gratefully acknowledge the Cetus Corporation, Emeryville, CA, for the gift of rhIL-2. We also thank Kathy Grenier for her excellent technical assistance with flow cytometry, members of the physics section, Department of Radiation Oncology for technical assistance and dosimetry and Ds run A. Boursy for his helpful discussionsand statistical analyses.
References 1 Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA. Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J Exp Med 1982;157:1356-1361. 2 Brooks CG, Holscher M, Urdal D. Natural killer activity in cloned cytotoxic T lymphocytes: regulation by interleukin 2, interferon, and specific antigen. J Immunol 1985;135:1145-1152. 3 Grimm EA, Robb RJ, Roth JA, Neckers LM, Lachman LB, Wilson DJ, Rosenberg SA. Lymphokine-activatedkiller cell phenomenon. III. Evidence that IL-2 is sufficient for direct activation of peripheral blood lymphocytes into lymphokine-activated killer cells. J Exp Med 1983;158:1356-1361. 4 Grimm EA. Human lymphokine-activatedkiller cells (LAK cells) as potential immunotherapeutic modality. Biochem Biophys Acta 1986;865:268-279. 5 Herberman RB. Interleukin-2 therapy of human cancer: potential benefits versus toxicity. J Clin Oncol 1989;7:1-4. 6 Hersey P, Bohlius R. “Nonspecific” MHC-unrestricted killer cells and their receptors. Immunol Today 1987;8:223-238. 7 Ottow RT, Steller EP, Sugarbaker PH, Wesley RA, Rosenberg SA. Immunotherapy of intraperitoneal cancer with interleukin 2 and lymphokine activated killer cells reduces tumor load and prolongs survival in murine models. Cell Immunol 1987;104:366-377. 8 Lotze MT, Rayner AA, Grimm EA. Problems with the isolation of lymphoid clones with reactivity to human tumors. Behring Inst Mitt 1985;77105-114. 9 Mule JJ, Yang J, Shu S , Rosenberg SA. The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: direct correlation between reduction of established metastases and cytolytic activity of lymphokine-activated killer cells. J Immunol 1986;136:3899-3909. 10 Ortaldo JR, Longo DL. Human natural lymphocyte effector cells: definition, analysis of activity and clinical effectiveness. JNCI 1988;80:999-1010. 11 Herberman RB, Hiserodt J, Vujanovic N, Balch C. Lymphokine-activatedkiller cell activity. Characteristics of effector cells and their progenitors in blood and spleen. Immunol Today 1987;8:178-181. 12 Itoh K, Tdden AB, Kumagai K, Balch CM. Leu-11+lymphocytes with natural killer (NK) activity are precursors of recombinant interleukin 2 (rIL-2)-induced activated killer (AK) cells. J Immunol 1985;134:802-807. 13 Ortaldo JR, Mason A, Overton R. Lymphokineactivated killer cells. Analysis of progenitors and effectors. J Exp Med 1986;164:1193-1205. 14 Phillips JH, Lanier LL. Dissection of the lymphokine-activated killer phenomenon. Relative contribution of peripheral blood killer cells and T-lymphocytes to cytolysis. J Exp Med 1986;164:814-825. 15 Moretta L, Pende D,Cossani R, Merli A, Bagnasco M, Mingari MC. T-cell nature of some lymphokine-activatedkiller (LAK) cells. Frequency analysisof LAK precursors within human T-cell populations and clonal analysis of LAK effector cells. Eur J Immunol 1986;16:1623-1625.
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16 Tilden AB, Itoh K, Balch CM. Human lymphokine-activated killer (LAK) cells: identification of two types of effector cells. J Immunol 1987;138:1068-1073. 17 Allavena P, Ortaldo JR. Specificity and phenotype of IL-2 expanded clones of human large granular lymphocytes. Diag Immunol 1983;1:162-167. 18 Anichini A, Fossati G, Panniani G. Clonal analysis the cytolytic T-cell response to human tumors. Immunol Today 1987;8:385-390. 19 Lotze MT, Chang AE, Seipp CA, Simpson C, Vetto JT, Rosenberg SA. High dose recombinant interleukin-2 in the treatment of patients with disseminated cancer: responses, treatment-related morbidity, and histologic findings. J Am Med Assoc 1986;256:3117-3124. 20 Miescher S, Whiteside TL. Moretta L, Von Fliedner V. Clonal and frequency analysis of tumor-infiltratingT lymphocytes from human solid tumors. J Immunol 1987; 138:4004-4011. 21 Rayner AA,Grimm EA, Lobe MT, Wilson DJ, Rosenberg SA. Lymphokine-activated killer (LAK) cell phenomenon. IV. Lysis by LAK cell clones of fresh human tumor cells from autologous and multiple allogeneic tumors. JNCI 1985;75:67-76. 22 Rayner AA, Grimm EA, Lotze MT, Chu EW, Rosenberg SA. Analysis of factors relevant to the immunotherapy of human cancer. Cancer 1985;55:1327-1333. 23 Hall EJ. Radiation damage and the dose-rateeffect in radiobiology for the radiologist. Radiobiology for the Radiologist.Philadelphia: JB Lippincott Company. 1988:108-136. 24 Munro, TR. The relative radiosensitivity of the nucleus and cytoplasmof the Chinese hamster fibroblasts. Radiat Res 1970;42:451-470. 25 Miyakoshi J, Tatsumi K, Tabebe H. Radiation sensitivityof T-lymphocytes grown with recombinant human interleukin-2. Mutation Res 1987;192:163-174. 26 Gerber M, Guichard M, Pioch Y, Dubois JB. The influence of interleukin-2, feeder cells, and timing of irradiation on the radiosensitivity of human T lymphocytes assisted by the colony-forming assay. Radiat Res 1989;120:164-176. 27 Joshi DS, Xck J, Murray D, Meistrich ML. Stage-dependent variation in the radiosensitivity of DNA in developing male germ cells. Radiat Res 1990;121:274-281.
Gamma-Irradiated Peripheral Blood Mononuclear Cells Can Express LAK Activity Anita S.-E Chong".b,c,D. E. Bier! U? J. Grimes', E. M. Hershb.' aDepartment of General Surgery, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois, USA; bDepartments of Internal Medicine, 'Biochemistry, dRadiation-Oncology, and eImmunology/Microbiology, The University of Arizona, Tucson, Arizona, USA. Key Words. Feeder cells ' LAK
IL-2
*
y-Irradiation
*
Mitogen-PHA
*
Cytotoxicity
Abstract. Peripheral blood mononuclear cells (PBMC) irradiated with high dose y-radiation (1000-5000 rad) are commonly used as feeder cells during the cloning of T lymphocytes, natural killer (NK) and lymphokine activated killer (LAK) cells. We report here that such y-irradiated PBMC can be stimulated with interleukin 2 (IL-2) to express the ability to lyse a variety of tumor cell targets. The non-major histocompatibility complex (MHC) restricted cytotoxicity demonstrated by irradiated PBMC is, however, lower than that expressed by their non-irradiated counterparts. The numbers of viable, ?irradiated LAK cells are significantly increased by the addition of the mitogen, phytohernagglutinin(PHA). Purification of the y-irradiated cells expressing cytotoxic activity by flow cytometry determined that the effector cells were predominantly CD3- cells, although some CD3' cells also expressed moderate LAK activity. The ability of y-irradiated cells to proliferate in the presence of PHA alone, or with IL-2 PHA, was maximal at day 4-5; but proliferation, as detected by 3H-thymidmeuptake, was not detectable beyond 12-15 days of in vitro culture. Because many of the LAK, T cell and NK cell cloning procedures require the presence of feeder layers, growth factors (usually IL-2) and mitogens, the presence of residual feeder cells expressing cytotoxic activity may affect the specificity of such clones. Thus, efforts should be made to ensure that such y-radiation-resistant cells capable of expressing cytotoxic activity are completely eliminated before the cloned cells are used for further experiments.
+
Introduction Peripheral blood mononuclear cells (PBMC) are stimulated by interleukin 2 (IL-2) to exhibit the ability to kill a wide range of tumor cell types in vitro [l-31. Correspondence: Dr. Anita S.-F. Chong, Department of General Surgery, RushPresbyterian-St. Luke's Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612, USA. Received June 27,1990; provisionally accepted August 8,1990; accepted for publication October 5, 1990. 0737-14541911$2.00/0 QAlphaMed Press
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The non-major histocompatibilitycomplex (MHC) restricted cytolytic activity is termed lymphokine-activatedkiller (LAK) activity [4-lo]. It is now clear that a variety of cell types from peripheral blood can exhibit LAK activity. The cell type that expressesthe highest level of LAK activity is the IL-2-stimulatedNK cell expressing the Leu 19(CD56),Leu ll(CD16),CD3- phenotype [ll-141. However, T lymphocytes that express Leu 19’have also been reported to exhibit significant LAK activity [fi, 161. It is known that irradiated cells retain some of their functions for a short period of time. We report here the PBMC pretreated with 1000-5000rad y-radiation can still be stimulated by IL-2 to exhibit LAK activity. The y-irradiated cell population expressing LAK activity is similar to that reported for non-irradiated PBMC, where the predominant cytotoxic cell population is CD3-. Since irradiated (2000-5000rad) PBMC, IL-2, and PHA have all been used in the generation of T, NK, and LAK cell clones [17-221, our findings indicate that steps should be taken to ensure that these y-radiation-resistantcells capable of expressing LAK activity are completely eliminated from the populations before they are used in further experiments. Materials and Methods
Cells and Culture Conditions PBMC were isolated from normal donor blood by Ficoll-hypaque separation (Pharmacia, Piscataway, NJ). Cells collected from the interface were washed with Hanks buffered salt solution (HBSS; GIBCO, Grand Island, NY) and resuspended to required concentrations. Complete medium consists of RPMI-1640 (GIBCO), 10% heat-inactivated fetal calf serum (FCS; GIBCO) supplemented with 100 U/ml each of penicillin and streptomycin (GIBCO). PBMC were resuspended at 1 X lo6cells/ml in complete medium or in defined serum-free medium (AIM-V; GIBCO). To stimulate PBMC, 1000 U/ml recombinant human (rh)IL-2 (a gift from Cetus Corp., Emeryville, CA) and/or 0.01 pg/ml purified PHA from Phaseolus spp. (HA 16; Burroughs Wellcome, Research Triangle Park, NC) were used, unless otherwise indicated. Cells were incubated in a humidified, COz incubator at 37” C. Non-adherent cells were collected after 5 days, washed and adjusted to the required concentrations for further use. The tumor cell lines K562 (erythroleukemia)and Daudi (Burkitt lymphoma), were maintained as suspension cultures, and MCF-7 (breast carcinoma), A375M (melanoma), and primary cultures of skin fibroblast cells were maintained as adherent cultures. All cells were maintained in RPMI-1640 (GIBCO), 10% heatinactivated fetal calf serum (FCS) with 100 U/ml each of penicillin and streptomycin (complete medium), and were subcultured every 3-4 days. All cell lines except A375M (gift from J. Fidier, M.D. Anderson Cancer Center, Houston), and skin fibroblastswere obtained from the American Type Culture Collection. Skin fibroblasts were cell outgrowths from normal skin (gift from Dr. S . Leong, Anzona Cancer Center, Tucson, AZ), cultured in RPMI, supplemented with 10% FCS and penicillin, streptomycin and gentamycin (50 pg/ml; GIBCO).
?-Irradiated PBMC Express LAK Activity
67
ylrradiation of PBMC PBMC were resuspended in complete medium or AIM-V, placed in a Petri dish (Falcon, Becton Dickinson, Lincoln Park, NJ), and irradiated with 1000-5000 rad, using a 4MV linear accelerator (Varian Associates, Pa10 Alto, CA). Calibration was performed weekly and technical assistance and dosimetry were provided by the Physics Section, Department of Radiation Oncology, University of Arizona. Irradiated cells were washed once and resuspended in the indicated culture medium at a concentration 1 X 10Vml with 1000 U/ml IL-2, or/and 0.01 pg/ml PHA, unless otherwise indicated. After 4-5 days of culture, the viable non-adherent cells were separated on Ficoll-hypaque, washed three times in HBSS, and resuspended in complete medium for further use. Assay for Cell-Mediated Cytotoxicity A modification of the standard Tr-release assay was used. Target cells were incubated overnight at 1 X 106/mlin complete medium containing 100 pCi/ml of W r . Target cells were washed, and 5 X lo3to 10 x lo3labeled target cells were incubated with the indicated ratios of effector cells for 4 h in a total volume of 200 pl in round-bottom microtiter plates (Costar, Boston, MA). After 4 h, 100 pl of supernatant were harvested and assayed for released T r with a y-counter. All assays were performed in triplicate. Percentage specific lysis (SL) was calculated as follows: % SL = cpm - SR x 100 MR - SR cpm being the mean counts-per-minute of the experimental sample, SR being the mean spontaneous release and MR being the mean maximum release of 1 X lo4 SICr-labeled cells incubated with 0.1 M HCI. Proliferation Assay Cells were plated in quintuplicate at a density of 1 x 10s cells/well, with 1000Ulml IL-2, or 0.1 pg/ml PHA, or both IL-2 and PHA. For long-term cultures, 100 pI/well of spent medium was removed and 100 &'well fresh medium, with the indicated additives, was added to each well every fourth day. Proliferation was assessed by measuring tritiated thymidine (3H-Tdr)uptake (2 mCi/mM; NEN/ Dupont, Boston, MA) during the last 8 h of incubation. Each well received 1 pCi of 3H-Tdr on the days indicated. Cells were harvested on a glass filter using a cell harvester (Cambridge Tech. Inc., Cambridge, MA), and the amount of 3H-Tdr incorporated by the cells determined by scintillation counter. Monoclonal Antibody Staining and Flow CytometricAnalysis of the Cloned
LA K Cell Population The surface phenotypes of the PBMC were determined by the binding of fluorescein isothiocyanate- and phycoerythrin-conjugatedmonoclonal antibodies (Becton Dickinson, Mountain View, CA).The test antibodies were Leu 19, Leu 7,
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Leu 11 (CD16), Leu 4 (CD3), Leu 2 (CD8), Leu 3 (CD4) and anti-IL-2 receptor (CD25). Controls using non-specific IgG were performed to determine non-specific binding. Cells were harvested and washed three times in PBS and made up to 2 x lo7cells/ml. Fifty microliters of the cell suspension were incubated with the conjugated antibodies, at concentrations recommended by the manufacturer, for 20-30 min at 4°C. Cells were washed twice in PBS then fixed in 1% formalin. Fixed cells were analyzed by flow cytometry on FACSCAN (Becton Dickinson). Purification of Subpopulations of Effector, ?Irradiated Cell Populations PBMC were irradiated at 3000 rad, and then stimulated with 1000 U/ml IL-2 and 0.01 pg/ml PHA for 5 days in complete medium. The cells were then centrifuged on Ficoll-hypaque to enrich for viable cells. Cells were then washed 2 x in HBSS, and resuspended at 1 X lo7celldm1 in HBSS. Viable cells were incubated with 100 ,ul/ml of antibody for 30 min on ice. Fluorescein isothiocyanateand phycoerythrin-conjugated monoclonal antibodies, Leu 4, Leu 2 and Leu 19 were purchased from Becton Dickinson. Excess antibodies were removed and cells washed 2 x in HBSS. Cells were then resuspended at 1 X lo7 cells/ml in complete medium before sorting under sterile conditions on FACSTAR (Becton Dickinson). Only strongly positive cells and strongly negative cells were collected on ice in 20% FBYRPMI. Cells were washed, counted and their cytolytic potential measured in a 4 h Wr-release assay. Cells pretreated with antibody, but not sorted, were also assayed as controls for the effects of antibody binding to the lymphoid cells. Statistics Paired, one-tailed, Student’s t test was performed on data to test their significance, using the Statsview 512+ program (Brainpower Inc., Calabasas, CA) for the Macintosh SE/30 computer.
Results y-Irradiated PBMC Are Stimulated by IL-2 to Exhibit LAK Activity PBMC, or y-irradiated PBMC, were cultured in complete medium, either alone or supplemented with IL-2 and/or PHA. After 5 days, the viable cells were assayed for their ability to lyse Daudi and K562 cells. Figure 1 demonstrates that both PBMC and y-irradiated PBMC were stimulated by IL-2 to exhibit LAK activity. Both non-irradiated PBMC and irradiated PBMC, co-stimulated with IL-2 + PHA, consistently expressed lower levels of cytotoxic activity for the tumor cells than when stimulated with IL-2 alone. The y-irradiated cells exhibited a lower cytolytic activity than their non-irradiated counterpart. Finally, K562 cells were more susceptible than Daudi cells to the cytolytic activities of the irradiated LAK cells ( p < 0.05). Data from one donor is presented in Figure 1, although similar results were observed with PBMC from 5 different donors.
69
y-Irradiated PBMC Express LAK Activity
100
'
1A:mn-irradlatmdMecion. Daudl tarpels
1 C non-radiatedaffecton. K562 targets
. 100
20 40
'
0
201
101
5:l
231
2O:l
5 1
101
2.5:1
x
0 'O
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'
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18:inidlated affecton. Dud1lug&
80
1 D irradiated Mecton. K562 tarpats
- 60
.#
40
201
l0:l 5:l 251 EFFECTOR : TARGET
2O:l
101 5:l EFFECTOR : TARGET
2.5:1
Fig. 1. Irradiated PBMC partially retain their ability to be stimulated by IL-2 to express LAK activity. Irradiated @ and D) and non-irradiated (A and C) PBMC are incubated for 5 days in medium (0, 1OOO U/ml IL-2 (0, R),0.01 pg/ml PHA ( X , A ) or both IL-2 +PHA (El, A). The viable cells are harvested and assayed in triplicate for LAK activity using a standard 4 h SICr-releaseassay. The tumor targets used were Daudi (A, B) and K562 (C, D) cells. Data obtained from one representative experiment of five are presented as mean percent cytotoxicity f SE.
o),
To investigate the radiation sensitivity of the LAK cell precursors, PBMC were subjected to increasing doses of y-irradiation. The cell viabilities, as determined by their ability to exclude trypan blue, and cytotoxic activities of these cells stimulated for 5 days with IL-2, were examined and the results presented in Figure 2. Increasing doses of y-radiation decreased the number of viable cells as well as their cytotoxic activities against K562 and Daudi cells. Specificiry of the LAK Activity in y-Irradiated PBMC IL-2-activated PBMC are reported to kill NK cell-resistant tumor cells and cultured primary-fibroblasts, but not fresh, unstimulated PBMC. We examined the ability of y-irradiated PBMC, stimulated with IL-2 PHA, to kill two NK cell-resistant cell lines (MCF-7 and A375M), fibroblasts and PBMC. Figure 3 demonstrates that y-irradiated PBMC, stimulated with PHA IL-2, exhibit LAK activity similar to, albeit lower, than non-irradiated IL-2 activated PBMC, and were able to kill primary fibroblast cells, MCF-7 and A375M cells, but not fresh PBMC (Fig. 3).
+
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ChonglBierlGrimeslHersh
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3000
4000
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0
1000
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GAMMA-RADIATION (RADS)
Fig. 2. The ability of PBMC to express LAK activity is radiosensitive. PBMC were irradiated at the indicated doses, then cultured in IL-2 (1000 U / d ) and PHA (0.01 pglml) for 5 days. The cells were centrifuged on Ficoll-hypaque and the number of viable cells determined by trypan blue exclusion on a hemocytometer (A). 100 % represents the nurnber of cells at the start of the experiment. The viable cells are then assayed in triplicate for LAK activity with a standard 4 h W3-release assay (B). The tumor targets used were Daudi (H)and K562 (0)cells. Data obtained from one representative experiment of two, are presented as mean percent cytotoxicity k SE.
LAK Activity by Irradiated PBMC is Dependent on IL-2 Concentration The effects of different concentrationsof IL-2 and PHA on y-irradiated PBMC were determined next. Irradiated cells were cultured with different concentrations of IL-2 and/or PHA for 5 days. The viable cells were counted and assayed for their ability to kill Daudi and K562 cells (Fig. 4). IL-2 only marginally increased the number of viable cells recovered from 8% to approximately 20%. However, the addition of increasing concentrationsof PHA, from 0 to 0.1 pg/ml, dramatically increased the number of viable cells recovered from 20% to approximately 70%; 100%represents the total number of PBMC at the start of the experiment. In contrast, the cytolytic activity of the surviving y-irradiated cells for K562 and Daudi targets was primarily dependent on the IL-2 concentration. Proliferation of y-Irradiated PBMC when Stimulated with IL-2 or PHA A time course to determine the survival of y-irradiated cells, measured by the uptake of 3H-Tdr,when stimulatedwith IL-2 andor PHA, was next performed. 3H-Tdr uptake by the irradiated cells was highest 4-5 days after y-radiation of the PBMC, beyond which the uptake of 3H-Tdr by the irradiated cells declined, and there was no detectable uptake after 15 days (Table I). These data indicate that the survival of the irradiated cells was limited. In contrast, the non-irradiated PBMC continued to proliferate beyond 20 days when cultured under similar culture conditions. Su$ace Phenotype of Surviving y-Irradiated Cells The phenotypes of surviving y-irradiated PBMC stimulated with IL-2 andlor
71
?-Irradiated PBMC Express LAK Activity
60
60
3: PBMC
3: FIBROBLAST
401
50
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c
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5:l
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;& 3: MCF-7
101
5:l 251 1.25:l EFFECTOR :TARGET
%
5:l
2.5:l
1.25:l
\
101
60
3: A375M
t
50 40
5:1 2.5:l 1.253 EFFECTOR : TARGET
Fig, 3. Specificity of LAK activity expressed by lL-2-activated, yirradiated PBMC. Irradiated PBMC are incubated for 5 days in loo0 U/ml IL-2 and 0.01 pglml PHA (A). Nonirradiated PBMC were cultured in medium (U)or with loo0 Ulml (H) for 5 days. The viable cells are then assayed in triplicate for LAK activity with a standard 4 h 51Crrelease assay. The targets used were fresh PBMC, primary-cultured fibroblasts, MCF-7 and A375M cells. Data obtained from one representative experiment of three are presented as mean percent cytotoxicity f SE.
PHA were determined by flow cytometric analysis. As expected, both startingPBMC and IL-2-activated PBMC population were heterogeneous, consisting largely of T cells of either CD4 or CD8 phenotype, with approximately 10%NK cells (Leu U+, Leu 19.) (Table XI). -,-irradiation consistently (4out of 4 donors tested) reduced the percent of surviving CD8 and Leu 11 expressing cells compared to their nonirradiated counterpart ( p < 0.05). Stimulation of the y-irradiated PBMC with IL-2 and/or PHA increased the number of surviving CD8 cells ( p < 0.05), but not the Leu 11 cells ( p > 0.05) (Table II). Considerable variation in the actual percentages of cells expressing each phenotype was observed among different donors (A. Chong, data not shown), and based on such phenotypic analyses we could not determine the phenotype of cells expressing LAK activity. Determination of y-Irradiated-LymphoidPopulation Expressing LAK Activity The cell that expresses high levels of LAK activity in peripheral blood is reported to be the NK cell. To determine the phenotype of cells that express LAK activity, -,-irradiated lymphocytes were incubated for 5 days in IL-2 and PHA.
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60
40
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0
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Fig. 4. Phenotypeof the irradiated PBMC that express LAK activity. Irradiated PBMC are incubated for 5 days in the indicated concentrationsof E-2 and 0.1 (E3) ,0.01 (8),0.001 (H)pgglml PHA. The viable cells were determined by trypan blue dye exclusion, and then counted by hemocytometer (A). The cells were also assayed in triplicate for LAK activity with a standard 4 h 50-release assay. The targets used were Daudi (B) and K562 (C) cells. Data obtained from one representativeexperiment of four are presented as mean percent cytotoxicity & SE.
The viable cells were then sorted into populations that expressed, or did not express, CD3,CD8 and Leu 19. The cytolytic activity of each of these cells for K562 tumor cells is presented in Figure 5. Leu 19- cells exhibited virtually undetectable cytolytic activity, and there were insufficient Leu 19' cells to perform a cytotoxicity
y-Irradiated PBMC Express LAK Activity
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Table I. Proliferative index of y-irradiated cells stimulated with IL-2 and/or PHA Mean cpmb (stimulation index)' Medium Day 4, non-irradiated" Day 4, yirradiated Day 8, non-irradiated Day 8, yirradiated Day 12, non-irradiated Day 12, yirradiated Day 15, non-irradiated Day 15, y-irradiated Day 20, non-irradiated Day 20, yirradiated
IL-2
787 1,512 1,881 589 2,355 465 5,423 970 4,063 343
PHA
35,548 (45.2) 1,759 (1.2) 56,652 (30.1) 2,291 (3.9) 61,483 (26.1) 973 (2.1) 85,682 (15.8) 1,248 (1.3) 68,541 (16.9) 459 (1.3)
PHA
+ IL-2
92,466 (117.5) 249,033 (316.4) 14,895 (9.9) 13,419 (8.9) 25,558 (13.6) 20,336 (10.8) 5,244 (8.9) 1,399 (2.3) 10,397 (4.4) 92,979 (39.5) 3,503 (7.5) 654 (1.4) 83,894 (15.5) 108,678 (20.0) 2,639 (2.7) 1,890 (1.9) 23,656 (5.8) 46,017 (11.3) 1,138 (3.3) 824 (2.4)
"Control and 7-irradiated (3000 rad) PBMC were cultured with lo00 Ulml IL-2 and/or 0.01 pglml PHA for 5 days in AIM-Vmedium. 100 pl of fresh medium with the indicated stimulators were added every 4 days of culture. bCells are pulsed with 1 pCi/well "-Tdr on the indicated days for 8 h. The amount of 3H-Tdr incorporated in the cells were determined by scintillation counter. 'Stimulation index is the ratio of the cpm of the stimulated cultures to the cpm of the cultures in medium only.
Table II. Surface phenotype of PBMC and y-irradiated PBMC after stimulation with IL-2 and/or PHA" Cell treatment -~
non-irradiated PBMC only PBMC + IL-2 PBMC PHA PBMC IL-2 i PHA y-Irradiated PBMC only PBMC + IL-2 PBMC PHA PBMC + IL-2 -t PHA
+ +
+
Leu 11 Leu 19 CD2
CD3
CD4
CD8 IL-2Rb
10.20 0.56 12.02 8.87
21.86 10.57 19.24 14.13
52.46 72.90 73.85 74.09
66.02 84.61 69.05 80.77
34.44 28.27 27.38 29.50
22.21 3.46 27.60 8.23 26.91 20.00 39.02 35.34
2.27 7.28 2.17 6.8
22.73 13.67 8.29 9.84
48.12 82.19 56.95 54.86
73.14 94.63 86.64 90.82
26.22 8.66 2.80 33.75 33.16 13.76 46.66 21.25 3.59 29.34 30.10 2.80
~
aControl and y-irradiated (3000 rad) PBMC were cultured with loo0 Ulml IL-2 andlor 0.01 pglml PHA for 5 days in RPMI containing 10%autologous serum. The surface phenotype of the viable cells was determined by monoclonal antibodies and flow cytometric analyses as described in the Materials and Methods section. bIL-2R = IL-2 receptor
assay (Figs. 5A and 5B). The LAK activity was highest in the CD3- population, although CD3+cells could also kill K562 tumor cells (Fig. 5A). Finally, both CD8+
74
Chong/Bier/Grimes/Hersh
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untreated --t CD3 pretreat CD3+
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-.-
Leu19 pretreat U CDlQ-
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+ LeulQ-
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EFFECTOR: TARGET RATIO
Fig. 5. Irradiated PBMC are incubated for 5 days in loo0 U/ml and 0.01 pglml PHA. The viable cells are then sorted into Leu 19, CD3 (A) and CD8 (B) expressing and nonexpressingpopulations by monoclonal antibodies and flow cytometry. The sorted cell subpopulationsare then assayed in triplicate for LAK activity with a standard 4 h Wr-release assay. The tumor targets used were K562 cells. Data obtained from one representativeexperiment of two are presented as mean percent cytotoxicity f SE.
and CD8- cells expressed comparable cytolytic activity for K562 cells (Fig. 5B), indicating that the increased numbers of CD8' cells, observed when IL-2 was added to the cell culture, were not responsible for the observed rise in cytotoxic activity. The y-irradiated cells expressingthe highest levels of LAK activity were the CD3cells. Similar results, but with considerably lower levels of cytotoxicity, were observed with Daudi targets.
Discussion The data presented in this paper demonstrate that PBMC, when irradiated with high dose (1000-5000 rad) y-radiation, partially retain their ability to be stimu-
y-Irradiated PBMC Express LAK Activity
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lated by IL-2 to express LAK activity ( p < 0.05). The addition of mitogen and PHA significantly increases the viability of these y-irradiated LAK cells ( p C 0.05). The phenotype of the effector cells mediating the radiation-resistantLAK activity cells were identified using monoclonal antibodies and flow cytometry. The CD3- cells expressed the highest, while the Leu 19' cells expressed the lowest levels of cytotoxic activity. Moderate levels of LAK activity were observed with the CD3+, and CDS' and CD8- subsets of T cells. These observations support the conclusion that the phenotype of irradiated cells can express LAK activity similar to those of the non-irradiated PBMC [3, 21, 221. The observationthat highdose (1000-5000 rad) irradiation did not completely eliminate LAK activity is somewhatunexpected. PBMC irradiated with 2000-3000 rad is a routine protocol for the generation of feeder cells [17-221. These feeder cells are used together in the presence IL-2 and/or mitogen, usually PHA, in the cloning of LAK, NK or T cells. Addition of feeder cells to the cloned population every 3-7 days is not uncommon. Under such cloning conditions and based on the data presented above, it is conceivable that some of the cytotoxic activity observed with cloned cells may be due to residual feeder cells. To eliminate such a contamination, cloned cells should be used in functional assays at least 12-15 days after the last addition of feeder layer, since most of the feeder cells should have disintegrated by then. The primary target of ionizing radiation in lymphocytes is believed to be DNA and the radiation results in interphase and/or mitotic cell death [23-251. Miyahoshi et al. have suggestedthat IL-2-stimulated lymphocyteshave some capacity to repair y-radiation-induced, sub-lethal damage [24]. Although the y-radiation doses used in our studies are greater than theirs, and were not split doses, IL-2 and PHA may act synergistically to generate some repair capacity in the irradiated lymphocytes. This repair mechanism may then act to prolong the survival of a larger percentage of lymphocytes, and to induce proliferativecell death, rather than mitotic cell death in the irradiated PBMC [25]. An alternativeexplanation for the ability of some irradiated PBMC to express cytolytic LAK activity is that induction of cytotoxicity is relatively radio resistant, and is not dependent on the ability of the replicative capacity of the LAK cell precursors. This is supported by reports that demonstrate that selected genetic repertoires are expressed, and certain cell functions are retained in heavily irradiated cells [26, 271. If this hypothesis was true, then it would imply that the IL-2 can directly induce activation and differentiation of LAK cell precursors to express non-MHC-mediated cytotoxicity.
Acknowledgments This work was supported by the following grants from the American Cancer Society, Illinois Division (#90-38),Arizona Disease Commission Grant ( 8 2 7 7 - ~ 1 4 - Y R - 9 3 0 1 ) and the National Institutes of Health (COREgrant #CA 23074 and MOPP grant #CA 17094).
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We gratefully acknowledge the Cetus Corporation, Emeryville, CA, for the gift of rhIL-2. We also thank Kathy Grenier for her excellent technical assistance with flow cytometry, members of the physics section, Department of Radiation Oncology for technical assistance and dosimetry and Ds run A. Boursy for his helpful discussionsand statistical analyses.
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