CELLULAR

IMMUNOLOGY

143,41-54

(1992)

Different Signaling Pathways Induced by a-CD3 Monoclonal Antibody versus Alloantigen on the Basis of Differential Ornithine Sensitivity’ PRITI MEHRO~RA NEE TANDON,’ D. SCOTT LIND, HARRY D. BEAR, AND BRIAN M. SUSSKIND~ Division c$Surgical Cbmnwnweal~h

Oncology, Depurirnrnt oj’.SzwgerJ~, Medicul Cbllqy University. 1101 Easr Marshall Street, Richmond, Rcceiwd

Jumury

6. 1992; accepted

April

of‘ I’irginia. Z’irginiu L’irginiu 23298

6, 1992

Previously we reported that 10 m,M ornithine (Orn) selectively inhibits the development of CD8+ CTL in MLC. Herein we show that induction by a-CD3 mAb of CD8’ killer cells which manifest antibody-redirected cytotoxicity (ARC) of FcR+ targets is not Om sensitive. Om resistance was independent of activation kinetics or a-CD3 mAb concentration. a-CD3 mAb added to the cytotoxicity assay did not reveal a cytolytic potential in CTL from an Orn-treated MLC when the target cells bore both the inducing alloantigen and FcR. Addition of a-CD3 mAb to MLC failed to overcome Orn inhibition of CTL and yet induced ARC activity in the same culture. Expression of mRNA for pore-forming proteins (PFP) and granzyme B was inhibited by Orn in CTL but not in ARC killer cells. Our results demonstrate differences in the T cell activation process stimulated by alloantigen or cu-CD3 mAb. icy 1992 Academic PW, IX

INTRODUCTION L-Ornithine (Orn) is an important amino acid in the regulation of lymphocyte growth and maturation. Early in the process of lymphocyte activation (within the first 10 min) Orn serves an essential role as a precursor in polyamine biosynthesis (1, 2). Functional maturation of lymphocyte subsets in viva and in vitro, however, may be differentially affected by the presence of extracellular Orn. We and others have shown that extracellular Orn suppresses development of the CTL response (3-6). Evidence indicates that Orn-mediated inhibition of CTL generation is exerted directly on the CTL, rather than being an indirect influence at the level of T helper (Th) cells or antigen-presenting cells (APC) (5). Furthermore, Orn treatment of CTL precursors (pTc) does not prevent their response to Th cell and APC signals in terms of lymphoblastoid transformation, expression of IL-2 receptors or cell division.4 Following transfer to fresh medium without Orn, fully activated effector CTL (eTc) emerged within 24’ This work was supported by Grant A124480 from NIH. * Current address: FDA/Center for Biologics Evaluation and Research, Building 29A, Room HFB 1820, 8800 Rockville Pike, Bethesda, MD 20892. 3 To whom correspondence should be addressed at current address: Department of Surgery. Box 8 109. Washington University School of Medicine, 4939 Audubon Ave., St. Louis, MO 63 I IO. 4 Schall, R. P., Lind, D. S., Mehrotra nee Tandon, P.. and Susskind. B. M., manuscript in preparation. 41 0008-8749/92 $5.00 Copyright C 1992 by Academic Press. Inc All rights of reproductmn in any form reserved

42

MEHROTRA

NEE TANDON

ET AL.

48 hr, coinciding with the expression of cytolytic granule-associated serine proteases (granzymes) and pore-forming proteins (PFP, “perform” or “cytolysin”) ((7) and see Footnote 4). By these criteria, according to Bach’s classification (8) Orn causesarrest of CTL development at the preeffector stage (peTc). Recent studies indicate that there are fundamental differences among T lymphocyte subsets in terms of their biochemical activation and regulation following triggering through the T cell receptor (TCR) (9-12). In this regard it is interesting to note that the in vitro inhibitory effect of Orn is selective for CTL in comparison to Th cells and T suppressor cells (4, 5, 13). TCR complex is composed of two major components, each containing multiple polypeptide subunits: a disulfide-linked c$ heterodimer that is the clonotypic antigen recognition structure (a/P-TCR) and the invariant CD3 component, consisting of y, 6, t, {, and 7 subunits (14, 15). The CD3 component of the TCR functions primarily in signal transduction. A monoclonal antibody (mAb) directed against the CD3-t subunit of the murine TCR complex, 145-2C11, is mitogenic for murine T lymphocytes and induces cytotoxic activity in CD8+ cells ( 16, 17). Evidence is emerging that distinct signaling pathways are evoked in T cells triggered via a/P-TCR vs CD3-c (15, 18-22). In this study we used mAb 145-2Cll (a-CD3 mAb) to probe further the selective effect of Orn on CD8+ cells. Interestingly, we found that in contrast to CTL activated by alloantigen, a-CD3-induced activation of CTL differentiation was resistant to Orn inhibition. Therefore, depending on the ligand bound (alloantigen or a-CD3 mAb), signaling through the TCR complex can trigger activation of cytolytic function in CD8+ lymphocytes by processesthat are discernable on the basis of differential susceptibility to Orn inhibition. MATERIALS AND METHODS Mice, cell lines, and mAbs. Virus-free female C57B1/6 (B6; H-2b), DBA/2 (H-2d), and AKR (H-2k) mice, 6 to 8 weeks of age, were obtained from Jackson Laboratory (Bar Harbor, ME). P815 mastocytoma cells (H-2d) were maintained by weekly ip passagein syngeneic DBA/2 mice. The YAC-1 (H-2a) and EL-4 (H-2b) lymphocytic cell lines were maintained in vitro in culture medium. Our subline of YAC-1 fails to express H-2Dd (5). The following mAbs were produced from hybridoma cell lines procured through the American Type Culture Collection: (1) a-CD8 from 56.3.72; (2) a-CD4 from GK1.5; (3) mouse a-rat Ig Klight chain from MAR 18.5; (4) a-FcyRII receptor (FcR) from 2.4G2; (5) a-CD3 from 145-2Cll. a-CD3, a-CD4, and a-CD8 mAbs were produced as ascitesin pristane-treated nude mice. a-CD3 was purified by protein G affinity column chromatography. MAR 18.5 was used in the form of culture supernatant from hybridoma cells. Generation of a-CD3 activated killer cells. The culture medium was composed of RPM1 1640 supplemented with 10% heat-inactivated FCS (GIBCO, Grand Island, NY), 2 mA4 glutamine, 10 mM Hepes, 5 X 10e5A4 2-mercaptoethanol, 100 U/ml penicillin, and 100 pg/ml streptomycin. Spleens were aseptically removed from mice and minced in Hanks’ balanced salt solution (HBSS) to prepare a single cell suspension. Cell debris was allowed to settle for 2 min on ice and then cells in suspension were washed three times with HBSS. Splenocytes (1.5 X lo6 cells/ml) in 2 ml of culture medium were cultured with various concentrations of purified a-CD3 mAb f 10 mM Orn at 37°C in an atmosphere of humidified air with 5% CO2 for l-4 days. Depletion of lymphocyte subsets. The details have been previously described (23). Briefly, splenocytes (10 X lo6 cells/ml) were incubated with a-CD8 or a-CD4 mAbs

CTL AND

ARC DIFFER

IN ORNITHINE

SENSITIVITY

43

(150 dilution of ascites)for 30 min at 4°C. The diluent used was RPM1 1640 plus 0.3% BSA. Cells were then resuspended in an equal volume of MAR 18.5 culture supernatant and incubated on ice for 30 min at 4°C. Cells were then washed with diluent, followed by incubation with a 1: 16 dilution of Low-Tox rabbit C (Accurate Chemical and Scientific Corporation, Westbury, NY) for 45 min at 37°C. Cells were washed, counted, and adjusted to the desired viable cell concentration. Viable cell recovery after a-CD8 or a-CD4 + C treatment was approximately 70%. Flow cytometeric analysis was performed to confirm the specific depletion of lymphocyte subsets after mAb + C treatment (data not shown). CTL stimzdution. Allospecific CTL were generated in MLC consisting of 5 X IO6 responder (B6 or AKR) splenocytes and 2 X lo6 y-irradiated (2000 R) allogeneic splenocytes (DBA/2 or B6) in 2 ml of culture medium per well in 24-well plates (No. 25280 Corning Glass Works, Corning, NY), cultured for 3 days. Prepuration Qfcytokine supernatants (SN). Con A SN was produced by culturing Sprague-Dawley rat spleen cells at 5 X 106/ml with 2 pg/ml of Con A (Calbiochem, La Jolla, CA) in 75-cm2 tissue culture flasks (Corning 25 115) in 50 ml of culture medium. After 48 hr of incubation, supernatant was collected, filter sterilized, aliquoted, and stored frozen. Before use, a-methyl-D-mannoside was added to a final concentration of 10 mg/ml to neutralize any residual lectin in the SN. A SN from a-CD3 mAb-activated spleen cells was prepared by stimulating normal B6 splenocytes with 0.3 pg/ml of a-CD3 mAb t- 10 mA4 Orn. Two days later cellfree culture SN was harvested and aliquots were passed over a protein G column (absorbed SN) to remove residual cu-CD3 mAb. Elimination of a-CD3 mAb was confirmed by inability of the SN to stain thymocytes, as detected in FACS by FITClabeled goat ol-hamster-IgG antibody (Kirkegaard and Perry Laboratories. Inc., Gaithersburg, MD). Cytoto.xicit?9assay. The cytotoxic activity of effector cells was assayed in a standard 4-hr 5’Cr release assay by using indicated tumor targets, as previously published (20). The percentage of lysis (% specific “Cr release) was calculated from the equation Experimental release - spontaneous release X 100 = % Lysis. Total release-spontaneous release Spontaneous release was determined from cultures containing labeled target cells only, whereas total releasable counts were determined from a lysate of target cells with 2% Triton X-100. Results given are the means of three replicates for each E:T ratio. Standard errors of the means (SEM) usually did not exceed 10% and therefore are not shown. Northern blot analysis. Total RNA was isolated by modification (24) of the acid guanidium isothiocyanate-phenol-chloroform extraction procedure (25). Total RNA was fractionated on a 1% formaldehyde agarose gel. The intrigrity and equal loading of the RNA was evaluated by staining the gel with ethidium bromide and inspection under uv transillumination. RNA was then transferred to a nytran membrane (Schleicher and Schuell, Keene, NH) using a vaccugene transfer machine (LKB Pharmacia) and covalently linked by baking at 80°C under vacuum for at least 2 hr. The probes for granzymes B and C (C 11 and B 10, respectively) were kindly provided by Dr. R. Chris Bleakly (Department of Biochemistry, University of Alberta, Edmonton, Canada). The probe for PFP was a gift from Dr. Pierre Henkart (NCI/NIH). Purified inserts were labeled by random priming using an oligolabeling kit (LKB Pharmacia).

44

MEHROTRA

NEE TANDON

ET AL

Blots were hybridized with labeled probe and washed as described (24) then exposed to Kodak XAR radiographic film (Eastman-Kodak, Rochester, NY) at -80°C. RESULTS Efect of Orn on a-CD3 activation of cytotoxic T lymphocytes. We have shown previously that antigen-specific CD8+ CTL are susceptible to inhibition by Orn (5, 20, 23). To study further the Orn sensitivity of TCR-triggered CD8+ cells, we selected a mAb against the CD3 component of the TCR complex. Virtually all splenic CDS+ cells express CD3 (26) and cr-CD3 mAb from the 145-2C 11 hybridoma induces highly cytotoxic killer cells that are CD@ (16, 17). In initial experiments the optimal concentration of soluble a-CD3 mAb for stimulation of T lymphocytes was titrated by culturing the spleen cells from B6 mice with different doses of purified a-CD3 mAb. Cytotoxic activity against NK-resistant P8 15 tumor cells was assayed after 2 days. 01CD3-activated killer cells were strongly cytotoxic against P8 15 and the activity reached a plateau at a concentration of 1.O pg/ml (Fig. 1). Killer cells were next induced with 0.01 to 1 pg/ml a-CD3 mAb in the presence or absence of 10 mM Orn. In contrast to alloantigen-specific (anti-H-2d) CTL development in MLC, which is completely inhibited at this Orn concentration (5), a-CD3-induced cytolytic activity was not affected by 10 mA4 Orn, even at suboptimal concentrations of a-CD3 mAb (Fig. 2). These results suggest a fundamental difference in the functional characteristics and/ or activation pathways of CTL induced by triggering with alloantigen or a-CD3. Kinetics of cz-CD3-activated killer cell generation. To determine whether the apparent resistance to Orn of cu-CD3 activation was merely a result of altered kinetics, we studied the effect of Orn on a-CD3 mAb-induced cytolytic activity over the course of several days. Spleen cells were incubated with a-CD3 mAb at 0.3 and 1.O pg/ml + 10 mM Orn, and cytotoxicity was tested on 4 consecutive days. As shown in Fig. 3,

60 % 60 : 7

40-

s 20 -

0

I 0.01

I 0.03

0.1

@CD3

0.3

1.0

3.0

10.0

(pg/ml)

FIG. I. Dose titration of wCD3. The optimal concentration of soluble a-CD3 mAb for T cell stimulation was titrated by culturing B6 spleen cells (1.5 X 106/ml) with different doses of protein G-purified a-CD3 mAb. After 2 days cytotoxicity was measured against “Cr-labeled P8 15 tumor cells. Results given are the mean percentages of specific lysis (n = 3) at the 50: 1 E:T ratio (SEM < 10%).

CTL AND

ARC DIFFER

IN ORNITHINE

SENSITIVITY

45

60

50 % :

40

s

30

g

20

10

0

0.01

0.03

0.1

a-CD3

0.3

1.0

(&j/ml)

FIG. 2. Effect of Orn on a-CD3 activation of cytotoxic T lymphocytes. wCD~ mAb-activated killer cells were induced by incubating B6 spleen cells (I .5 X 10h/ml) with 0.01 to I Kg/ml ru-CD3 mAb in the presence or absence of 10 mM Orn. Two days later cells were tested for cytolytic activity against P8 15 tumor cells in a 4-hr 5’Cr release assay. Results given are the mean percentages of specific lysis at the 50: 1 E:T ratio (SEM < 10%).

cytolytic activity of cu-CD3-activated T cells was inappreciable at 24 hr, peaked by 48 hr. and declined substantially between 72 and 96 hr; cytotoxicity of ol-CD3-activated T cells was not inhibited by Orn at any time point. Thus, our initial studies establish that the Orn resistance of a-CD3 activation is not conditional upon the extent or duration of stimulation. Cytotmic spectrum of killer cells. The spectrum of cytotoxicity of the a+CD3-activated killer cells was examined against a variety of tumor targets: P8 15 (FcR+). EL4

60

-

%

:

40-

7

S 20 -

0’

Y 1

I 2

3

4

DAYS IN CULTURE FIG. 3. Kinetics of killer cell generation. Cultures were initiated by incubating B6 spleen cells (I .5 X 106/ ml) with soluble a-CD3 mAb at 0.3 and 1 pg/ml f 10 mW Orn. Lytic activity of killer cells was assayed on Days 1 to 4 of culture. Results given are the mean percentages of specific lysis (n = 3) at the 50: 1 E:T ratio (SEM < 10%).

46

MEHROTRA

NEE TANDON

ET AL.

and YAC- 1 (FcR- targets), and the 145-2C 1 I hybridoma. Spleen cells were activated with 0.3 pg/ml of a-CD3 mAb + 10 mM Orn, and after 48 hr lytic activity was measured. ol-CD3-activated killer cells lysed the 145-2Cll hybridoma and the FcR+ P8 15, but did not kill the FcR- tumors, YAC- 1 and EL4 (Fig. 4). Induction of cytotoxicity against P8 15 and 145-2Cll was not inhibited by Orn. Cytolysis of P8 15 was blocked when a-FcR mAb (2.462) was added to the assay. This inhibition was not due to a general effect of the c+FcR mAb on the effector cells because killing of 1452C 11 hybridoma cells, which express surface a-CD3 Ig, was not affected. These results thus demonstrate that cytolysis of FcR+ P8 15 was due to antibody-redirected cytotoxicity (ARC). Furthermore, Orn-resistant cytolytic activity of cr-CD3-activated cells (ARC cells) cannot be accounted for by LAK cells, which might have been generated in the cultures (20) since highly NK- and LAK-sensitive YAC- 1 tumor cells were not lysed by these cells. Moreover, LAK activity against P815 is not blocked by a-FcR mAb (data not shown). Serologic phenotype of precursors and efectors of cu-CD3-activatedkiller cells. Since Orn sensitivity in previous studies correlated with CD8+ phenotype, we next wanted to determine whether ARC cells were CD8+ cells. Normal B6 spleen cells were depleted of CD4+ or CDs+ cells by Ab + C treatment and then cultured with 0.3 pg/ml of CXCD3 mAb + 10 mA4 Orn. In the groups depleted of CD4+ or CD8+ cells, Con A SN (12.5%) was added as a source of cytokines. After 2 days, cytotoxicity was determined against P8 15 tumor cells. As shown in Table 1, depletion of either CD4+ or CD8+ cells from the precursor population extinguished the activation of cytolytic activity (Experiments I and II). The addition of exogenous Con A SN restored the cytolytic activity in the group depleted of CD4+ cells but not in the group depleted of CD8+ cells (Experiment II). Furthermore, the cytolytic activity replenished by Con A SN in the group depleted of CD4+ cells was not susceptible to Orn inhibition. These data thus indicate that the ARC cells were derived from CD4- CD8+ cells and that their

P815

EL-4

YAC-1

1452Cll

FIG. 4. Target spectrum of the a-CD3 activated killer cells. B6 Spleen cells (1.5 X 106/ml) were activated

with 0.3 fig/ml cr-CD3 mAb +- IO mM Om and 48 hr later cytotoxicity was assayed against P8 15, EL4, and YAC-1 tumor cells, and the 145-2Cll hybridoma (a-CD3 mAb producing) in the presence or absence of cu-FcR mAb. Results given are the mean percentages of specific lysis (n = 3) at the 50: I E:T ratio (SEM < 10%).

CTL AND

ARC DIFFER

IN ORNITHINE TABLE

47

SENSITIVITY

I

Precursor Phenotype of oc-CD3-Activated

Killer Cells 4%Specific 51Cr release’

Progenitor cell treatment” Experiment I Complement Complement wCD~ + C o-CD8 + C (y-CD4 + C a-CD4 + C Experiment II Complement Complement @-CDS + C n-CD8 + c a-CD4 + C a-CD4 + C n-CD4 + C + Om

50: I

6:l

62.8 56.4 8.9 5.9 16.1 10.8

36.7 27.9 2.1 1.6 3.4 2.3

72.8 80.1 7.2 14.5 46.2 75.5 69.8

63.4 83.0 4.1 10.1 23.9 79.8 72.3

Om + + + Con A SN + + + +

’ Progenitor spleen cells from C57Bl/6 mice were treated with a-CD8 or (u-CD4, MAR 18.5. and finally with C. Treated and untreated cells were cultured with 0.3 fig/ml of a-CD3 mAb in the presence or absence of Om (I 0 m M) or Con A SN ( I :8 dilution). * Cytotoxicity was determined on Day 2 of culture by testing against P8 I5 tumor cells: results given are the mean percentages of specific lysis (n = 3; SEM < 10%).

induction was Orn resistant, but dependent upon the production of cytokines by CD4+ cells. To determine the effector cell phenotype, cells cultured with a-CD3 -t Orn for 2 days were depleted of CD4+ or CD8+ cells by treatment with a-CD4 or (r-CD8 mAb + C and then residual cytotoxicity was tested against P8 15 tumor cells. Results from these studies demonstrate that the ARC cells were CD4- CD8+ in both the control and the Orn-treated groups (Table 2). Therefore, Orn-resistant ARC precursor and effector cells are CD8+.

Sensitivity to Orn cf MLC-derived CTL cannot be reversed at the eTc stage by addition qfcu-CD3 to the “Cr release assaql.CTL utilize the o(//3-TCR component of the TCR complex to recognize target cells, whereas ARC cell-to-target conjugation involves a+CD3 mAb on the surface of the ARC effector cell which attaches to the FcR of the target cell. Therefore, it was possible that the apparent Orn resistance of ARC cell activation resulted from antibody-redirected binding that could bypass the normal mechanism of CTL-target recognition. To test this hypothesis, n-CD3 mAb was added to the cytotoxicity assay of eTc from an Orn-treated B6 anti-DBA/2 MLC. This procedure, which would enable H-2d-specific CTL to bind FcR+ P8 15 target cells (H-2d) by an ARC-like mechanism, did not result in target lysis (Table 3). Therefore, facilitation of target cell binding by a-CD3 does not account for the Orn resistance of cy-CD3-activated ARC cells.

48

MEHROTRA NEETANDON ETAL. TABLE 2 Serologic Phenotype of a-CD3-Activated

Killer Cells % Specific 5’Cr release’

a-CD3 + Om”

Effector cellh treatment

5O:l

6:1

-

Complement cr-CDS + C a-CD4 + C Complement a-CD8 + c a-CD4 + C

50.9 6.8 61.2 46.1 7.1 57.9

13.1 2.0 24.2 13.7 1.9 19.8

+ + +

a Spleen cells from C57Bl/6 mice (1.5 X IO6 cells/ml) were activated with 0.3 &ml of a-CD3 mAb + 10 mMOm. b On Day 2, the effecters were treated with a-CD4 or a-CD8, MAR 18.5, and then C. c Cytotoxicity was determined against P8 I5 tumor cells; results given are the mean percentages of specific lysis (n = 3; SEM < 10%).

Orn resistance of ARC activation is not the result of cytokines produced upon w CD3 activation. We have shown previously that addition of various cytokines (e.g., IL-l, -2, -3, -4, -6, -7, and Con A SN) to MLC will not override Orn inhibition of CTL differentiation ((27) and see Footnote 4). To determine whether Orn-resistant a-CD3 activation results from secretion of a unique cytokine or combination thereof, cytokine SN from a-CD3 mAb-stimulated cultures (see Materials and Methods for details) were tested for their ability to abrogate Orn inhibition of allo-CTL. Protein G-absorbed SN was added to (B6 a-DBA/2) MLC + Orn, and cytolytic activity was measured after 72 hr against P8 15 target cells. Similar results were obtained with SN generated in the presence or absence of Orn and are shown in Table 4. Addition of absorbed a-CD3 SN (12.5%) to MLC + Orn failed to reverse the effect of Orn. This suggeststhat Orn resistance of ARC cells is not due to a unique array of cytokines produced as the result of a-CD3 activation, but rather is a unique property of the cyCD3 activation pathway relative to alloantigen stimulation of CTL. TABLE 3 Addition of n-CD3 mAb to the Assay Does Not Overcome the Omithine

Inhibition

of CTL

% Specific release’ Culture condition0

o-CD3 mAb in assay b

25:l

12:1

MLC MLC + Orn MLC MLC + Om

+ +

40.5 10.2 49.1 18.4

29.5 5.4 37.1 12.9

0 CTL were generated in 3-day MLC F 10 mM Om (B6 X DBA/Z). h 1 pg/ml of a-CD3 mAb was added to cytotoxicity assay. c Cytotoxicity was assayed against “Cr-labeled P8 15 tumor cells; results given are the mean percentages of specific lysis (n = 3; SEM < 10%).

CTL

AND

ARC DIFFER

IN ORNITHINE

49

SENSITIVITY

TABLE 4 Effect of Addition of a-CD3-Activated

Lymphocyte

Supernatant on Ornithine

Inhibition

of MLC

D?o Cytotoxicity’ MLC f Orn”

Additions to MLC”

50: I

25:l

-

-

+ + +

Absorbed cKD3 SN Absorbed (u-CD3 + Orn SN

41.4 9.8 7.2 7.1

28.9 7.3 5.8 4.5

’ MLC consisted of spleen cells from B6 (5 X 106) mice as responder and y-irradiated (2000 R) spleen cells from DBA/2 mice (2 X IO”) as antigen in 2 ml of culture medium + 10 rn*U Orn. ’ SN was prepared by activating B6 spleen cells with 0.3 pg/ml of wCD3 mAb k 10 m.kl Orn. Two days later cell-free SN was collected, ahquoted, and passed over protein G to remove residual mAb. Protein Gabsorbed SN was used at 1:s final concentration. ’ After 3 days cytotoxicity was tested against P8 I5 tumor cells: results given are the mean percentages of specific lysis (n = 3: SEM < 10%).

Distinction between a-CD3 and alloantigen activation pathways. ARC that is resistant to Orn might result from a-CD3 being a more potent stimulus than alloantigen. In order to test whether Orn sensitivity was a function of the strength of the stimulatory signal, AKR (H-2k) splenocytes were stimulated with y-irradiated B6 (H-2b) splenocytes t Orn and + a-CD3 mAb (0.03 pg/ml). Controls included AKR splenocytes activated with a-CD3 mAb alone + Orn. Cytotoxicity was tested on Day 3 against EL4 (alloCTL target, FcR-) and P8 15 (ARC target, FcR+) tumor cells (Figs. 5A and 5B). AlloCTL induction was inhibited by Orn (Fig. 5A). As before, ARC cells were Orn resistant (Fig. 5B). Both allo-CTL and ARC cells were activated in MLC with added a-CD3 mAb; however, in MLC + ORN + a-CD3 only ARC activity was notable. Since pilot studies had shown that the normal allo-CTL response may be somewhat suppressed by the addition of a-CD3 mAb at T = 0 hr, we also tried adding it at T = 24 hr and at two doses (0.03 and 0.1 pg/ml). Allo-CTL induction was not inhibited by the addition of a-CD3 mAb at 24 hr, and remained Om sensitive. Therefore, boosting the activation signal by stimulation with both alloantigen and a-CD3 mAb failed to abrogate the Orn sensitivity of allo-CTL response. That the addition of w-CD3 mAb to MLC + Orn failed to overcome inhibition of allo-CTL and yet induced ARC activity suggests CTL and ARC cells either differentiate through two different pathways or constitute two distinct subpopulations of CDS+ lymphocytes with cytolytic potential. Expression qj”granzyme and per@in mRNA in CTL and ARC cells. Total RNA was isolated from ARC cells 48 hr after induction with a-CD3 mAb (0.3 @g/ml) k Orn, and from CTL generated in 72 hr of MLC k Orn (Fig. 6). The samples were subjected to Northern blot analysis using probes for granzyme B and PFP. Orn-inhibited CTL showed lower steady-state levels of mRNA for granzyme B and PFP, whereas Orn did not reduce granzyme B and PFP mRNA expression in ARC cells. Similar results were obtained for granzyme C (data not shown). Therefore. both CTL and ARC cells transcribe mRNA for PFP and granzyme B, but Orn differentially regulates the expression depending on the ligand binding to the TCR complex. DISCUSSION Several years ago we and others reported that Orn selectively inhibits the induction of the allo-specific CDS+ CTL response in murine MLC (3-6). It recently has been

50

MEHROTRA

NEE TANDON

ET AL.

EL4 Target

40

MLC+ a-CD3 ~3Ong/rnl; T-Oh)

%

L Y s i S

50 MLC+ &CD3 (30n~/ml: T=24h) a0 ML0 (1oonQ/ml,

atCD3 T-24h)

10 (30ng/ml:

No ORN

T=Oh)

10 mM ORN

CTL Activity P815 Target

MLCt OCCD3 (30ng/ml; T=Oh) MLC+ at-CD3 (30ng/ml: T=24h) MLC* (Y-CD3 (lOOng/ml: T=24h) (30ng/ml:

No ORN

T=Oh)

10 mM ORN

ARC Activity FIG. 5. Distinction between a-CD3 and allo-Ag activation pathways. CTL were generated in MLC (2.5 X 106/ml AKR spleen cells and 106/ml y-irradiated B6 splenocytes) +- 10 mM Om. Soluble a-CD3 mAb was added either at the initiation of the culture (T = 0 hr) or after 24 hr (T = 24 hr). ARC cells were induced by incubating AKR splenocytes (1.5 X 106/ml) with 0.03 pg/ml a-CD3 mAb + Om. Cytotoxicity was tested on Day 3 against (A) EL4 (Ag-specific target, FcR-) and (B) P8 I5 (ARC target, FcR+) tumor cells. Results given are the mean percentages of specific lysis (n = 3) at the 50: 1 E:T ratio (SEM < 10%).

shown that interaction of the CD3 component of the murine TCR complex with mAb 145-2Cll induces cytotoxic lymphocytes that are CD8+ and which kill FcR+ targets via ARC ( 16, 17). Results reported herein demonstrate that, in contrast to CTL stimulation by alloantigen, ARC cells were not susceptible to inhibition by Orn, thus suggesting a fundamental difference in the process by which T cell activation triggered by alloantigen or a-CD3 is induced, mediated, or regulated. Differential susceptibility of CTL vs ARC cells to inhibition by Orn cannot be accounted for on the basis that they are from separate cell lineages. Phenotypically, CTL and ARC cells are both CD8+ at the precursor and effector cell stage (Tables 1 and 2). This fact signifies that the cytolytic activity which develops in a-CD3-stimulated splenocyte cultures is not due to Orn-resistant LAK cells, which are primarily derived from CDS- NK in the spleen (20). a-CD3 mAb is a polyclonal activator of CDS+ cells ( 16, 17,26). As in the case of Con A-induced cytotoxic T cells, ARC cells are apparently comprised of antigen-specific CTL subsets (28). Therefore, ostensibly the same CTL precursor cell may respond to alloantigen by an Orn-sensitive pathway and to a-CD3 by an Orn-resistant pathway.

CTL

AND

ARC DIFFER

IN ORNITHINE

GRANZYME MLC

SENSITIVITY

51

B

a-CD3

ORN - +

- +

PFP MLC ORN

-

+

ct-CD3 -

+

FIG. 6. Effect of Om on granzyme B and PFP mRNA expression in allo-CTL and ARC cells. Spleen cells were activated with 0.3 pg/ml of (u-CD3 mAb -+ 10 mM Orn for 48 hr; CTL were generated in 72 hr MLC (B6 X DBA/Z) k 10 mM Orn. Total cellular RNA was isolated from the cultured cells and Northern blot analysis was performed. The intrigrity and equal loading of the RNA was evaluated by staining the gel with ethidium bromide and inspection under uv transillumination. Granzyme B. 10 pg/lane RNA: PFP. 30 fig/ lane RNA.

Several factors were examined in an effort to uncover the basis for the differential ORN sensitivity of CTL activated by alloantigen vs a-CD3 binding to the TCR complex. Time period of activation and concentration of a-CD3 were not elements that could account for the Orn resistance of ARC reactivity relative to allo-CTL (Fig. 3). The latter is an important observation because memory CTL (mTc), which are preferentially activated by low concentrations of a-CD3 mAb (29) are Orn insensitive (B.M.S., unpublished results). These factors cannot explain the Orn-resistant characteristic of ARC cells, however, because naive pTc (Orn-sensitive), as well as mTc (Orn-resistant), are stimulated at higher concentrations of a-CD3 mAb (29). The fact that we do not observe any Orn inhibition at the higher doses of a-CD3 mAb therefore argues against a selective activation of mTc as the basis of ARC resistance to Orn inhibition. Another consideration was that Orn resistance might result from additional cytokines produced as a consequence of a-CD3 stimulation. Failure of the protein G-absorbed SN from splenocytes activated with a-CD3 (+ Orn) to abrogate Orn sensitivity of CTL generated in MLC indicated that the Orn resistance of a-CD3 activation was not due to secretion of a unique cytokine or combination of cytokines (Table 4). We also analyzed whether the differential Orn susceptibility of pTc activated by alloantigen vs a-CD3 mAb could be explained by a difference in the potency of the activation signal. Our results do not support this explanation because ARC cells induced using suboptimal concentrations of a-CD3 mAb retained resistance to Orn inhibition (Fig. 2). Furthermore, addition of a-CD3 mAb to MLC failed to overcome Orn inhibition of CTL and yet induced ARC activity in the same cultures (Fig. 5; an observation that also argues against the role of a soluble product). Taken together, these

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results indicate that differential susceptibility to Orn of pTc activated by alloantigen vs a-CD3 mAb is due to a qualitative, not quantitative, difference in the pathway leading to effector function. Our findings indicate that TCR occupancy by antigen/MHC vs a-CD3 mAb delivers instructive signals for the activation of lymphocyte functions that are processed differently. Support for this suggestion can be derived from several recent studies. Klausner et al. (18) detected biochemical differences in the process of T cell activation due to stimulation by antigen and (u-CD3, revealed by the finding that CAMP inhibits CD3 l-chain tyrosine phosphorylation induced by antigen but not by a-CD3 mAb. Schlitt et al. (19) have reported differences related to activation of human lymphocytes by mAbs directed against al/P-TCR and CD3, as evidenced by sensitivity to cyclosporin and IL-2 dependence. Hengel et al. (22) recently showed that CTL effector function triggered via cr/&TCR requires PKC activity whereas CTL depleted of PKC retain their ability to lyse target cells when triggered via CD3. The activation pathway initiated as a consequence of the ligand bound to the TCR complex may be associated with the use of different signal-transducing elements of CD3. Ashwell and co-workers have reported that in T cell hybridomas, inositol phosphate hydrolysis correlated with the expression of TCR complexes containing (7 heterodimer subunits (a/P-TCR/CD3{q); inositol phosphate hydrolysis was induced in ol/fi-TCR/CD3-rv-negative cells, however, by triggering with a-CD3 mAb but not with antigen (30,3 1). Other evidence also indicates that a/P-TCR/CD3-