radioresistance of culture-induced augmented natural killer-like activity

Radioresistance of Culture-induced Augmented Natural Killer-like Activity

Charlotte Brovall,* Stuart M. Levitz,t Jerrold J. Ellner, and Bernice Schacter

A B S T R A C T : Human NK actk,ity is radiosensitive under the control of X-hnked genes. We have evaluated the expression of these genes in other forms of cdlular cytotoxicity. The NK radioresistant and radiosensitk;e phenotype is expressed in ADCC. Specific cellular eytotoxiciu, generated in a MLC with a radiosensitive donor as responder, was radioresistant. NK-like activity recruited from nonadherent cells of radiosensitive subjects stimulated with allogeneic cells, mitogens (PHA, Con A or PWM), or recall antigens (TT or PPD) was radioresistant. The acquisition of radioresistance was relatively rapid, beginning within 24 hr after exposure to PHA, prior to detectable proliferation. Radioresistance of MLR augmented NK-like activity was maximal 3 days after initiation of the culture. MLR augmented NK-like activity was spared by the immunosuppressive polypeptide antibiotic CsA at doses up to 1 tzm/ml. CsA did. however, reduce acquisition of radioresistance by the NK-like activity at doses above 0.01 tzgm/ml, a concentration which does not inhibit uptake of ~H-thymidine but does reduce the level of specific CML. These data suggest that mitogens and antigens, inchtding allogeneic cells, are recruiting radioresistant NK-like activity which can be distinguished from the radiosensitive spontaneous NK activity of the cell donor. Further, in the MLR, both radiosensitk,e and radioresistant NK-like activity may be recruited.

A B B R E V I A T I O N S ACDD antibody-dependent cellu- MLR mixed lymphocyte reaction

lar cytotoxicity c~NK augmented natural killer CML cell-mediated lympholysis N K natural killer CsA Cyclosporin A NSE nonspecific esterase Con A Concanavalin A PBMNC peripheral blood mononu- E/T effector to target ratio clear cells FCS fetal calf serum PHA phytohemagglutinin HRPMI Hepes buffered RPMI PPD purified protein derivative

1640 PWM pokeweed mitogen IL-2 Interleukin 2 TT tetanus toxoid MLC mixed lymphocyte culture

From the Departments of Pediatrics, Medicine. and Pathology. Case Western Reserve University, Cleve- land, OH 44106.

~Present Address." Department of Pediatric Oncology. Johns Hopkins Hospital. Baltimore, MD 21205. ~Present Address: Department of Medicine, Boston University. Boston, MA. Address requests for reprints to Dr. Bernice Schacter. The Institute of Pathology. 2085 Adelbert Road,

Cleveland, OH 44106. Received November 24. 1982," accepted March 4, 1983.

Human Immunology 7, 151-162 (1983) © Elsevier Science Publishing Co., Inc., 1983 52 Vanderbilt Ave., New York, NY 10017

151 o198-8859/83/$3.oo

152 C. Brovall et al.

I N T R O D U C T I O N

N K a cells have recently been characterized as large granulated lymphocytes with a kidney-shaped nucleus, azurophilic cytoplasmic granules, and a high cytoplasmic to nuclear ratio [1]. They comprise 1-2% of the total peripheral lymphocytes. N K activity is determined and defined by the ability to kill a variety of cultured tumor target cells in vitro. The biological function of N K cells in man has not been firmly established but murine studies implicate N K cells in the immuno- surveillance against spontaneously occuring tumors [2] and in recovery from certain viral infections [3,4]. Interferon augments N K activity both in vivo [5] and in vitro [6,7]. Interferon enhanced N K activity is mediated by cells of the same phenotype as those that mediate unstimulated NK activity [8].

In vitro stimulation with mitogens [9,10], PPD ~ [9], FCS ~ [9,11], allogeneic cells, and autologous lymphoblastoid cell lines [12,13] result in enhancement of N K activity. MLR ~ induced NK-like activity can be achieved with lymphocyte preparations depleted of cells expressing the Fc gamma receptor--that is, depleted of N K cells suggesting that the cell mediating MLR enhanced N K activity is different from the cell which mediates spontaneous NK activity [12,13].

We have previously shown that a gene on the X-chromosome controls the resistance of spontaneous N K cell activity to gamma irradiation [ 14]. Lymphocyte functions are known to be variably radiation sensitive [15], and MLR generated specific alloreactive cytotoxic T cells have been reported to be relatively radiation resistant [16]. In this study, we have evaluated the expression of the X-linked gene which confers radiosensitivity or resistance to spontaneous N K activity in other cell mediated cytotoxicities--ADCC ~, MLR induced specific and nonspecific cytotoxicity, as well as mitogen and antigen induced nonspecific cytotoxicity, in order to determine if radiation sensitivity can be used to distinguish these activities and the cells which effect them. In order to further evaluate the relevance of radiosensitivity in exploring the relationship between NK, ~NK ~, and specific cytotoxicity, we have evaluated the effect of the immunosuppressive polypeptide antibiotic CsA * on the MLR induced specific and nonspecific cytotoxicity and the radioresistance of the MLR induced augmented nonspecific cytotoxicity. In vitro CsA exerts a selective effect on T lymphocyte responses sparing suppressor functions [17-21]. The effects of CsA on lymphocyte activation may reflect, in part, interference with expression of receptors for IL-2 ~ [22]. The sensitivity of MLR induced ~xNK to CsA, and the radioresistance of the oeNK may thus provide information with regard to the generation of radioresistant ~NK.

M A T E R I A L S A N D M E T H O D S

Subjects. Healthy adult caucasian male and female volunteers between the ages of 24-40 with genetically determined radiation sensitive N K cells were studied [14].

Cell preparation. PBMNC* were isolated by Ficoll-Hypaque gradient centrifu- gation, washed 3 times in RPMI 1640 supplemented with HRPMI* and antibiotics (penicillin 100 unit/ml and streptomycin 100 mg/ml), followed by adherence to plastic dishes to deplete macrophages. The nonadherent cells were enriched further by passage through a nylon wool column [23]. The T cell enriched population was <0 .5% positive for nonspecific esterase and contained 75-80% E-rosetting cells. All experiments reported here were carried out with lymphocytes depleted of adherent cells.

Radioresistance of Natural Killer-like Activity 153

C u l t u r e s

Mitogens, antigens, CsA. 3 x 106 cells of the T cell enriched population suspended in 2 ml of HRPMI with 20% pooled heat inactivated human AB serum were cultured alone or with the mitogens, such as 2.5 /zg/ml P H A * (HA16, Wellcome, England), 10/.tg/ml Con A s (Pharmacia, Sweden), and l:100/ml PWM* (Gibco, Grand Island, NY) or with antigens such as 100/.tg/ml PPD ~ (Lederle, Pearl River, NY) or l:10/ml TT * concentrate (Lederle). CsA was the gift of Dr. J.F. Borel Dr. and E. Wiskott of Sandoz LTD, Basel, Switzerland. Stock solution was prepared by dissolving 1.0 mg CsA in 0.1 ml of absolute ethanol containing 20% Tween 80 (Sigma, St. Louis, MO); then, HRPMI (Microbiological Asso- ciates, Rockville, MD) was added dropwise to a final volume of 1 ml. Solvent controls were used for the CsA. CsA was added at the beginning of the MLC. Interferons [~ Interferon (Interferon Sciences, New Brunswick, NJ): ultrapuri- fled, 5 x 106 unit/mg protein, 1.5 x 106 unit/ml (Lot #2015.2) , Cat #1500); ~, Interferon purified, 1 x 106 unit/mg protein, 1 x 106 unit/ml (Lot #2006 , Cat #3200) ] were diluted in RPMI1640 to working stocks of 105 unit/ml, stored at - 70°C and added to cultures in appropriate volumes to achieve 102-103 unit/ ml.

MLC. 3 x 106 cells/ml responder T cells were cultured with equal numbers of stimulator PBMNC (allogeneic or autologous), which had been irradiated with 3000 rads, suspended in 2 ml of HRPMI and 20% AB serum. The cells cultured with the various stimuli were harvested at various times after initiation of the culture (see Results), washed 3 times in HRPMI, and adjusted to a final effector cell concentration of 1.5 x 106 cells/ml in HRPMI with 20% FCS.

CML. For assay of culture induced specific cytotoxicity, bulk MLC were established in 20% AB serum in RPMI 1640 at 2 x 106 cells/ml and 1 : 1 ratio of responders to stimulators. Stimulators received 3000 fads prior to culture. Target cells were cultured at 2 x 106/ml without PHA in 20% AB serum in RPMI 1640.

Determination of proliferation. 1 x 105 cells, suspended in 0.1 ml of HRPMI and 20% AB serum, were added to each of triplicate microtiter plate wells. Mitogens, antigens, and allogeneic cells were added in the same concentrations as indicated above for bulk cultures and the plate was incubated at 37°C and 5% CO2 in air. At the end of the culture period, the wells were pulsed with 3H-thymidine (Amersham, England) 25 Ci/ml, 5 ~Ci/well for 5 hr, and harvested onto filter discs with a cell harvester and 3H-thymidine incorporation quantified by scintil- lation spectroscopy.

Target cell. The N K target cell was the human erythroleukemia cell line K562, kindly provided by Dr. Juan Minowada of Roswell Park Memorial Institute. K562 and the lymphoblastoid cell line CCRF-CEM (American Type Culture Collection) were maintained in suspension culture in HRPMI and 20% FCS. K562 was 51Cr- labeled by incubating 2 -3 x 106 cells in 0.150 mCi sodium 51Chromate (New England Nuclear, Boston, MA) in normal saline for 90 rain at 37°C. The labeled target cells were washed 3 times and resuspended in HRPMI and 10% FCS at a concentration of 105 cells/ml. Target cells for the CML were lymphocytes, cultured as described in the CML assay, labeled as described for K562. For ADCC, cells were labeled with 51Cr, then sensitized for 30 rain at 37°C with 1 : 20 dilution of rabbit antibody raised against CCRF-CEM.

Effectors. Fresh effector cells were suspended in HRPMI with 20% FCS at 1.5 x 106 cells/ml. Prior to assay, cells which had been precultured were washed 3 times in HRPMI and similarly resuspended in HRPMI-FCS at 1.5 x 106/ml.


Irradiation. Aliquots of the effector cell suspensions were irradiated at room temperature by lowering the cells in capped plastic tubes into a cylinder of a U.S. Nuclear 6°Cobalt irradiation Model GR-9 providing approximately 1600 rads/min. Correction for decay of 6°Cobalt was made during the period of these studies. An aliquot of the cells was held at room temperature without receiving irradiation. All cells were assayed within 1 hr after irradiation.

In vitro cytotoxiciu assay. Irradiated and nonirradiated effector cells were loaded in triplicate into round bottomed wells of a microtiter tray with 5 × 10351 Chromium- labeled target cells at an E/T of 30: 1, 15: 1, and 7.5:1 in a total of 150/xl. The tray was spun at 50 × g for 5 min, incubated for 4 hr at 37°C in 5% CO2 and air, and centrifuged at 150 g for 10 min. An aliquot (50/xl) of the supernatant was then harvested into glass tubes for counting in a gamma counter. Cytotoxic activity was quantitated as percent cytotoxicity:

cpm (experimental release) - cpm (spontaneous release) × 100

cpm (total release) - cpm (spontaneous release)

Spontaneous 51Chromium release from target cells was determined in medium alone, whereas total release was determined by lysis with detergent (1% sodium

x o o

FIGURE 1 Expression of NK radiosensitivity phenotype in ADCC and NK assays. Data are expressed as percent cytotoxicity at an E/T of 30:1 for ADCC to antibody sensitized CCRF-CEM and for NK to K562. The ADCC target CCRF-CEM was sensitized with a 1:20 dilution of a rabbit serum raised against CCRF-CEM. This antibody will sensitize ADCC for other human targets including K562. Spontaneous NK activity against CCRF- CEM at 0 rads was 22.0% _+ 1.5% for the resistant subject, 16.0% -+ 3.0% for the sensitive subject; at 3000 rads 25.6% + 1.4% for the resistant subject, 3.1% -+ 0.4% for the sensitive subject. With antibody sensitized K562, ADCC at 0 rads was 75.0% -+ 1.5% for the resistant subject, 61.7% _+ 1.7% for the sensitive subject; at 3000 rads 71.5% -+ 4.0% for the resistant subject, 9.8% + 0.1% for the sensitive subject. (D) ADCC sensitive subject, (©) ADCC resistant subject, (ll) NK-sensitive subject, (Q) NK- resistant subject.

60

' ' ' o' 'o 5 0 0 I 0 0 0 2OOO 5 O0 50 0

RADS


dodecyl sulfate). In all experiments, the spontaneous release was less than 10% of the total releasable chromium.

R E S U L T S

Express ion o f Radiosens i t iv i ty in ADCC*

ADCC, mediated by peripheral nonadherent lymphocytes against CCRF-CEM, a relatively poor N K target sensitized with rabbit antibody raised against it, reflected the distinction between radioresistant and radiosensitive subjects seen with spontaneous N K activity. (A representative experiment is shown in Fig. 1.)

I n d u c t i o n o f N K Ac t iv i ty and Rad ia t ion Resis tance by P H A

Peripheral blood lymphocytes with N K activity sensitive to irradiation with 3000 rads, depleted of adherent cells and enriched for T cells, were cultured with and without P HA for up to 3 days, and N K activity, radiation sensitivity to 3000 rads of the N K activity and proliferation determined by 3H-thymidine incorporation were measured daily (Fig. 2). On day 1, the N K activity of the PHA-stimulated cells had increased significantly above the activity of the control cells and the cells assayed were partially resistant to 3000 rads (60% cytotoxicity without radiation, 30% after). The N K activity of the PHA culture peaked on day 2, at which time the activity was totally resistant to radiation. The cells remained irradiation resistant on day 3. The N K activity of the unstimulated cells did not increase above the initial cytotoxicity, and the activity remained sensitive to the radiation dose of 3000 rads throughout the culture period. The induction of radiation resistance in the PHA culture preceded proliferation, which was not detected prior to 48 hr.

100"

90"

80"

7 0 -

~ 60 -

50-

40-

30-

20 .-'" -

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0 1

....-"~

. ...-"

/ / i)> z .

i

/ .. / i

J i i

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DAYS

- 5 0 , 0 0 0

- 4 0 . 0 0 0

FIGURE 2 Augmentation of NK-like activity and induction of

-ao, ooo radiation resistance by PHA during 3 day culture. The data are expressed in percent cytotoxicity of unstimulated control cells receiving 0 (-) and 3000 fads ( - - - ) ;

-2o,ooo PHA-stimulated cells receiving 0 ( .... ); and 3000 rads ( .... ) after culture. Proliferation in response to PHA expressed in cpm

-lO,OOO of 3H-thymidine incorporated ( .... ).


I n d u c t i o n of N K Act iv i ty and Radiat ion Resistance by Other Lectins and A n t i g e n s

Similar experiments were carried out with Con A, PWM, PPD, and TT testing stimulated cells daily for up to 3 -5 days (Table 1). Cells for stimulation with PPD were obtained from an individual with a positive skin test for PPD. All stimuli resulted in augmentation of N K activity above that of unstimulated cells within 2 4 - 4 8 hr after the initiation of the culture and there was a significant increase in radiation resistant N K activity after 48 hr of culture. The ~ N K activity was totally resistant to 3000 fads at the end of the culture period (3 days for the mitogens; 5 days for the antigens).

I n d u c t i o n of N K Act iv i ty and Radiat ion Resistance after Culture w i th A l l o g e n e i c Cells in MLC

Culture with allogeneic cells in MLC produced analogous results (Fig. 3). There was a marked decline of N K activity of allogeneic and autologous effector cells on day 1 which most likely was due in part to dilution by inactive irradiated stimulator cells (which had not yet died) among the effector cells. On day 3, the activity of the effector cells from the allogeneic cultures was greatly enhanced above that of the effector cells from autologous cultures and the activity continued to be elevated on day 6. The N K activity of the cells from the autologous cultures remained sensitive to radiation with 3000 rads during the culture period, while the activity of the cells from the allogeneic cultures had become almost totally resistant (73.2% remaining activity after radiation) on day 3 and was totally

T A B L E 1 Induction of NK-like activity and radiation resistance by mitogens and antigens a

Exp. # 1 Day 0 Day 1 Day 2 Day 3 Day 5

Control

Con A

PWM

Exp. #2

Control

PPD

% cytotoxicity 40.1 ± 0.6 % cytotoxicity 2.3 ± 0.4 after 3000 rads (5.7%)

% cytotoxicity % cytotoxicity after 3000 rads


% cytotoxicity 56.2 ± 1.3 % cytotoxicity 4.7 ± 0.9 after 3000 rads (8.4%)


% cytotoxicity Tetanus

% cytotoxicity Toxoid

after 3000 rads

30.4 ± 1.3 36.4 ± 2.1 35.6 ± 0.6 1.4 ± 0.2 0.9 ± 0.6 1.4 ± 0.4 (4.6%) (2.5%) (3.9%)

57.9 ± 1.9 61.5 ± 2.5 49.2 ± 2.6 10.0 ± 1.0 48.8 ± 2.9 43.7 ± 1.7

(17.3%) (79.3%) (88.8%)

36.3 ± 1.3 54.5 ± 0.9 54.0 ± 1.6 3.2 ± 0.3 17.8 ± 1.5 39.8 -+ 0.9 (8.8%) (32.7%) (73.7%)

36.6 --+ 2,2 38.0 + 10.5 38.8 --+ 3.4 1.9 ± 0.3 0,7 ± 0.2 1.9 ± 2.1 (5.2%) (1.8%) (4.9%)

44.2 + 2.4 52.0 ± 1.8 72.2 ± 1.7 5.2 ± 0.7 35.1 ± 2.8 68.7 ± 3.0 (11.8%) (67.5%) (95.2%)

34,1 ± 1.3 45.7 -+ 5.8 68.1 ± 1.7 2.1 ± 0.5 18.0 ± 1.2 60.5 ± 3.3 (6.2%) (39.4%) (88.8%)

aCultures were established in 20% AB serum in RPMI 1640 at 2 x 10/' nonadherent cells/ml. Cells were harvested at times indicated, washed, and assayed for K562 cytotoxicity at an E/T of 30:1. Irradiation of effectors was done just prior to assay.


60 t 4 0 .

3 0 .

2 0 - , . J " ' - - - . . . . . . . . . .

10- / " ~ /

I l I I I I

0 1 2 3 4 5 6

DAYS

FIGURE 3 Augmentation of NK-like activity and induction of radiation resistance in MLC during 6 day culture. The data are expressed in percent cytotoxicity ofK562 mediated by effector cells from allogeneic culture receiving 0 (-) or 3000 rads ( - - - ) ; and effector cells from autologous culture receiving 0 ( .... ) and 3000 rads ( .... ).

resistant on day 6. Specific cytotoxicity generated in the MLC was also found to be relatively resistant to 3000 rads of radiation (Table 2).

CsA has been repor ted to spare spontaneous and interferon enhanced N K activity as well as MLR enhanced N K activity (~NK) at doses completely abro- gating the MLR and the generation of specific allocytotoxocity, suggesting a distinction between specific T cell cytotoxicity and ~ N K [25]. We have similarly observed a sparing of a N K at doses inhibiting proliferation and CML generation by CsA but have also noted that CsA, at doses not inhibitory of a N K , diminished the radioresistance of the ~NK.

T A B L E 2 Radioresistance of MLR-induced specific and nonspecific cytotoxicity

Percen t cytotoxici ty

T a r g e t

Cu l tu re I r rad ia t ion C G K 5 6 2

C x G 0 4.7 _+ 1.8 26.6 +_ 2.6 36.5 ± 1.9

3000 n.t. 22.1 +- 1.6 30.6 -+ 3.7

C × C 0 0.2 -+ 1.1 1.6 + 2.3 22.4 ± 1.5

3000 n.t. n.t. 1.6 -+ 0.2

G × C 0 20.1 -+ 1.8 - 1 . 1 -+ 2.0 23.8 -+ 1.9

3000 15.6 ± 2.5 n.t. 15.6 ± 1.0

G × G 0 1.5 ± 1.3 - 1 . 2 ± 1.6 12.7 ± 1.0

3000 n.t. n.t. 0.5 ± 0.1

Cultures were established in 20% AB serum in RPMI1640 at 2 × 10 ~' cells/cc and 1:1 ratio of stimulator to responders. Stimulators received 3000 rads prior to culture. Cytotoxicity was assayed after .7 days in culture with E/T in all cases at 30:1 in a 4 hr assay. Allogeneic targets were cultured alone without PHA. Effector cells were harvested from cultures, washed twice, and adjusted to the appropriate concentration in RPMI1640 + 10% FCS. Irradiation of effectors was done just prior to assay.


Figure 4 shows a representative experiment evaluating the effect of CsA on blastogenesis as well as generation of c~NK activity and CML in human allogeneic MLR. 3H-thymidine incorporation in allogeneic cultures was inhibited by CsA in a dose-dependent fashion with a significant effect first noted at 0 .1/ ,g/ml. The generation of CML was exquisitely sensitive to the effects of CsA as has been previously reported. Concentrations of CsA of 0 .01/ ,g/ml inhibited CML activity by 63%, and >0 .1 / , g /ml completely abolished it. The autologous cultures showed no CML activity. CsA did not affect the development of c~NK activity except at higher concentrations (10 /*g/ml; p < 0.005). Thus, the concentration of CsA required to inhibit ~ N K activity was 100- and 1000-fold greater than that required to depress blastogenesis and CML, respectively. The autologous cultures dem- onstrated reduced N K activity (p < 0.005) which also was unaffected by CsA. We also studied the effects of including CsA in the MLC on the radiosensitivity of a N K activity (Fig. 5). Following bulk MLC, with and without CsA ( .01-1.0 /,g/ml), effector ceils were irradiated with 3000 rads immediately before assay of ~ N K with no further addition of CsA. CsA partially inhibited the emergence of radioresistance in a dose-dependent fashion (p < 0.05 at 0.01 /,g/ml CsA; p < 0.005 at 0.1 /,g/ml CsA; and p < 0.0005 at 1.0/~g/ml CsA-Student 's t test). Autologous cultures, with or without CsA, failed to develop significant radioresistance. Interferon, an activator of N K activity, is produced in antigen and mitogen activated mononuclear cell cultures. We evaluated the ability of both alpha and gamma interferon to confer radioresistance to N K activity (Table 3). Both alpha and gamma IFN augment N K activity, but did not confer radioresistance.

FIGURE 4 Effect of CsA on blastogenesis and generation of CML and c~NK in allogeneic (A × Bx) and autologous (A × Ax) one-way MLC. -~H-thymidine incorporation is expressed as the mean of quadruplicate wells _+ S.D. CML (solid bars) and c~NK (dotted bars-lower graph) were determined in a 4 hr 5~Cr release assay. Results shown are at an effector cell:target cell ratio of 30:1 for oeNK and 60:1 for CML and represent the mean of triplicate determinations _+ S.D.

o

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, 2 0 I

~ 0

6o i .~ 40 n "

3 ~ 2O

0 0 0.01 0.1 1.0

i

AxBx

I0.0 0 1.0 i i i

AxAx

Concentration of C s A ( p g / m l )


6O

40 Q~

20

t-.

¢' 60

40 .9 ~: 20

g 0 , , 0 0.01 0.1 1.0

Concentration of CsA (pg/ml)

FIGURE 5 Effect of irradiation on aNK generated in bulk MLC in the presence of CsA. Effector cells harvested from 6 day bulk allogeneic one-way MLC, were irradiated with 3000 rads before assay of ~NK using 51Cr-labeled K562 target cells at an effector target ratio of 30:1. In the upper panel, c~NK activity without (-) and with ( .... ) irradiation are presented as mean of triplicate determinations _+ S.D. In the lower panel, the data are presented as percent radioresistance defined as:

NK after irradiation x 100%

NK without irradiation

D I S C U S S I O N

We have previously shown that sensitivity to gamma irradiation with 3000 rads of spontaneous N K activity of normal healthy individuals is controlled by an X- linked gene with a gene frequency of the resistant allele in caucasions of 10% [14]. We propose to call this gene the XNR. In this report , we demonstrate that the XNR gene is expressed by cells effecting ADCC activity. We also repor t that stimulation of lymphocytes initially expressing radiation sensitive N K activity with mitogens (PHA, Con A, and PWM) and antigens (PPD, TT, and allogeneic cells) will not only augment NK-l ike activity after various periods of stimulation, but will also confer radiation resistance to the nonspecific cytotoxicity or ~ N K

T A B L E 3 Interferon recruited N K activity retains radiation sensitivity phenotype

D o n o r

p h e n o t y p e A d d i t i o n to cu l tu re Tota l N K N K after 3 krad

% cyto toxic i ty

Sens i t ive 0 17.4 + / - 0.3 1.6 + / - 0.4

100 U a lpha I F N 2 4 . 8 + / - 0 . 6 1.3 + / - 0 . 6 Sens i t ive 0 12.5 + / - 0 . 5 1.2 + / - 0 . 4

100 U a lpha I F N 1 9 . 9 + / - 0 . 7 3 . 1 + / 1.9 Sens i t ive 0 8.5 + / - 1.3 0.1 + / - 0.1

1000 U a lpha I F N 22.3 + / - 1.0 1.3 + / - 0 . 8

100 U g a m m a IFN 12.2 + / - 0 . 8 1 . 8 + / - 0 . 5 1000 U g a m m a I F N 18.2 + / - 1.2 0.1 + / - 0.6

0.5 m i c r o g r a m P H A 40.4 + / - 0.8 19.7 + / - 1.9 Res i s t an t 0 15.1 + / - 2.3 26.7 + / - 2.3

100 U a l p h a l F N 3 0 . 1 + / 1.8 3 4 . 1 + / - 1 . 2 Res i s t an t 0 6.5 + / - 1.6 7.5 + / - 4 . 3

100 U a lpha IFN 2 2 . 0 + / - 0 . 8 1 7 . 0 + / - 0 . 5

NK activity was assayed at an E/T of 20:1 against K562 after 18 hr of culture of peripheral blood mononuclear cells in RPMI I640 plus 20% AB serum and gentamicin with or without addition as indicated per ml of culture. Cells were washed prior to assay.


during the culture period. The percent remaining cytotoxic activity after treat- ment of culture stimulated cells from radiosensitive subjects with 3000 rads ranged from 50 -95%. The mean remaining activity of unstimulated spontaneous N K activity of 41 normal genetically sensitive individuals is 9 .1% -+ 4 .3% (X -+ S.D.) [14].

The induction of radiation resistant NK-like activity was evident early within the first 24 hrs of culture with PHA at a time when no proliferation of the cells determined by 3H-thymidine incorporation could be detected. The mechanism by which radiation sensitive NK-like activity is rendered resistant is not known. Recrui tment of a cell to express radioresistant NK-like activity is a likely hy- pothesis. Allogeneic cells have been shown to recruit cells with NK-like activity when cultured with lymphocytes depleted of cells expressing N K activity [13]. The MLR culture kinetics are consistent with recruitment of a new cell with NK- like activity resistant to 3000 rads irradiation. In the cultures stimulated with PWM, PPD, TT, and allogeneic cells in MLC, there was a drop of NK-like activity at 24 hr of culture followed by increase in N K activity and induction of radiation resistance at 48 hr, consistent with the recruitment of a new cellular nonspecific cytotoxic activity supplementing the loss of spontaneous N K activity. TT which has been shown to activate only cells reactive with the monoclonal antibody OKT4 [25], which has been associated with helper cell function [23], induced radiation resistant NK-like activity. Spontaneous N K cells are OKT3 negative, OKT8 negative, and OKM1 positive [26,29t. It is possible that the antigen activated OKT4 positive cells can exert nonspecific cytotoxic activity themselves or the OKT4 cell may activate a second cell type to express nonspecific cytotoxicity which is radioresistant. It is unlikely to be identical to the spontaneous N K cells. Zarling and Kung reported that the MLC induced N K activity was resistant to lysis by complement and OKM1, as well as OKT3 and OKT8 [26].

Experiments combining the single cell N K assay with fluorescent staining with monoclonal antibodies will be necessary to establish the nature of the recruited cell in view of the results of Zarling and Kung. Experiments in mice have sug- gested that N K activity may be mediated by as many as four different cell types [27]. CsA at concentrations inhibiting both proliferation and generation of specific cytotoxicity in the MLR spared the c~NK activity but caused the level of radioresistance of the c~NK to diminish. These results suggest that at least two types of ~ N K are recruited in the MLR-one is radiosensitive and one is radioresistant. The radioresistant c~NK activity is not spared at doses of CsA > 0.01 ~g/ml although overall c~NK activity is maintained. CsA has been reported to act by inhibition of the development of receptors for IL-2. These results with o~NK may thus implicate IL-2 in the process of recruitment of the radioresistant ~NK. IL-2 has been shown to enhance N K activity [28]. The IL-2 enhanced N K activity in the mouse is mediated by Thyl positive cells and thus can be distinguished from the Thyl negative N K cells which respond to interferon [27]. We have observed that purified human alpha and gamma interferon enhanced N K activity remains radiosensitive, allowing further distinction between interferon enhanced N K activity and culture augmented NK-like activity.

The data in this report suggest that radiation sensitivity may be a useful tool to distinguish spontaneous N K activity from proliferation induced o~NK activity, as well as a tool to study the aspects of the N K mechanism distinctive of the spontaneous N K cell.

A C K N O W L E D G M E N T S

This work was supported in part by grants from the Cuyahoga County Chapter of the American Cancer Society and the Clark Dunlap Fund.


Dr. Jerrold J. Ellner is the recipient of a Young Investigator's Award from the National Institute of Allergy and Infectious Disease of the National Institutes of Health.

We would like to acknowledge the assistance of Kay Edmonds, Susan Hansal, and James Kadushin in the performance of these experiments and Jean Nagy in the preparation of the manuscript.

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radioresistance of culture-induced augmented natural killer-like activity

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