CELLULAR

IMMUNOLOGY

133,4 l-54 ( I99 1)

Differential Sensitivity of Cytotoxic T Lymphocytes and Lymphokine-Activated Killer Cells to Inhibition by L-Ornithine’ BRIAN M.SUSSKIND,~JAYASRISEKAR,PRITIMEHROTRANEETANDON, D.SCOTTLIND,ANDHARRYD.BEAR Division of Surgical Oncology, Department of Surgery, Medical College of Virginia, Virginia Commonwealth University, 1101 East Marshall Street, Richmond, Virginia 23298 Received July II, 1990: acceptedSeptember 30, 1990 The selective inhibition of murine cytotoxic T lymphocyte (CTL) differentiation in C57B1/6 (B6) anti-DBA/Z mixed leukocyte cultures (MLC) by the amino acid L-omithine (Om) could not be reversed by addition of up to 1000 U/ml IL-2. Analysis of the effects of Orn on induction of lymphokine-activated killer (LAK cells), using dosages of IL-2 from IO- 1000 U/ml and measuring cytolytic activity against two tumor targets (P8 15 and YAC- I) over the course of 5 days, indicated that LAK cells were not suppressed by Orn. LAK precursors and effector cells were CDS- and ASGM I+, indicating that they were derived from natural killer (NK) cells. We also found that the growth and maintenance of cloned CTL lines were not sensitive to inhibition by Orn; nor was their acquisition of nonspecific cytolytic activity in the presence of high lymphokine concentrations. Thus, induction of naive CTL shows differential susceptibility to Orn inhibition relative to LAK and LAK-like activities by NK and cloned CTL lines in response to IL-2. o 1991 Academic Press. Inc.

INTRODUCTION Small molecular weight organic cations modify the immunologic functions of lymphocytes in vivo and in vitro. The best known biogenic amine, histamine, is a potent mediator of Type I and Type III hypersensitivity reactions, and the ability of this monamine to modulate cell-mediated immune responseshasbeen widely demonstrated (1). Other biogenic amines involved in the regulation of lymphocyte functions include polyamines (e.g., putrescine, spermidine, and spermine) and their precursors (arginine and L-ornithine (Orn)). Polyamine biosynthesis is an essential and early process in lymphocyte activation and development (2-5). Exogenously supplied polyamines also influence lymphocytes, mediating both augmentative and, somewhat paradoxically, suppressive effects (6- 11). Lymphocyte subsetsare differentially affectedby polyamines (7, 10-l 3). Putrescine, spermidine, and spermine inhibit T cell proliferation in responseto phytohaemagglutinin (8, 9, 14). Proliferation of cells in mixed leukocyte cultures (MLC) and the induction of cytotoxic T lymphocytes (CTL) are also inhibited by spermidine and ’ This work was supported by Grant AI24480 from the NIH. ’ To whom ah correspondence and reprint requests should be addressed at Department of Surgery, Medical College of Virginia, Box 466, Richmond, VA 23298. 41 000%8749/9 1 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form resewed.

42

SUSSKIND

ET AL.

spermine (8) but only CTL function is inhibited by putrescine (12). The presence in the tissue culture medium of serum containing specific amine oxidases is essential for inhibition by spermidine and spermine (8, 9, 14) but not for putrescine (12). The cause of inhibition of cell proliferation by spermidine and spermine is purported to be the production of aminoaldehydes ( 15, 16) or acrolein ( 17). Spermidine and spermine also inhibit lipopolysaccharide-induced B cell mitogenesis in the presence of ruminant serum (8). The acetylated polyamine derivatives tetramethylenebisacetamide and hexamethylenebisacetamide inhibited polyclonal B cell proliferation and differentiation in the absence of serum ( 10, 1 l), whereas proliferation of transformed B cell lines, of activated B cells to B cell growth factors, and of T cells to phytohaemagglutinin were not inhibited, suggesting that the diacetyl diamines are selective for the activation of naive B cells. The polyamine precursors arginine and Orn also modulate immune cell functions. Dietary supplementation with arginine increased T cell activation in terms of response to mitogens ( 18, 19), generation of CTL ( 18), delayed type hypersensitivity reaction (20), and tumor immunity (20,2 1). The mechanism of arginine augmentation in vivo may be mediated by polyamines since levels of arginase, which converts arginine to Orn (the precursor of the “parent polyamine,” putrescine), are elevated in activated macrophages (22,23). Furthermore, production of Orn by different categories of macrophages (e.g., resident, elicited) correlated with their capacity to serve as antigenpresenting cells in vivo for the priming of subsequent secondary in vitro CTL responses (6). Macrophages with weak immunogenicity were enhanced by injection of exogenous Orn with the stimulating cells. Orn and putrescine were also found to augment the in vivo priming for secondary in vitro CTL responses against minor histocompatibility antigens (22). It has been shown earlier that in contrast with their net augmenting effects in vivo, arginine, Orn and putrescine inhibit the in vitro development of CTL (7, 12,24). The inhibitory effect of Orn was selective, since cell proliferation in the MLC, the mitogenstimulated production of IL-2, IL-3, and interferon-y, and IL-Zdependent proliferation of T cell clones or mitogen-activated thymocytes were not inhibited (12, 24). Furthermore, we observed that in contrast to CTL, generation of radioresistant, antigenspecific T suppressor cells was not blocked by Orn (24). Orn-mediated inhibition of CTL generation, however, is not attributable to T suppressor cells ( 12,24). Thus, Orn shows differential effects on lymphocyte subsets. In this report we present the results from studies that examined the effects of Orn on the activation of another class of cytolytic effector cells, lymphokine-activated killer (LAK) cells. MATERIALS

AND

METHODS

Mice. C57B1/6 NCrIBR (B6; H-2b) and DBA/2 mice NCrIBR (H-2d) were supplied through the National Cancer Institute and were used at 8 to 12 weeks of age. Preparation of cellsfor in vitro culture. Spleens were aseptically removed and teased apart in Hanks’ balanced salt solution (HBSS). After allowing large debris to settle out, the cells in suspension were washed three times with HBSS. Tissue culture medium used for the induction of CTL from murine splenocytes, cell line maintenance, and cytotoxicity assays was composed of RPM1 1640 (Grand Island Biological Co. (GIBCO), Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS; GIBCO), 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 5 X lop5

ORNITHINE

SENSITIVITY

OF CTL

AND

LAK

43

M 2-mercaptoethanol, and 10 mM Hepes, and is henceforth referred to as complete medium (CM). CTL generation in MLC and induction of LAK. MLC consisted of 5 X lo6 B6 splenocytes, 2 X lo6 y-irradiated (2000 R) DBA/2 splenocytes and 2 ml of CM per well of a 24-well tissue culture plate (Corning 25820). LAK were induced by culturing normal B6 splenocytes at 5 X 106/ml in 2 ml of CM supplemented with the indicated concentrations of IL-2. Depletion of lymphocyte subsets with antibody and complement. Monoclonal antibody 4 l-3-48 [anti-mouse Lyt 2.2 (CD8)] was the kind gift of Dr. E. A. Boyse (Sloan Kettering Institute, New York) and was used at a 1:500 dilution of the ascites.Rabbit anti-asialo-GM 1 (ASGM 1) serum was purchased from Wako Pure Chemicals (Osaka, Japan) and used at a 1:50 dilution. The diluent used was HBSS-1% FCS. Cells were incubated ( 10 X 1O6cells/ml) with antibody for 30 min at 4°C. Cells were then washed in diluent and resuspended in an equal volume of a 1:12 dilution of Low-Tox-M rabbit complement (C; Accurate Chemical and Scientific Corporation, Westbury, NY) for 45 min at 37°C. Following two washes with diluent, cells were adjusted to the desired viable cell concentrations. Controls consisted of cells treated with C alone. Tumor cell fines. P8 15 mastocytoma cells (H-2d) were maintained by weekly ip passagein syngeneic DBA/2 mice. The YAC- 1 (H-2”) lymphocytic cell line was maintained by in vitro passagein CM. Our line of YAC-1 is a variant that fails to express H-2Dd (12). Lymphobfasts. Lipopolysacchride (LPS)-stimulated blasts were prepared for use as target cells by incubating DBA/2 splenocytes at 106/ml with 10 pg/ml LPS in 15 ml CM in a 25-cm2 tissue culture flask. After 48 hr the cells were washed once in HBSS, resuspended in 3 ml HBSS, layered over 5 ml of Ficoll-Hypaque (specific gravity = 1.077 g/ml) and centrifuged for 20 min at 400g at room temperature. Blast cells collecting at the interface were washed twice with HBSS, counted using trypan blue, and labeled with 5’Cr. Cytotoxicity assay. Cytotoxic activity was assayedby the method of Cerottini and Brunner (25). Target cells (2-4 X 106)were mixed with 200 PC1Na2 5’Cr04 (sp act 300 to 400 mCi/mg, New England Nuclear Corp., Boston, MA) in 0.5 ml RPMI10% FCS and incubated at 37°C for 1 hr. The 5’Cr-labeled cells were washed three times by centrifugation through RPMI-20% FCS and finally resuspended in RPMI10% FCS. Cultured lymphocytes were harvested and washed in HBSS. Viable cells were counted using trypan blue and resuspendedat 2.5 X 106/ml in RPMI- 10%FCS. Four twofold dilutions of effector cells were made and 100 ~1plated into conical well microculture plates (Dynatech Laboratories, No. 00 1-O1o-2602). One hundred microliters containing 5 X 103 “Q-labeled target cells were added per well. The plates were centrifuged for 3 min at 30g and incubated at 37°C in humidified air containing 5% co*. After 4 hr the plates were centrifuged at 4008 for 5 min and the radioactivity released was determined by analyzing 0.1 ml of each culture-well supernatant in a Beckman 4000 y-counter. Percentageof specific 5’Cr releasewas calculated as experimental release - spontaneous release x 100%. total release - spontaneous release Total releasable counts were determined from a lysate of target cells with 2% Triton X- 100. Spontaneousreleasewas determined from cultures containing target cells alone.

44

SUSSKIND

ET AL.

Results given are the mean of three replicates for each effector-to-target (E:T) ratio. The standard error of experiments reported did not exceed 10% of the mean. CTL clones. Cells from 3-day MLC (B6 anti-DBA/2) were harvested, suspendedin HBSS at lO’/ml, layered over an equal volume of Ficoll-Hypaque (specific gravity = 1.077) and centrifuged at 4008 for 20 min at room temperature. Cells from the interface were collected and cultured at 10 cells/well in CM supplemented with 10% T cell growth factor supematant (TCGF) in 0.2-ml flat-bottom microtiter plates (Linbro Scientific, No. 76-003-05) containing 2.5 X lo6 irradiated stimulator cells. After 1 week wells were tested for cytolytic activity against P8 15 target cells. Wells displaying cytolysis were cloned by limiting dilution at 0.3 cells/well in the same manner as the CTL lines were originally established. Clones were grown in 24-well tissue culture plates at an initial density of 1.25 X lo5 cloned cells/ml and 1.25 X 106/ml irradiated allogeneic stimulator cells in 2 ml of CM-lo% TCGF. Reagents. Human recombinant IL-2 was obtained from Cetus Corp. (Emeryville, CA). Orn, Ficoll-Hypaque, and LPS (Escherichia coli, 055:B55) were obtained from Sigma Chemical Co. (St. Louis, MO). TCGF was produced by culturing Sprague/ Dawley rat spleen cells (5 X 106/ml) with 2 pg/ml of Concanavalin A (Con A; Calbiochem, La Jolla, CA) in 75 cm2 tissue culture flasks (Corning No. 25 115) in 50 ml of CM. After 48 hr the supernatant was collected, sterile filtered, aliquoted, and frozen. Before use, a-methyl Dmannoside was added to a final concentration of 10 mg/ml. RESULTS High doses of IL-2 fail to block suppression of CTL by Om. Previously we have shown that 10 mA& Orn inhibited the generation of CTL in MLC selectively, i.e., without affecting T helper cell or T suppressor cell functions (12, 24). The inhibitory effect of Orn could not be abrogated by adding 10 U/ml IL-2 to the MLC, a dose chosen becauseit can replace T helper cells (26, 27) and can also negate the effects of antigen-specific suppressor T cells (12). In subsequent experiments we attempted to overwhelm the Orn-mediated blockade of CTL development by boosting the level of IL-2. B6 anti-DBA/2 MLC were established in CM supplemented with 0 or 10 mM Orn and 0, 100, or 1000 U/ml IL-2. Cytolytic activity was measured after 3 days against P8 15 target cells. As shown in Fig. 1, CTL activation was inhibited by 10 mM Orn. Adding 100 or 1000 U/ml IL-2 to MLC boosted the CTL response by an equal degree, and the addition of Orn to IL-2-containing cultures lead to a substantial inhibition of CTL activity. The decreasein lytic capacity observed in the presence of IL-2 and Om, however, was of a smaller proportion than that observed in the absence of IL-2 in the presence of Orn, suggesting that IL-2 may partially reverse the Ornmediated inhibition. The P8 15 mastocytoma cell line used as the specific target in the CTL assay, syngeneic for the DBA/2 mouse strain used as alloantigen in the MLC, is NK-resistant (28) but LAK-sensitive (29). Hence, it was possible that the partial recovery of CTL activity in the Om-inhibited MLC with added IL-2 was an artifact due to activation of LAK. Therefore, we tested the specificity of the cytotoxic cells recovered from MLC against a nonspecific target, the highly NK- and LAK-sensitive cell line, YAC- 1. Our subline of YAC-1 fails to express H-2Dd and therefore can be used to address this question in the B6 anti-DBA/Z system (12). As shown in Fig. 2a and 2b, CTL from normal MLC that lysed P8 15 (Fig. 2a) failed to kill YAC-1 (Fig. 2b) target cells.

ORNITHINE

SENSITIVITY

OF CTL

AND

45

LAK

80 -*- MLC

70

-O- MLC+Orn

% 60

-W MLC+l OOU/ml IL-2 -O- MLC+l OOU/ml IL-P+Orn

I s 30

-A- MLC+lOOOU/ml

20

IL-2

-A- MLC+l OOOUlml IL-2+0rn

10 0

25:1 12:l 6:l 3:l 5O:l EFFECTOFtTARGET CELL RATIO FIG. 1. Effect of IL-2 on Om-mediated inhibition of MLC anti-P8 15 cytolytic activity. MLC were set up in CM with and without 10 mMOm, and received 0, 100, or 1000 U/ml IL-2. After 3 days cytolytic activity was measured against P8 15 target cells.

Cytolytic activity in the Orn-inhibited MLC was negative against both targets. Cells derived from MLC + 100 U/ml IL-2 killed both P8 15 and YAC- 1 targets, and nearly as effectively. The level of cytolytic activity against P8 15 was increased by the addition of 100 U/ml IL-2 to the Orn-inhibited MLC, although it was still far less than in the normal MLC; cytolytic activity against YAC- 1; however, was nearly equivalent to the normal MLC + IL-2. These results suggestedthat a portion of the cytolytic activity against P815 recovered in the Orn-inhibited MLC fortified with IL-2 might be due to LAK. To determine the extent to which antigen-specific CTL may also be activated in MLC-supplemented with IL-2, we conducted a third experiment analogous to those described above, only this time using DBA/2 LPS blasts as the antigen-specific target cells because of their insensitivity to LAK-mediated cytolysis. As shown in Fig. 3, CTL from normal MLC were allospecific, lysing only the DBA/2 LPS blasts and not the YAC-1 target cells. In the MLC with 100 U/ml IL-2 added, CTL activity against DBA/2 LPS blasts was enhanced, and there was also nonspecific cytolytic activity against YAC- 1. As expected, the MLC + Om lacked cytolytic activity against both targets. In the MLC + Om + 100 U/ml IL-2, the level of nonspecific cytotoxicity of the YAC- 1 target cell was equivalent to the normal MLC + 100 U/ml IL-2, while the antigen-specific cytolytic responseagainst the DBA/2 LPS blasts was still completely inhibited. Two conclusions that can be drawn from the results depicted in Figs. 1, 2, and 3 are: (i) Om-mediated inhibition of CTL development in MLC cannot be reversed by up to 1000 U/ml IL-2; and (ii) stimulation of LAK with IL-2 is less sensitive than CTL to inhibition by Orn. Lack of sensitivity to Orn of LAK cells. Experiments were conducted to test directly the effect of Orn on LAK. Normal B6 lymphocytes were cultured with IL-2 over a 2-

46

SUSSKIND ET AL.

a

y?$

s $;

:i___i

q‘\

20 -10 -.

O-0

O-0

O5O:l

12:l 6:i 25:l EFFECTOFKTARGET CELL RATIO

311

b 90 80 -: % 7o 60 --

k

50 -. sL 40-Y

l+q;$,

-O- MLC+Orn+lL-2

1 30 -. s 20 -. lo--

0 woO--a

O5O:l

25:l 12:l EFFECTORTARGET

6:l CELL RATIO

3:l

FIG.2. Development of nonspecific cytolytic activity in MLC supplemented with IL-2. MLC were initiated in the presence or absence of 10 mM Om, and in the presence or absence of 100 U/ml IL-2. After 3 days cytolytic activity against P8 15 (a) and YAC- 1 (b) tumor target cells was measured.

log range, from 10 to 1000 U/ml, in the presence or absence of 10 mM Orn, and assayedfor cytolytic activity against YAC- 1 and PS15 target cells over the course of 5 days. The results at the 50: 1 E:T ratio are illustrated in Figs. 4a and 4b. When the data for all of the E:T ratios were analyzed by a modification of the three-dimensional dose-responsesurface model of Taswell(30), using a regressionmodel basedon ranks3 the results showed what appears obvious from that depicted in Fig. 4: LAK induction was not inhibited, irrespective of the dose of IL-2 utilized, the number of days allowed for LAK induction, or the choice of target cell. In the previous experiment LAK cells were removed from CM containing Orn and assayedin fresh CM not containing Om. To address the question of whether LAK effector cells might be Orn sensitive, 10 mM Om was added to a cytotoxicity assayof normal LAK cells. LAK cytolytic activity against YAC- 1 and P8 15 target cells was not Om sensitive (Table 1). 3Gennings, C., B. M. Susskind, M. P. Crockett, and L. C. Eby. Analysis ofthe Cell Mediated Lympholysis Assay Using a RegressionModel Based on Ranks. Manuscript in preparation.

ORNITHINE

SENSITIVITY

OF CTL AND

47

LAK

70 -

60 -50 --

40 -% LYSIS (5O:l) 30 --

MLC

MLC+Orn

MLC+IL-2

MLC+Orn +IL-2

EFFECTOR CELLS FIG. 3. Cytolytic cells which develop in Om-inhibited MLC supplemented with IL-2 are not antigen specific and fail to kill allogeneic LPS blasts. MLC were initiated in the presence or absence of 10 mA4 Om, and in the presence or absence of 100 U/ml IL-2. After 3 days cytolytic activity against DBA/Z LPS blasts and YAC- I target cells was measured.

Phenotype of B6 splenic LAK. LAK are known to be derived from both T cells and NK cells (3 1,32). To determine the source of the Om-resistant LAK cells, the responder lymphocyte population was treated with anti-CD8 monoclonal antibody + C or antiASGM 1 antiserum + C before the induction of LAK activity. After 3 days of cultivation with or without Orn in the presence of 1000 U/ml IL-2, cytolytic activity of the surviving cells was assayed against YAC- 1 and P8 15 target cells. As shown in Table 2, LAK development was not reduced by depletion of CD8+ splenocytes, but was greatly reduced by depletion of ASGM 1+ cells. The effects of antibody pretreatment were the same for LAK generated in the presence or absence of Orn, and the results obtained were the same with both target cells. In contrast, depletion of CD8+ cells but not ASGMl’ cells eliminated the CTL response. Although flow cytometric analysis reveals the presence of ASGMl on CTL precursors (33) the relative insensitivity of CTL to cytotoxic elimination by anti-ASGMl serum + C is a characteristic that distinguishes CTL from NK (33,34). Our results demonstrate that the Om-resistant LAK activity induced in our system was generated from CD8-, ASGMl’ cells, i.e., cells expressing the classical NK phenotype (3 1). Assessment of LAK effector cell phenotype was based on negative selection of 5day cultured LAK cells by treatment with anti-CD8 + C or anti-ASGMl + C, and determination of their residual cytotoxic activity. As shown in Table 3, LAK effector cells induced in the presence or absence of Orn were not affected by treatment with anti-CD8 + C just prior to assay, but were partially sensitive to anti-ASGMl + C treatment. Taking into consideration that cultured cells are less susceptible to cytolytic antibody than naive cells (35), and the variable expression of phenotypic markers on

48

SUSSKIND ET AL.

a 100 90 _ 80 -

-*- 10 U/ml

70 -

% LYSIS (50:1)

-+

10 U/ml + Orn

60 -

-.- 100 U/ml

50 -

-O- 100 U/ml + Orn

40 -

*

1000 U/ml

30 -

*

1000 U/ml + Orn

+

0 U/ml IL-2

20 10 0

. r-x1

2

3 DAY

4

5

b 80 70 -*- 10 U/ml

60

-o- 10 U/ml + Om 50

-‘-

% LYSIS 4. (5O:l)

100 U/ml

-O- 100 U/ml + Orn *

30

1000

U/ml

*

1000 U/ml + Orn

-x-

0 U/ml IL-2

10 0 1

2

3 DAY

4

5

FIG. 4. Effects of Om, days, and IL-2 on LAK induction. Normal B6 splenocytes were stimulated with 0, 10, 100, or 1000 U/ml IL-2 in the presence or absenceof 10 mM Om; cytolytic activity that developed over the course of 5 days was measured against YAC-1 (a) and PSI5 (b) target cells.

activated cells (29, 36, 37), these results are consistent with our conclusion from the preceding study (Table 2) that LAK activity from the spleensof adult B6 mice against P8 15 and YAC- 1 target cells was derived from IL-2 stimulated NK. LAK cells are insensitive to arginine and putrescine. We have formerly shown that arginine and putrescine also selectively inhibit CTL in a manner similar to Orn (12). Consistent with the differential sensitivity between CTL and LAK to Orn inhibition, we have also observed that development of LAK activity was not inhibited by arginine or putrescine (Fig. 5).

ORNITHINE

SENSITIVITY TABLE

OF CTL AND

49

LAK

1

Effect of Om on LAK Effector Cells” 9%Specific lysis Target cell

f10 mM Om

5O:l

25:l

YAC- I YAC- 1 P815 P815

+ +

77.8 73.8 28.3 31.4

65.6 61.6 17.1 17.5

’ LAK were induced from B6 splenocytes by cultivation

for 3 days with 1000 U/ml IL-2.

Acquisition of LAK-like cytolytic capacity in CTL clones is not inhibited by Orn. Antigen specific anti-H-2d CTL clones cultured in medium containing 10% TCGF maintained their cytolytic activity when cultured in the presence of 10 mM Orn for 4 days (Table 4). In medium supplemented with 50% TCGF, CTL clones exhibited major histocompatibility complex (MHC)-unrestricted (LAK-like) cytotoxic activity within 48 hr against YAC-1 tumor cells, as others have previously shown (38, 39). Under conditions of high lymphokine concentrations in the presence of 10 rnt4 Orn, CTL clones still acquired LAK-like cytotoxic activity (Table 4). Similar results were obtained with arginine and putrescine (not shown). Thus, CTL incubated under the same conditions of antigen and TCGF exhibit the same profile of cytolytic activity whether maintained in the presence or absence of Orn. DISCUSSION In the present study, CTL generation that was inhibited by the addition of 10 rnJ4 Orn to MLC at first appeared to be partially restored with high doses of IL-2 (100 or

TABLE 2 Phenotype of LAK and CTL Precursor Cells % Specific lysis YAC- I Effector cells induced

Treatment of precursor cells”

LAK LAK LAK LAK LAK CTL CTL CTL

C Anti-CD8 + C Anti-CD8 + C Anti-ASGM I + C Anti-ASGM I + C C Anti-CD8 + C Anti-ASGMI + C

fOrn (10 mM)

+ + -

P815

25:l

6:l

25:l

6:l

81.4 83.0 83.2 16.8 8.1 2.6 2.8 1.4

72.3 68.1 75.9 5.0 2.6 I.0 2.4 0.7

36.9 27.8 30.9 7.8 1.2 74.2 5.5 72.1

23.5 16.9 16.4 4.0 0.0 68.8 2.0 61.6

a Splenocytes were treated with monoclonal anti-CD8 + C or anti-ASGM 1 serum + C prior to induction of LAK or CTL.

50

SUSSKIND

ET AL.

TABLE 3 Phenotype of LAK and CTL Effector Cells 90 Specific lysis YAC- 1

P815

Effector cells induced

Treatment of effector cells”

+Om (10 mM)

25:l

6:l

25:l

6:l

LAK LAK LAK LAK LAK CTL CTL

C Anti-CD8 + C Anti-CD8 + C Anti-ASGM I + C Anti-ASGMl + C C Anti-CD8 + C

+ + -

61.8 58.9 68.4 35.8 49.3 ND ND

43.3 41.1 41.0 18.9 14.6 ND ND

15.3 14.2 13.6 6.9 7.0 29.9 3.6

8.1 5.1 6.9 2.8 3.7 15.3 2.1

a Splenocytes were treated with monoclonal anti-CD8 + C or anti-ASGM 1 serum + C prior to use in the cytotoxicity assay.

1000 U/ml) when assayed against a tumor cell line (P815) of the stimulating H-2 haplotype. Effector cells capable of exerting nonspecific anti-tumor activity, however, were also detected in the MLC supplemented with IL-2, with or without Orn. Further studies with LAK-resistant, allogeneic lymphoblast target cells demonstrated that generation of antigen-specific cytotoxic activity was completely inhibited by Orn even in the presence of exogenous IL-2. These results suggestedthat LAK cells could become activated in MLC supplemented with exogenous IL-2 under experimental conditions where CTL were inhibited. Experiments in which spleen cells were cultured for 1 to

90

80 I

70 -% 60 -L 50.Y s 40.I s so-20 --

-*- Control -o- Ornithine -.- Arginine -o- Putrescine

10 -. 0’ 5O:l

6:l 12:l 25:l EFFECTORTARGET CELL RATIO

3:l

FIG. 5. Effects of Om, arginine, and putrescine on LAK induction. Normal B6 splenocytes were stimulated with 100 U/ml IL-2 in the absence or presence of 10 m.44 Om, arginine, or putrescine. Cytolytic activity was measured after 3 days against YAC-1 target cells.

ORNITHINE

SENSITIVITY

OF CTL AND

51

LAK

TABLE 4 Effect of Om on Antigen Specific CTL Clones and LAK-like

High lymphokine doseb % specific lysis

Low lymphokine dose” % specific lysis P815

YAC- I CTLL clone

IC9 2G6

L?CTL absence ’ CTL absence

YAC-I

P815

fOrn (10 mM)

2:l

1:l

2:l

I:1

2:l

I:l

2:1

I:1

+ + + +

7. I ND 0.2 ND 5.1 ND 10.4 ND

4.3 ND 0.1 ND 1.7 ND 8.8 ND

ND ND 61.3 58.5 70.5 64.3 21.6 20.9

ND ND 44.5 42.8 59.1 50.1 11.3 12.9

79.7 71.6 89.4 82.1 79.9 52.3 55.6 42.0

45.0 36.5 55.2 50.5 49.3 30.9 22.8 16.6

82.9 77.1 89. I 87.2 76.0 75.6 68.5 59.3

64.7 61.5 86.3 83.8 63.0 60.6 36.1 29.0

lA7 IC8

CTL

clones were grown for 4 days in of IO mA4 Om prior to assay for clones were grown for 2 days in of IO mA4 Om prior to assay for

CTL medium supplemented with IO% TCGF in the presence or cytolytic activity. CTL medium supplemented with 50% TCGF in the presence or cytolytic activity.

5 days with a broad range of IL-2 concentrations (lo- 1000 U/ml) and assayed for cytolytic activity against YAC- 1 and P8 15 target cells confirmed that LAK induction was not an Orn-sensitive process. Thus, IL-2 did not overcome the inhibition of CTL by Orn, but rather elicited a separate population of LAK cells that are not suppressed by Orn. There has been disagreement in the literature about whether LAK belong to the T cell or NK cell lineage. Reasons for the discrepancies have been recently reviewed, and some may be attributed to variations in culture conditions, choices of tumor target cells, and variable expression of phenotypic markers on cells in different stages of maturation (3 1,32). Other variables relevant to mice include the age, strain, and organ source for inducing LAK.4 In our studies, LAK activity against both tumor targets was sensitive to treatment with anti-ASGMl + C before and after culture in IL-2. Depletion of cells with the CD8+ phenotype was without an effect. It would thus appear that the anti-YAC- 1 and anti-P8 15 activity of LAK in the spleens of adult B6 mice was derived from CD8-, ASGMl’ cells, the phenotype most often associated with NK cells (31). The LAK response of NK to IL-2 therefore is not sensitive to inhibition by Orn, in contrast to the allogeneic CTL response of CD8+ T cells. The differential sensitivity to Orn of CTL and LAK extends to the closely related amines, arginine, and putrescine (Ref. (12) and Fig. 5). Because the mechanism whereby Orn inhibits CTL differentiation-whether due to polyamines or by other means-remains to be elucidated, possible reasons for the differential effect of Orn on CTL and LAK production are not easily surmised. In 4 Mehrotra nee Tandon, P., H. D. Bear, and B. M. Susskind. Phenotypic and Functional Heterogeneity of Thymic and Splenic Lymphokine Activated Killer Cells Related to Ornithine Sensitivity. Cellular Immunology, in press.

52

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contrast to LAK induction (40) CTL generation involves cell-cell interactions among several different subpopulations of leukocytes (41) that might be affected by Or-n. The inhibitory effect apparently is exerted directly on the CTL precursor cell, however, since (i) inhibition by Orn cannot be blocked by a variety of cytokines (12); (ii) T helper cells in the presenceof Orn produce as much or more IL-2, IL-3, and interferony as in its absence (7, 12); and (iii) MLC depleted of accessory cells so that CTL generation was dependent upon addition of lymphokines remained susceptible to inhibition by Orn (12). An important consideration relevant to the differential effects of Orn on CTL and LAK activation is that distinct progenitor cells were involved in the two responses. Cell differentiation in CTL and NK may proceed via different pathways. Induction of LAK activity from NK involves acquisition of additional target cell reactivities by cells with inherent cytolytic activity for some sensitive tumors (mostly leukemias and lymphomas), whereas CTL precursors in normal spleen are completely cytolytically inactive. Oftentimes drugs that are active in suppressing immunologic functions in naive cells display little or no effect in immunologically competent cells (42). In addition, although both CTL and NK require IL-2 for induction of cytolytic activity, T cells also require signals transduced through the T cell receptor (43). Signaling by the T cell receptor proceedsvia a mechanism that is protein kinase C dependent, produces hydrolysis of inositol phosphates,and is cholera toxin sensitive, in contrast to activation through the IL-2 receptor (44-46). Also, activation via T cell receptor and IL-2 receptor lead to different events at the level of cellular proto-oncogene expression (47). Since particular stimuli (e.g., antigen plus IL-2 vs IL-2) may induce distinct mechanisms of cell activation, it is conceivable that unique biochemical events occur during activation of CTL that are sensitive to Orn. One possibility is that the mechanism whereby Orn inhibits CTL generation may be related to the expression of the cytolytic machinery. CTL-mediated lysis is associated with the secretion of certain CTL-specific serine proteases (38, 48-5 1). Orn inhibits transcription in CTL of B 10, a transcript encoding a CTL specific serine esterasenot present in fresh NK (48), and the expression of serine esteraseactivity (52). Acquisition of theseactivities correlates with the development of cytotoxicity following the removal of Orn (52). Although NK express serine esterasegenesin response to IL-2 in culture (53) cytolysis mediated by NK and NK-derived LAK occurs in the absenceof serine esterasesecretion (38, 49). Furthermore, LAK-like cytolytic activity in cloned CTL after exposure to high concentrations of IL-2 was not associatedwith serine protease secretion (3849) and we have now shown that LAK-like cytolytic activity in cloned CTL, which developed within 48 hr in response to elevated TCGF, was not Om sensitive (Table 4). Combined, these observations suggestthat Orn inhibition may be associatedwith the failure to develop a functional, serine-esterase-dependentcytolytic mechanism. In conclusion, results from these studies indicate that LAK cytotoxicity is unaffected by concentrations of Orn that abolish CTL generation. In the present experiments, CTL development in MLC was completely inhibited by 10 mM Om, even in the presence of 1000 U/ml IL-2. In contrast, induction of LAK activity by stimulating NK with IL-2 was not affected. At what point in the sequence of events following antigen stimulation Om interferes with development of the CTL responseis not clear, but lack of sensitivity to Om indicates that acquisition of LAK activity by NK occurs

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by a processresistant to Orn and therefore different from the development of cytolytic activity by CTL. ACKNOWLEDGMENT The authors gratefully acknowledge Ms. Fay Akers for expert help in the preparation of the manuscript.

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