Characterization and activation requirements of bovine lymphocytes acquiring cytotoxic activity after interleukin-2 treatment

Veterinary Immunology and Immunopathology, 32 (1992) 205-223 205 Elsevier Science Publishers B.V., Amsterdam

Characterization and activation requirements of bovine lymphocytes acquiring cytotoxic activity

after interleukin-2 treatment

Manuel Campos a, Charles R. Rossi b, Helle Bielefeldt Ohmann a'c, Terry Beskorwayne ~, Noreen Rapin a and Lorne A. Babiuk ~'d

• Veterinary Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, Sask. STN OWO, Canada

bAnimal Health Research, Alabama Agricultural Experiment Station, and Department of Microbiology, College of Veterinary Medicine, Auburn University, Alabama, AL 36849, USA CDepartments of Veterinary Pathology and dMicrobiology, College of Veterinary Medicine,

University of Saskatchewan, Saskatoon, Sask. S7N OWO, Canada

(Accepted 17 July 1991 )

ABSTRACT

Campos, M., Rossi, C.R., Bielefeldt Ohmann, H., Beskorwayne, T. Rapin, N. and Babiuk, L.A., 1992. Characterization and activation requirements of bovine lymphocytes acquiring cytotoxic activity after interleukin-2 treatment. Vet. Immunol. Immunopathol., 32: 205-223.

Interleukin-2 (IL-2) treatment of cells and generation of non-major histocompatibility complex (MHC)-restricted cytotoxic cells from peripheral blood mononuclear leukocytes (PBML) was studied. Effector-target conjugate assays demonstrated that bovine PBML bound but did not lyse K562, HL60S and HL60R cells unless activated with IL-2. The magnitude of IL-2-activated killing of tumor cells as well as the magnitude of antibody-dependent cellular cytotoxicity depended on the IL-2 concentration. A short treatment ( 12-18 h ) of effector cells with IL-2 was sufficient for development of cytotoxic activity. Withdrawal of IL-2 from the culture resulted in a reduction of cytotoxic activity that could be restored by further addition of IL-2. Cytotoxic activity of IL-2-activated populations obtained after nylon wool or Sephadex G-10 passage, and Percoll gradient centrifugation of PBML suggests that lymphokine-activated killer (LAK) cell activity in PBML is mainly mediated by a non- adherent lymphocyte lacking markers for B-cells. Positive and negative selection experiments using cell sorting confirmed these findings and demonstrated that the cell responsible for LAK cell activity in cattle belongs to a non-monocyte, non-B, CD2 + lymphocyte population. Furthermore, cytotoxic activity could not be generated in CD2 + populations enriched for cells expressing molecules equivalent to human and murine CD4 and CDS. These findings suggest that effector cells mediating non MHC-restricted cytotoxicity in cattle prevail in a population beating a CD2 +, CD4-, CDS- phenotype and that this population depends on the continuous presence of IL-2 for optimal cytotoxic function.

ABBREVIATIONS

ADCC, antibody-dependent cell cytotoxicity; Con A, concanavalin A; FALS, forward angle light

Present address: c/o Menzies School of Health Research, P.O. Box 41096, Casuarina, Darwin, NT 081 l, Australia.

© 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05.00

206 M. ('AMPOS E ] al .

scatter, IL-2, intcrleukin-2: LI 90, 90 ~' light scatter: LAK, lymphokinc-activatcd killcr: mAb, monoclonal antibody; MHC, major histocompatibility complex; NK, natural killer: NWE, nylon wool effluent: PBML. peripheral blood mononuclear leukocytes.

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

The possibility that cytotoxic mechanisms may be involved in defence against certain neoplasms and intracellular parasites has been widely studied. Cytotoxicity mediated by bovine leukocytes has been studied by numerous investigators in recent years. The mechanisms described have included T- cell-mediated antigen-specific cytotoxicity (Eugui and Emery, 1981; Emery et al., 1981 ; Campos and Rossi, 1986a ), antibody-dependent cell cytotoxicity (ADCC) mediated by macrophages and neutrophils (Rouse et al., 1976; Wardley et al., 1976; Duffus et al., 1978; Townsend and Duffus, 1982), lectin-dependent cell-mediated cytotoxicity (Campos and Rossi, 1987), allo- reactivity (Stott et al., 1975; Teale et al., 1985) and a variety of natural or spontaneous antigen-nonspecific cytotoxicities (Stott et al., 1975; Probert et al., 1977; Honma et al., 1980; Campos et al., 1982; Yamamoto et al., 1985; Brigham and Rossi, 1986; Chung and Rossi, 1987). However, cells compa- rable to human and murine natural killer (NK) cells, the most notable cell capable of mediating antigen-nonspecific non-major histocompatibility complex (MHC) restricted cytotoxicity, have been difficult to detect in cattle, although lymphokine-activated killer (LAK) cells have been reported (Cam- pos and Rossi, 1986b; Fong and Doyle, 1986 ). LAK activity differs from NK activity in that the latter are able to lyse only certain types of cells (cultured tumor cells of the hematopoeitic lineage, and certain viral-infected cells), whereas LAK cells are able to lyse NK-susceptible and NK-resistant cells, including fresh tumor cells. The relation of LAK to NK cells has been investigated extensively. While there is disagreement regarding the relationship between these cells it is relatively certain that NK precursors and effector cells can mediate LAK activity after treatment with IL-2 (Itoh et al., 1985, 1986). What has not been determined is the extent to which cells other than NK cells contribute to the total LAK activity (Phillips and Lanier, 1986; Ortaldo et al., 1986; Ballas et al., 1987; Ferrini et al., 1987; Tilden et al., 1987; Van de Griend et al., 1987). Although we have attempted to detect NK cells in bovine peripheral blood mononuclear leukocytes (PBML), we have been una- ble to positively identify them. However, we have been able to demonstrate non-specific cytotoxicity under a variety of circumstances (Campos et al., 1982; Brigham and Rossi, 1986; Campos and Rossi, 1986b; Campos and Rossi, 1987; Chung and Rossi, 1987). In cattle a cell with functional characteristics similar to human and murine NK cells (i.e. direct lyses of a variety of tumor cells, virus-infected cells, and ADCC) can be demonstrated after

ACTIVATION AND CHARACTERIZATION OF BOVINE LAK CELLS 207

treatment of PBML with IL-2 (Campos and Rossi, 1986b). Because of our interest in detecting NK cells and the possible relationship between LAK and NK cells, we decided to investigate some of the cellular characteristics and activation requirements of bovine LAK cells.

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

Human target cells

The erythroleukemia cell li~e K562, the lymphoblastoid T-cell line HSB-2, the myeloblastic cell line KG-l and 2 sublines spontaneously generated from the promyelocytic cell line HL60 (HL60R and HL60S) were obtained from Dr. A. Tilden (Department of Surgery and Cellular Immunology Unit of the Cancer Center, University of Alabama at Birmingham, AL). Other than HL60R, the cell lines are sensitive to human NK cell lyses. The tumor cell lines were cultured at 38 °C in a humidified atmosphere containing 5% CO2 and maintained in RPMI- 1640 containing 10% FCS, 200 U ml- ~ of penicil- lin, l 0 #g ml- ~ of streptomycin, and 2 mmol L-glutamine.

Antiserum

Antiserum against HSB-2 cells was produced in calves by repeated intra- muscular injection of 10 s cells at monthly intervals. Positive serum, as determined by agglutination, was heat-inactivated (50 ° C, 30 min) and stored at - 20 ° C until used.

lnterleukin-2 and assay for IL-2 biological activity

Lymphocyte culture supernatant fluids containing IL-2 were prepared as previously described (Campos and Rossi, 1986b). Briefly, PBML were cultured in 25-30 ml in 75 c m 2 tissue culture flasks at 3.5× 10 6 cells ml -~ in Opti-MEM I medium (GIBCO, Grand Island, NY) supplemented with 3% FCS, antibiotics, and 0.1 mmol 2-mercaptoethanol. Culture flasks were incubated for 24 h at 38°C in an atmosphere containing 5% CO2. After incubation, PBML were supplemented with 5/zg ml- ~ of concanavalin A (Con A ) and 10 ng ml- ~ of phorbol myristate acetate (Sigma Chemicals, St. Louis, MO ). Supernatant fluids were harvested after 18 h of further incubation. Re- combinant human IL-2 (Hoffman-La Roche, Nutley, NJ and from Boehrin- get Mannheim Canada, Dorval, Que.) was assayed on bovine IL-2-dependent lymphoblasts which were obtained either by Con A-stimulation or by one-way mixed lymphocyte culture. The IL-2-dependent lymphoblasts were washed free of growth medium, suspended at l × 105 cells ml- ~ in medium ( R P M I - 1640 supplemented with 15% FCS, antibiotics, and 2 mmol L-gluta-

208 M. CAMPOS ETAL.

mine), added to 96-well Microtiter plates ( 100/~1 well- 1 ) and cultured for 18 h with serial two-fold dilutions of IL-2. Six hours before cultures were termi- nated, 1/~Ci of 3H-thymidine was added to each well. Cultures were harvested onto glass fiber filter strips and 3H-thymidine uptake was determined to as- sess cellular proliferation using a liquid scintillation counter.

Preparation of effector cells

Peripheral blood mononuclear leukocytes were obtained from blood of normal 12- to 24-months-old steers by venipuncture as previously described (Campos and Rossi, 1986a, b).

Sephadex G-10 effluent and nylon wool effluent (NWE) populations were obtained after passage of PBML through Sephadex GoI0 (Pharmacia, Dor- val, Que. ) and nylon wool (Fenwal Laboratories, Deerfield, IL) respectively as previously described (Campos et al., 1982; Campos and Rossi, 1985). In some experiments, the NWE cells were further fractionated using Percoll (Pharmacia) discontinuous gradient centrifugation as described by Timonen et al. (1982). Final concentrations of Percoll were prepared, at 44.2, 50.8, 55.0 and 66.7%. The fractions were carefully layered into 15 ml conical cen- trifuge tubes and 1 ml of HBSS containing 5 × 107 to 1 × 108 NWE cells was layered onto the top layer of the gradient. Tubes were centrifuged at 550×g for 20 min at room temperature and individual layers were collected with a Pasteur pipette. The fractions collected in this way were numbered from top to bottom and washed three times in HBSS before use.

When effector populations were activated with lymphokine supernatant fluids, 25 mmol a-methyl-D-mannoside (Sigma) was added to the culture medium to bind free Con A (Sitkousky et al., 1982). In instances in which activation with IL-2 was continued over a 3-day period, culture medium was removed on Day 2 and fresh medium and IL-2 were added. After culturing the cells, they were layered onto a polymer-sucrose-diatrizoate gradient (Histopaque, specific gravity 1.077 g c m - 3) (Sigma) and centrifuged to sep- arate dead from live cells.

Flow cytometric analysis and fluorescence-activated cell sorting of effector populations

All analysis and cell sorting were performed with an EPICS CS (Coulter Electronics, Hialeah, FL). Flow cytometer data from 20 000 cells were collected to analyze the patterns of monoclonal antibody (mAb) reactivity. Two parameter analyses of forward angle light scatter (FALS) vs. 90 ° light scatter (LI 90) were used to gate out debris and doublets from the population under analysis. The fluorescence data were collected both as dot scatter plots of FALS vs. fluorescence and histograms of fluorescence vs. cell number. The percent-


age of positive cells was determined using the immunoprogram (Coulter Electronics, MDAPS system version 3. l ) for the analysis of immunofluoresc- ence histograms.

Specific mAb to bovine leukocytes were purchased from the Washington State University Monoclonal Antibody Center (Pullman, WA). These mAb had been previously characterized (David et al., 1987; Davis et al., 1988) and the following lineage specific mAb were used: B26A (CD2 equivalent antigen), CACT84 B (CD4 equivalent), BA¢ l l l (CD8 equivalent), B7A (non-T non-B-lymphocyte antigen), DH59B (granulocyte/monocytes antigen), BAO44A (B-lymphocyte antigen ). Appropriate controls to detect nonspecific labeling, including FITC-label F (ab ' )2 goat anti-mouse Ig alone and in combination with isotype matched mAb specific for irrelevant antigens, were run in parallel. The indirect labeling procedure used for flow cytometric analysis has previously been described in detail (Griebel et al., 1988). For cell sorting, staining was done at 4 ° C at a cell concentration of 5 × 107 cells ml- 1. All mAb dilution and washing were done in HBSS with 0.3% BSA. After staining was completed, cells were washed three times in HBSS and resuspended at 4 × 106 cells ml - t. Analysis for green fluorescence was performed selecting green fluorescent-positive cells for one direction and non-fluorescent cells for the other direction. Preparative sorting was conducted of a rate of 1200-1500 cells s-t until a minimum of 2 × 10 6 cells was obtained (2-5 h ) in all fractions. Analysis was performed on unsorted, positive and negative fractions after all preparative sortings to determine purity of all cell populations. Viability of sorted cells always exceeded 95%.

Cytotoxicity assay

Cytotoxicity assays were performed in quadruplicate in 96-well, round-bottom microtiter plates using 5~Cr-labeled targets (l04 cells per well) as described previously (Campos and Rossi, 1986b). For ADCC assays, anti-HSB- 2 antibody (25/~l) was added to the wells before addition of effector cells. In all experiments, 100/zl of effector cells were added to each well to obtain specified effector :target cell ratios. Eight wells were treated with 100 #l of Triton X-100 to determine maximum releasable 5~Cr. At the termination of the assay, supernatant fluids were collected with the Titertek harvesting device (Skatron, Stealing, VA) and the amount of 5 ~Cr release was determined in a gamma counter. Standard deviations of quadruplicate wells were usually less than 6%. Specific cytotoxicity was calculated as follows: % specific S~Cr release = 100 × [ (mean cpm exper iment- mean cpm spontaneous release) / (mean cpm detergent lys is -mean cpm spontaneous release) ].

2 lO M. (AMP()S El .S,l.

Binding assay

The ability of PBML to form conjugates with target cells was tested by mix- ing effector and target cells at a 10:1 ratio. After centrifugation (200×g) of the effector: target cell mixture for I 0 min, cell pellets were incubated at 37 °C for 10 min. Cells were subsequently resuspended and wet cell preparations analyzed microscopically to determine the percentage of target cells capable of binding effector cells (% effector-target conjugates ).

RESULTS

Concanavalin-A supernatant fluids containing IL-2 have previously been shown to activate PBML to become cytotoxic. Thus, initial experiments were performed to compare activation by 1L-2 with that by Con A supernatant fluids. Exposure of PBML to either Con A supernatant fluids ( 10% v/v) or to IL-2 (5 and 25 U ml - I ) induced cytotoxic activity in PBML (Fig. 1). Development of LAK activity was similar in both instances. Direct lysis of K562 cells in a 4-h 5~Cr-release assay was not detected in non-stimulated lymphocytes (time 0). However, after 8 h of activation with either Con A supernatant fluids or IL-2, cytotoxicity against K562 target cells was detectable and increased thereafter until termination of the experiment ( 18 h).

0

20 ] Con A SUP. 10% V V r~=4

15

, ° i ,

40 i~[~ ~-UNITS;rnl I • IL-2 S UNITS ml

n=6

20

10 ~ ~

o . . . .

0 4 8 ~2 ~6

T I M E (HR.)

/

T

/ / ±

. ,

, r ~ / a t ." . ' ~" ~

Fig. 1. Comparison between the activation of PBML by Con A supernatant fluids and IL-2. Cytotoxicity of peripheral blood leukocytes from three different animals against K562 target cells was evaluated at different times after treatment of PBML with Con A supernatant fluids ( 10% v/v) or with IL-2 (5 and 25 U ml-~ ).


TABLE l

Effect of different concentrations of IL-2 on the activation of cytotoxic cells from precursors from peripheral blood mononuclear leukocytes of cattier

IL-2 Specific 5tCr-release from target cells Proliferation ~ U ml -~ c p m × 10 -4

K562 HSB-2 HSB-2+Ab

1000 26.4±4.62 32.3±13.1 73.2±20.1 7.36±2.9 250 19.2±8.9 21.5±6.3 56.1±15.5 6.75±2.7 50 11.0±6.5 13.4±8.3 51.8±13.5 6.00±2.7 10 4.0±3.1 1.0±3.3 24.5±5.9 3.60±1.5 None - 1 . 0 ± 1 . 1 0.6±2.3 0 .5±1.8 0 .44±3.4

tPBML from six animals were cultured for 3 days in complete medium supplemented with IL-2; cytotoxic capabilities of the cultured cells was measured in a 4-h ~tCr-release assay at effector: target cell ratio of 10: I. Antibody-dependent cellular cytotoxicity was evaluated using HSB-2 cells in the presence of 25/tl of anti-HSB-2 antibody. 2Results are expressed as mean_+ SD from six animals. 3Proliferation was evaluated in parallel cultures of PBML (3.5 × 105 per well) in round-bottom microtiter plates. 3H-thymidine incorporation was used as indication of cellular proliferation.

To further evaluate the effect of IL-2 on bovine lymphocytes, PBML from six animals were cultured at 38 °C in the presence of different concentrations of IL-2 and their proliferation and cytotoxic capacity was evaluated 3 days later. Degree of proliferation and cytotoxicity (direct and ADCC) was pro- portional to the concentration of IL-2 used up to 1000 U ml -~. ADCC was demonstrated with lower doses of IL-2 than required for induction of direct target cell lysis (Table 1 ).

Cytotoxicity of lL-2 activated PBML against cultured tumor cells

In order to evaluate the cytotoxic capacity of PBML against cultured tumor cells, non-stimulated PBML from three animals were tested for conjugate for- mation with target cells and target cell lysis in the presence and absence of IL- 2 in an 16-h 5~Cr-release assay. Non-stimulated PBML formed conjugates with K562, HL60S and HL60R, but not with KG-I target cells. Only PBML that were cultured in the presence of IL-2 lysed conjugated target cells. KG- l target cells were resistant to lysis even after IL-2 treatment of effector cells (Ta- ble 2).

To test the possibility that cytotoxicity of IL-2-treated PBML cultures was due to an increased binding capacity of effector cells, PBML from six animals were treated with IL-2 (50 U ml- ~ ) for 18 h and their binding to K562 cells was evaluated. Under these conditions the percentage ofeffector: K562 target conjugated was 20.4 +_ 3.0 and 20.0 +_ 3.2 in IL-2-treated and nontreated cul-

212 M. CAMPOS EI" AL.

TABLE 2

Sensitivity of various target cell lines to lysis mediated by IL-2 activated bovine PBML ~

Target cell lines Pre-IL 2 treatment effeclor- target conjugates (%)

Specific 5~Cr release mediated by PBML (%)

Without IL-2 With IL-2

K562 15.0±3.2 ~ - 0 . 1 ± 1 . 6 18.4±5.5 HL60S 9.7±3.4 1.8±0.9 15.8±8.3 HL60R 8.6±1.0 0.5±1.8 17.5±7.1 KG-I 2 .3~2.2 - 2 . 9 ± 0 . 2 2 .4±4.6

~PBML from six animals were tested for their ability to bind and lyse different target cells. Cytotox- icity assays were run for 16 h in the presence and absence of ILo2 ( 50 U ml - ~ ). 2Results are expressed as mean + SD from six different animals.

TABLE3

Comparison between the cytotoxic capabilities of human and bovine PBML before and after activation with IL-2 ~

Species Donor IL-2 ( U m l - t )

Specific 5 ~Cr-release from target cells (%)

HL60R HL60S K562 HSB-2 HSB-2 + Ab 2

Human CR

MC

Bovine 120

128

25 10.0 65.8 75.2 30.1 60.1 5 7.3 49.6 66.0 31.5 55.7

None 1.3 36.2 66.0 28.2 45.1 25 26.2 79.9 77.9 29.9 62.2

5 27.3 62.5 66.8 28.9 67.0 None 14.0 60.5 63.8 21.7 63.0

25 28.2 17.4 33.6 18.9 55.2 5 - 0 . 2 - 1.3 14.5 - 0 . 9 26.1

None - 0 . 9 -7 .1 - 2 . 9 - 5 . 2 12.3 25 16.3 6.9 26.1 24.4 44.4

5 19.1 3.1 24.9 15.3 31.9 None - 3 . 2 - 5 . 4 2.8 - 6 . 0 7.4

tPBML from bovine and human donors were tested for their ability to mediate cytotoxicity against different target cells in the absence and presence of IL-2. Cytotoxicity/activation assays were run for 16 h at effector: target cell ratio of 50: 1. - 'Antibody-dependent cellular cytotoxicity was evaluated using HSB-2 cells in the presence of 25/zl of antiHSB-2 antibody.

tures, respectively, suggesting that the observed cytotoxicity was not due to an increase in number of target-binding effector cells.

To investigate the need for IL-2 activation for cytotoxic function of bovine PBML, the cytotoxic capabilities of human and bovine PBML were compared. In all instances, human effector cells efficiently lysed NK-susceptible


target cells in the absence of IL-2 stimulation, whereas non-stimulated bovine PBML were not capable of performing such a function. Addition of IL-2 was sufficient to activate bovine effector cells to lyse otherwise resistant targets (Table 3 ).

Effect of different IL-2 stimulation protocols on the generation of cytotoxic cells from PBML

To further establish activation requirements of LAK cells, PBML from three animals were incubated in the presence and absence of IL-2 (50 U ml - ~ ). After 10 and 18 h of incubation, effector cells were tested for mediation of direct lysis of K562 cells and direct lysis and ADCC of HSB-2 cells in a 4-h 5~Cr-release assay. The effect of continuous exposure to IL-2 on cytotoxicity was evaluated by adding 25 U ml-~ IL-2 to the cytotoxic assay. Maximal ADCC and direct lysis of target cells were obtained when effector cells were continuously exposed to IL-2 (Table 4). PBML cultured and tested for cytotoxicity in the absence of IL-2 had little cytotoxic activity. PBML cultured with IL-2 before the assay but without IL-2 during the assay produced inter- mediate levels of cytotoxicity. ADCC was the only cytotoxic activity detected in cultures in which IL-2 was only present during the assay period.

To evaluate the effect of IL-2 withdrawal on LAK activity, PBML from three animals were incubated with IL-2 ( 50 U ml- ~ ). After 24 h incubation, stimulated cultures were further cultured for an additional 24 h in the presence and absence of IL-2 ( 50 U ml- ~ ). Cytotoxicity was measured at the end

TABLE 4

Effect of different IL-2-stimulation protocols on the generation of cytotoxic cells from bovine PBML ~

Time of preincubation Activation during IL-2 during before assay preincubation assay

Specific ~Cr-release from target cells (%)

K562 HSB-2 HSB-2 + Ab

10h None + 2.4___1.42 3.1+_2.1 27.3+_7.3 - 0.3+_1.2 0.5_+2.5 8.6_+4.9

IL-2 + 14.2+-1.2 10.3+_3.7 45.9+_3.4 - 8.9+_2.3 5.8+_3.4 34.5+_4.1

18h None + 2.7+_2.3 0.5+_0.6 17.3+_7.6 - 0.5+_0.6 0.6+_0.3 11.6+_1.4

IL-2 + 15.9___3.1 8.4+-3.1 40.2+-7.5 - 12.3+-4.2 5.7+_2.2 29.4+_8.1

~PBML from three animals were incubated before the cytotoxicity assay for the specified presence and absence of 50 U m l - ' of IL-2. Cytotoxicity assays were run for 4 h in the absence of II_,-2 (50 U ml- ] ). 2Results are expressed as mean _+ SD from three animals.

times in the presence or

214 M ('AMPOS E'I" '\L.

TABLE 5

Effect o f IL-2 withdrawal on the cytotoxic capabilities o f IL-2-activated bovine peripheral blood mononuclear leukocyles ~

Stimulation protocol IL-2 during belbre assay assay

Specific ~Cr-releasc from target cells (%)

Day 1 Day 2 K562 HSB-2 H S B - 2 + A b 2

IL-2 Medium + 19.1±6.2 11.7~2.4 52 .8±3.2 - 10.1±1.3 6 .1±1 .4 33.3±4.1

IL-2 IL-2 + 20 .5±4 .8 13.7±4.3 63 .8±11.2 - 15.3±2.5 7 .7±0.3 49 .9±9 .4

~PBML from three animals were incubated in the presence o f IL-2 ( 50 U m l - ~ ). After 24 h incubation, s t imulated cultures were cultured in the presence and absence of IL-2 ( 50 U ml ~ ) for an additional 24 h. The cytotoxic capabilities o f the cultured cells were evaluated using a 4 h S~Cr-releasc assay at effector: target cell ratio o f 50: l in the presence and absence o f l L-2 ( 50 U m l-~ ). -'Results arc expressed as mean ± SD from three different animals.

of culture time in a 4-h 5~Cr-release assay in the presence or absence of IL-2 (25 U ml - t ) . Maximal direct cytotoxicity of K562 and HSB-2 and maximal ADCC against HSB-2 were obtained in cultures that were exposed to IL-2 throughout the experiment (Table 5). IL-2 withdrawal caused a marked reduction in the cytotoxic capacity of the effector populations. However, cytotoxicity was rapidly restored by the addition of IL-2 during the 4-h assay.

Effect of different cell separation protocols on the generation of cytotoxic cells from bovine PBML

The involvement of adherent cells in the generation of cytotoxic function after IL-2 activation of PBML was investigated using different cell separation protocols. Cells recovered after passage of PBML through Sephadex G- 10 columns retained their ability to mediate direct lysis of tumor cells after IL-2 treatment (Table 6 ). Similarly, removal of adherent cells by nylon wool (Ta- ble 7 ) and plastic adherence during the activation procedure (Table 1 ) were capable of mediating direct lysis of target cells as well as ADCC, suggesting that adherent monocytes were not responsible for the cytotoxic activity detected under the present experimental conditions. Further experiments using PBML from two animals confirmed that nylon wool cell separation enriched PBML with effector cells responsible for cytotoxic activity in PBML, and depleted PBML of cells belonging to the monocyte (DH59B ÷) and B-cell (BA044A + ) lineages (Table 8).

To further characterize the cell responsible for cytotoxic activity in the cow, PBML were passed through nylon wool and layered on top of Percoll gra- dients. After centrifugation, four distinct interface fractions were obtained.

ACTIVATION AND CHARACTERIZATION OF BOVINE LAK CELLS

TABLE 6

Effect of Sephadex G-l 0 separation on cytotoxic activity generated after I L-2 activation ~

215

Animal number Effector cell population IL-2 activation Specific ~Cr-release (%) from HSB-2

147 PBML No 7.3 Yes 47.7

G-10 effluent No 4.8 Yes 49.5

149 PBML No 8.2 Yes 54.6

G-10 effluent No 4.8 Yes 64.8

150 PBML No 7.2 Yes 48.9

G- l 0 effluent No 3.4 Yes 79.3

~PBML were passed through Sephadex G-10 columns and evaluated for cytotoxic potential at an effector: target cell ratio of 50: I after 3 days of culture in the presence or absence of rHuIL-2 ( 100 U ml-~).

TABLE 7

Effect of nylon wool separation on cytotoxic activity generated after IL-2 activation ~

Animal number Effector cell population 2 Specific S~Cr-release (%)

K562 HSB-2 HSB-2 + Ab 3

120 PBML 12.4 N.D.* N.D. NW effluent 31.1 N.D. N.D.

125 PBML 12.3 13.4 23.0 NW effluent 33. I 42.4 57.0

128 PBML 17.7 8.6 23.1 NW effluent 62.6 32.7 89.7

~The cytotoxic ability of IL-2 activated effector cell populations before and after passage of PBML through nylon wool columns was evaluated. Direct cytotoxicity was assessed using K562 and HSB-2 target cells. 2Effector cell populations were activated during the 18-h cytotoxic assay with 100 U ml-~ of rHuIL- 2. The effector target:cell ratio used was 50: 1. 3Anti-HSB-2 antibody was included in order to evaluate the potential of effector cells to mediate ADCC. 4N.D., not done.

From top to bottom, fraction 1 contained the majority of dead cells and fraction 4 was devoid of cells with cytotoxic activity. Cells capable of mediating cytotoxic function were present in fractions 2 and 3. However, the levels of cytotoxicity obtained from fraction 2 were always higher than those obtained

216 M. ('AMPOS ET AI.

TABLE 8

Effect of monocyte and B-cell depletion by nylon wool columns on cytotoxic activity generated after IL-2 activation ~

Effector cell Positive cells (%)-" Cytotoxicity population

DH59B BAQ44A K562 + IL-2 K562

Calf I PBML 8.6 26.6 31.1 - 3.0 NW effluent 2.0 I 1.7 62.5 - 3.2

Calf 2 PBML 7.7 26.4 41.0 - 2.1 NW effluent 3.2 12.2 57.0 0.0

~PBML were passed through nylon wool columns and assayed for cytotoxicity against K562 target cells in the presence or absence of 100 U ml-~ of rHulL-2. The effector:target cell ratio used was 50: 1. 2The percentage of monocytes (DH59B ÷ ) and B-cells (BAQ44A ÷ ) in the effector cell population was evaluated using flow cytometry.

TABLE 9

Enhancement of IL-2-generated cytotoxic activity by Percoll gradient centrifugation ~

Effector cell Positive ceils (%) Cytotoxicity population-"

CD2 ÷ %over CD2 ÷v

CD4* +CD8 ~ C D 4 - / C D 8 -

Calf I

Calf2

NW effluent 68.5 76.7 23.3 63 Percoll fraction 2 71.0 37.7 62.3 79 Percoll fraction 3 73.5 80.7 19.3 51 Percoll fraction 4 70.8 84.4 15.6 3

NW effluent 75.8 75.5 24.5 64 Percoll fraction 2 80.5 55.2 44.8 88 Percoll fraction 3 78.4 85.4 14.6 40 Percoll fraction 4 65.5 94.9 5.0 7

~Nylon wool effluent cells were fractionated using Percoll discontinuing gradient centrifugation. Three of the four fractions obtained were evaluated for cytotoxicity against K562 in an 18-h cytotoxic assay in the presence of 100 U ml-~ ofrHuIL-2. The effector: target cell ratio used was 50: 1. :Effector cell phenotype was assessed using flow cytometry.

from fraction 3. Flow cytometric analysis o f the cells recovered in each of the Percoll fractions revealed that the major phenotypic difference between cells obtained from the different fractions was the percentage of CD2 ÷ or CD8 + phenotype, respectively. Thus a large percentage of CD2 ÷ cells in fraction 2 lacks CD4 or CD8 molecules (Table 9).


Effect of cell type specific sorting on cytotoxic activity

Peripheral blood mononuclear leukocytes (PBML) were labeled with FITC using an indirect staining technique with antibodies specific for monocytes, B-cells, and lymphocytes bearing CD2-, CD4- and CD8-equivalent molecules. After staining, cell populations were separated based on fluorescent in-

TABLE 10

Effect of cell type-specific sorting on cytotoxic activity generated after IL-2 activation ~

MoAb used Cell population specificity Cell % Positive cells on PBML population after cell sorting

obtained after cell sorting 2

% Cytotoxicity 3

DH59B Monocytes DH59B + 43.2 4.5 DH59B- 2.2 48.5

BAQ44A B-cells BAQ44A ÷ 87.3 6.1 BAQ44A- 1.1 39.4

B26A CD2-cells CD2 "~ 90.1 50.4 CD2- 10.1 20.6

HT23A None PBML N.A. 45.7

tFluorescent antibody cell sorting was used to select cell populations bearing or lacking cell-specific cell surface markers. ~Effector cell populations were activated during the 18-h cytotoxicity assay with a 100 U mi-~ of rHuIL-2. The effector:target cell ratio used was 25: 1. 3Cylotoxic capacity of effector cells was assessed in a standard ~lCr-release assay using K562 cells as largets.

TABLE 11

Effect of cell type-specific sorting on cytotoxic activity generated after IL-2 activation ~

MoAB Cell type specificity Cell % Positive cells used on PBML population after cell sorting

obtained after cell sorting 2

% Cytotoxicity 3

CACT83B CD4-cells CD4 ÷ 78.8 7.7 CD4- 0.7 21.3

BAQ 111 CD8-cells CD8 ÷ 64.5 6.2 CDS- 4.8 21.0

HT23A None PBML N.A. 22.3

~Fluorescent antibody cell sorting was used to select cell populations bearing or lacking specific cell surface markers. 2Effector cell populations were activated during the 18-h cytotoxicity assay with 100 U ml - t of rHulL-2. The effector: target cell ratio used was 10: 1. ~Cytotoxic capacity of effector cells was assessed in a standard ~ tCr-release assay using K562 cells as targets.

2 1 8 M. ( " ~ M P ( ) S E q A [ .

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~ ~ r ~ " "' I ¢~lo,..=i~ill 111% "~- I I c >. , - .<,,~ ~.~:i~,,. ~

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FLUORESCENCE INTENSITY

Fig. 2. Flow cytometric profile and cytotoxicity of 11.-2 activated effeetor cells against K562 target cells. PBML were stained with FITC using a monoclonal antibody specific for the bovine CD2 equivalent molecule (B26A) and sorted for CD2 ÷ and CD2- populations using FACS. The recovered cells were further analyzed by flow cytometry to obtain the percentage ofCD2 ÷ cells and for cytotoxicity against K562 in the presence of 100 U ml -~ of rHulL-2. The effector: target cell ratio used for the cytoloxic assay was 50: 1.

tensity into posit ive and negative populations, using an EPIC CS fluorescent cell sorter as described in Materials and Methods. Populat ions obtained in this manner were tested for cytotoxicity in a 18-h 5~Cr-release assay against K562 target cells in the presence or absence of IL-2. Using these procedures we demonst ra ted that populat ions enriched for monocytes (43.2% DH59B ÷ cells) and B-cells (87.3% BA¢44A ÷ cells ) were devoid of cytotoxic potential, whereas populat ions depleted of monocytes (2.2% DH59B ÷ cells) or B-cells (1.1% BA¢44A +) retained cytotoxicity (Table 10). In contrast, cells enriched for erythrocyte receptor-positive cells (CD2 ÷) retained cytotoxic function, whereas the depleted fraction showed reduced cytotoxic potential (Table 10, Fig. 2). Since CD4 ÷ and CD8 ÷ cells are contained within the CD2 ÷ populat ion in bovine PBML, a cell-sorting experiment was designed to evaluate the cytotoxic potential o f CD4 ÷ and CD8 ÷ cells. In both instances, cells enriched for positive cells (78.8% CD4 + and 64.5% CD8 + cells) had a reduced level of cytotoxicity, whereas the depleted fractions (0.7% CD4 ÷ and 4.8% CD8 ÷ cells) retained cytotoxic function (Table l l ).

D I S C U S S I O N

The present studies were designed to analyze the IL-2 requirements for activation of bovine effector cells capable of performing NK-like activities (i.e. A D C C and non-MHC restricted lysis of target cells) and to characterize the


effector population using available reagents for the cow. In previous studies, failure to detect functionally active N K / K cells in fresh bovine PBML, even at high effector to target:cell ratios, suggested a lack of mature NK cells (Campos and Rossi, 1985 ). Although active NK cells were not detectable in fresh peripheral blood, cells capable of direct non-MHC-restricted lysis and ADCC of tumor and virus-infected cells could be generated after cultivation of the mononuclear cell fraction with supernatants obtained by Con A-stimulation of PBML (Campos and Rossi, 1986b). In the present report we ex- tended our observations on the activation of cytotoxic cells in cattle and demonstrated that IL-2 is sufficient to induce non-cytotoxic precursors in peripheral blood to develop cytotoxic activity.

With regard to the nature of the precursor cell in peripheral blood capable of functioning as cytotoxic cells after IL-2 treatment, there are various possi- bilities that should be considered. First, a small undetectable population of active cells proliferates and becomes detectable. Studies in cattle suggest that activation of cytotoxic cells from inactive precursors in peripheral blood could occur after 5 days but not after l day of IL-2 treatment (Fong and Doyle, 1986). However, in the present report we demonstrated that lysis of tumor cells can be detected as soon as 12 h after treatment with IL-2, before significant proliferation occurs. Thus, a mere proliferative response to IL-2 cannot account for the cytotoxic activity detected after treatment with IL-2. Second, cytotoxic activity could be due to maturation of existing precursors. We have shown in this report that, in contrast to human PBML, bovine PBML are devoid ofcytotoxic cells capable of performing ADCC or direct lysis of tumor cells. In human beings and mice, IL-2 boosts the cytotoxic capacity of NK cells, not only in regard to an increased level of lysis against NK-susceptible targets (Kuribayashi et al., 1981; Trinchieri et al., 1984; Shaw et al., 1985; Tilden et al., 1987) but also by expanding their target cell range (Trinchieri et al., 1984; Itoh et al., 1985, Ortaldo et al., 1986). Besides the capacity of IL- 2 to boost NK activity of endogenous NK cells, IL-2 is responsible for the induction of NK cytotoxic function in instances where immature NK cells devoid of cytotoxic activity have been used (Shau and Golub, 1985; Seki et al., 1985; Ueno et al., 1985; Yung et al., 1985 ). Therefore, the possibility ex- ists that inactive pre-NK cells in bovine PBML await the IL-2 signal to become active and perform cytotoxic functions. Third, bovine cells capable of performing N K / K cell-like activities depend on IL-2 for activation and main- tenance of cytotoxic function. Results showing that treatment with IL-2 did not increase the percentage of effector-target conjugates when compared with non-treated controls, and the fact that fresh bovine PBML were able to bind to target cells but did not lyse the bound target cell unless they were activated with IL-2 suggest that the effect of IL-2 on cytotoxic precursor cells in cattle is at a stage subsequent to the initial binding event. In mice, a similar situa- tion has been found in bone marrow, where a population of immature NK-

220 M. ( 'AMPOS E'I AL.

1 + cells bind, but do not kill YAC-I cells, but can become cytotoxic after culturing with IL-2 (Koo et al., 1984). Studies on NK activity of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex have also demonstrated that the cytotoxic ability of target-bound, non- cytotoxic NK cells can be restored by treatment with IL-2 (Bonavida et ai., 1986).

Since IL-2 was required for demonstrat ion of cytotoxic activity in bovine PBML, the most descriptive term for the effector cell shown in these experiments would be LAK cells. Phenotypic analysis of the bovine cytotoxic population showed that the effector cells described in these experiments belong to a subpopulation of CD2 + lymphocytes lacking CD4 and CD8 molecules. Furthermore, these effector cells are also capable of mediating ADCC. Using cold-target competit ion experiments, we have previously shown that the cell population responsible for ADCC after IL-2 activation is the same as that responsible for direct lysis of K562 cells (Campos and Rossi, 1986b), indi- cating that the bovine effector cells are Fc-receptor-positive. These characteristics closely resemble phenotypic properties described for NK cells in other species (reviewed in Ritz et al., 1988). Non-stimulated bovine lymphocytes are not able to mediate natural killer-associated activities (i.e. direct lysis of tumor and virus-infected cells, and ADCC) and direct comparison of the effector population described in this experiment with NK cells is difficult. However, the fact that IL-2-activated bovine PBML have functional and phenotypic characteristics similar to those described for NK cells in other species suggests that a cell similar or related to NK cells is a major contributor to the activity described in these experiments.

Based on these observations we postulate that a cell capable of mediating NK-like cell activity in cattle requires the continuous presence of IL-2 to obtain maximal levels ofcytotoxicity and that withdrawal of IL-2 from the stimulated lymphocyte cultures causes a decrease in cytotoxicity. In this respect, it has been demonstrated that the addition of monocional antibodies against human IL-2 causes a significant inhibition of human spontaneous NK activity. suggesting that IL-2 is constantly required for the expression of NK cell cytotoxic activity (Domzig et al., 1983 ).

In summary, our results show that cytotoxic cells in bovine peripheral blood with characteristics similar to NK cells are inactive until stimulated with IL- 2. The ability to generate cytotoxic cells from non-cytotoxic precursors in peripheral blood of cattle should prove useful for further analysis of cytotoxic cell activation by IL-2 in vivo.

A('KNOWLEDGEMENTS

This work was supported in part by the Alabama Agricultural Experiment Station, US Department of Agriculture Science and Education, Grant No. 83-


CRSR-22146, CIBA-GEIGY Canada, and the Natural Science and Engineer- ing Research Council of Canada. Published as Publication No. 1882 of the College of Veterinary Medicine and Alabama Agricultural Experiment Sta- tion Journal, Series No. 5-861160, Auburn University, Alabama, and published with the consent of the Director of VIDO as Journal Series No. 83. We thank Roger Bridgman for technical assistance and Irene Kosokowsky and Marilee Hagen for secretarial help.

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Characterization and activation requirements of bovine lymphocytes acquiring cytotoxic activity after interleukin-2 treatment