Tumor cell lysis by T cells distinct from NK cells and alloantigen-specific cytotoxic T cells

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

49,405-423

(1988)

Tumor Cell Lysis by T Ceils Distinct from NK Cells and Alloantigen-Specific Cytotoxic T Cells DWAIN Liver

and Rheumatic

L. THIELE

AND PETER E. LIPSKY

Diseases Units, Department of Internal Southwestern Medical Center, Dallas,

Medicine, The University Texas 75235

of Texas

The generation and mechanism of tumor cell lysis by cytotoxic T cells derived from natural killer cell (NK) and allospecific cytotoxic T cell (CTL)-depleted precursors were examined. NK cells and the precursors of alloantigen-specific CTL were deleted from human peripheral blood lymphocytes by preincubation with L-leucyl+leucine methyl ester (Leu-Leu-OMe). Following phytohemagglutinin activation, CD3( + ), CD4( + ) or CD8(+), CDllb(-), CD16(-), and NKHl(-) killer cells capable of lysing a broad spectrum of tumor targets were generated. Cytolysis was not strictly lectin dependent as similar killer cells were generated by activating Leu-Leu-OMe-treated T cells with immobilized monoclonal antibodies to the CD3 molecular complex. The rate of tumor cell lysis by these mitogen-activated T cells was slower than that mediated by CD3( -) NK cells. Tumor cell lysis by mitogen-activated killers was inhibited by anti-CD3 but was not restricted by major histocompatibility complex antigen expression on target cells or by CD4/CD8 expression on effecters. Although similar to NK cells in susceptibility to anti-LFA-l inhibition of killing, these mitogen-activated killer cells were more sensitive to the inhibitory effects of anti-CD2 than were CD3( -)-activated NK-like cells. Thus, tumor cell lysis by CD3( +) cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes appears to be mediated in part by mechanisms distinct from those employed by CD3( -) NK cells or antigen-specific CTL. o 1988 Academic press, tnc.

INTRODUCTION

Human cytotoxic lymphocytes are functionally and phenotypically heterogeneous. Thus, antigen-committed cytotoxic T cells (CTL) recognize target cells by means of classic T cell receptors and are restricted in recognition of targets by products of major histocompatibility complex (MHC) genes. In contrast, natural killer cells (NK) are defined by their ability to recognize and lyse a variety of malignant or virus-infected cells in the absence of MHC restriction. Furthermore, NK activity is spontaneously present in nonimmunized hosts whereas induction of antigen-specific CTL requires in vivo or in vitro immunization. A number of investigators, however, have noted that after repeated stimulation of antigen-specific CTL clones or after nonspecific activation of freshly isolated lymphocytes, clonal populations of CD3-positive cytotoxic cells can be generated that possess the ability to lyse a broad spectrum of tumor targets in an MHCunrestricted fashion (l-6). Recently, small subsets of peripheral blood CD3positive T cells have also been shown to possess NK-like activity (7, 8). Because such freshly isolated cytotoxic T cells or cultured T cell clones exhibit a similar spectrum of target specificity and may express certain markers such as CD16 (Leu-11, B73.1), CDllb (LeulS, OKMl), or NKH-1 (N901) that are found on 405 0090- 1229/88 $1.50 Copyright AI1 rights

B 1988 by Academic Press. Inc. of reproduction in any form reserved.

406

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LIPSKY

almost all human peripheral blood NK cells, they have been studied as models of NK cell function (1, 2, 9, 10). However, because these cytotoxic cells, unlike the majority of peripheral blood NK cells, express CD3 and a functional T cell antigen receptor (2, ll), the lineal relationship of such tumoricidal T cells to peripheral blood NK cells or other peripheral blood CTL is unclear. Previous studies from our laboratory suggested that differences in sensitivity to the toxic effects of Leu-Leu-OMe could be used to characterize tumoricidal T cells derived from peripheral blood precursors that are distinct from NK cells or from the vast majority of alloantigen-specific CTL. Thus, when peripheral blood mononuclear cells (PBM) were incubated with this agent, NK cells, monocytes, and the T cell precursors of alloantigen-specific cytotoxic T cells are killed, whereas B cells and T helper/inducer cells remain viable and functionally intact (12). However, lymphocytes cytotoxic for tumor targets could still be generated from Leu-Leu-OMe-resistant lymphocytes by culturing them for 2-6 days with the mitogen PHA (13). The present studies were designed to analyze the phenotype and function of these cytotoxic cells in greater detail. The data demonstrate that T cells distinct from MHC-restricted CTL or NK cells can be activated in short term culture to lyse tumor targets via cell-mediated mechanisms that are similar but not identical to those utilized by either alloantigen-specific CTL or by CD3 negative large granular lymphocytes (LGL). MATERIALS

AND METHODS

Medium

Except where noted, medium RPM1 1640 (Inland Laboratories, Fort Worth. TX) supplemented with 10% fetal bovine serum, penicillin G (200 U/ml), gentamicin (10 kg/ml), and L-glutamine (0.3 mg/ml) was used for cell culture. Lymphokines

and Reagents

Recombinant human interleukin 2 (rIL-2) was a gift of Cetus Corp. (Emeryville, CA). Phytohemagglutinin was obtained from Wellcome Reagents Div., Burroughs Wellcome Co. (Research Triangle Park, NC). t-Leucyl-L-leucine methyl ester was synthesized by esterification of leucyl-leucine (Sigma Chemical Co., St. Louis, MO) as previously described (14). Monoclonal

Antibodies

(Mab)

The Mab OKT3 ((15) anti-CDS), OKT4 (( 15) anti-CD4), OKTS (( 15) anti-CD@, OKTll ((15) anti-CD2), OKMl ((16) anti-CDllb), HNK-I ((17) Leu-7), MB40.5 ((18) anti-HLA A,B,C), and PA2.6 ((19) anti-HLA-A,B,C) were purified from hybridoma cells obtained from the American Type Culture Collection (Rockville, MD). Mab 60.3 ((20) directed against a common determinant of LFA-I. CR,, p150,95), 64.1 ((21) anti-CD3), and 9.6 ((22) anti-CD2) were gifts of Dr. P. Beatty, Seattle, Washington. Leu-llb ((23) anti-CD16), Leu-llc (24), and Leu-15 (antiCD 11b) were purchased from Becton-Dickinson Monoclonal Center, Inc. (Mountain View, CA). Anti-NKH-1 (25) was purchased from Coulter Immunology (Hialeah, FL). Phycoerythrin conjugates of Leu-llc, Leu-15, anti-NKH-I, and

MECHANISMS

OF

TUMOR

CELL

LYSIS

BY

T CELLS

407

mouse IgG control (MsIgGl-RDl) were used for cell staining followed by analysis with an Ortho System 50HH flow cytometer. Cell Preparation

PBM were obtained from healthy adult volunteers by centrifugation of heparinized venous blood over sodium diatrizoate/ficoll gradients (Isolymph; GallardSchlesinger Chemical Mfg. Corp., Carle Place, NY). Monocyte (M+)-enriched populations were prepared from glass or plastic adherent cells and M+depleted lymphocytes were prepared by passage of PBM through nylon wool columns as detailed (12, 13). In experiments where Leu-Leu-OMe-treated cells were used, lymphocytes were suspended in phosphate-buffered saline at 2.5 x 106/ml and incubated for 1.5 min at room temperature with 250 PM Leu-Leu-OMe as previously described (12, 14). Following Leu-Leu-OMe treatment in this manner, spontaneous lytic activity against K562 targets was always reduced by >99% as previously detailed (12, 13). In other experiments, LGL were depleted by incubation with anti-Leu-1 lb, HNK1, OKMI , and complement as previously described (13). The LGL-depleted lymphocytes prepared by either of these methods contained < 1% Leu-1 lb, HNK-1, or OKMl( +) cells. CD3( - ), LGL-enriched populations were prepared by incubation of nylon column passed lymphocytes with OKT3, OKT4, and L243 (26, anti-HLA-DR) followed by panning on goat anti-mouse immunoglobulin-coated petri dishes as previously described in detail (12). After being panned twice, the cells were incubated with additional OKT3, OKT4, L243, and complement for 75 min at 37°C. The resulting LGL-enriched population was ~5% OKT3( +). In some experiments, T8- and T4-enriched T cell subsets were obtained by negative selection panning as previously described (12). Such TCdepleted cell populations were 75-88% OKT8( +) and ~2.5% OKT4( +), whereas T8-depleted cells were >92% OKT4( +) and ~1.5% OKTS( +). In other experiments, cells were stained with OKT8 or OKT4 and separated with a fluorescence-activated cell sorter (FACS III, Becton-Dickinson) as previously described (12). The highly purified T8- or T6enriched cells positively selected by this technique were >98.5% OKT8( +) or OKT4( +), respectively, whereas OKT8( -> or OKT4( -) cell populations had ~1% contamination by positively stained cells. Culture Conditions for Generation of Activated

Killer Cells

Cells (0.25 x lO?ml) were cultured with mitomycin C-treated M+ (0.1 x 106/ml) in upright 25-cm2 tissue culture flasks with PHA (0.5 p.g/ml) for generation of PHA-activated killer cells. In other experiments, lymphocytes (0.5 x lO?ml) were cultured in 17 x IOO-mm polypropylene tubes with rIL-2 in the presence or absence of PHA. When immobilized 64.1 Mab was used to activate LeuLeu-OMe-treated peripheral blood lymphocytes, 100 rig/well 64.1 in 50 ~1 phosphate-buffered saline, pH 7.4, was added to flat-bottom microtiter wells (Costar No. 3799, Cambridge, MA) and incubated for 2 hr at 22°C. Wells were then washed extensively and 25,000 responder cells in 200 p.1 culture medium were

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added per well. Except where indicated, assay for cytolytic activity. Culture Conditions

for Generation

LIPSKY

cells were cultured for 4-5 days before

of Allountigen-Specijic

CTL

LGL-depleted lymphocytes (1 x 106/ml), prepared by anti-NK antibody and complement treatment, were cultured with mitomycin C-treated allogeneic M+ and 50 U/ml of supplemental rIL-2 for 5 days as previously described in detail (12).

Tumor Cell Lines K562, an erythroleukemia cell line (27, Cur, a renal cell carcinoma line (28, a gift renal cell carcinoma line (28, gift of Dr. M. and U937 (30, ATCC) were maintained in mented with 10% FBS. Cytotoxicity

a gift of Dr. Prager, culture

of Dr. M. Bennett, UTHSCD); M. Prager, UTHSCD); Caki, a UTHSCD); Daudi (29, ATCC); in medium RPMI 1640 supple-

Assays

Prior to cytotoxicity assay, nonadherent target cells were washed, resuspended in 1 ml fresh medium, and incubated with 100 pCi 51Cr for l-2 hr. Cells were then washed and counted. Flasks containing adherent Cur or Caki target cells were washed and cells were covered with 2 ml of Puck’s EDTA solution and incubated for 1 hr with 100 PCi 51Cr. Trypsin (50 l.~l of 2.5% solution; GIBCO Labs, Life Technologies, Inc., Chagrin Falls, OH) was then added and after another 2-3 min, detached cells were washed and counted. Lymphocyte killing of these targets was then assessed in 5’Cr release assays and the percentage of specific lysis or lytic units of cytotoxic activity was calculated as previously described (12, 31). In the experiments reported in this manuscript, spontaneous 51Cr release from Cur targets was <20%, K562 targets <25%, and Caki and Daudi targets ~35% during 18to 20-hr assays. RESULTS

Phenotype of the Precursors of PHA-Activated Leu-Leu-OMe-Resistant Lymphocytes

Cytotoxic

Cells Generated fkom

Previous studies have demonstrated that exposure of freshly isolated human peripheral blood cells to 100 FM or greater concentrations of Leu-Leu-OMe results in irreversible loss of all spontaneous NK function. As shown in Fig. 1, this loss of NK function was associated with loss of all Leu-llc, NKH-1, or Leu-15( +) cells. Furthermore, after 4 days of PHA stimulation of LeuLeu-OMe-treated lymphocytes (Fig. 1, right), no cells which stained above control levels with Leu-1 lc, NKH-1, or Leu-15 could be detected. In contrast, a discrete subset of NK antibody-positive cells was still readily detectable in PHAstimulated cultures of control lymphocytes. Despite the absence of cells bearing CD16, CD1 lb, or the antigen recognized by NKH-1, PHA activation generated potent lytic activity (Table 1). As previously reported, >97.5% of PHA-activated Leu-Leu-OMe-treated cells were OKT3( + )

MECHANISMS

MrfpG

OF

TUMOR

LWllC

NKH-1

CELL

k”35

LYSIS

MITgG hulk

BY

NKH-I

T CELLS

409

LP”15

FIG. 1. NK antigen phenotype of control and Leu-Leu-OMe-treated lymphocytes before and after PHA activation. Freshly isolated peripheral blood lymphocytes were incubated for 15 min in the presence or absence of 250 PM Leu-Leu-OMe. Cells were then analyzed within 2 hr by flow cytometry or were cultured with PHA and autologous MI$ for 4 days prior to monoclonal antibody staining.

by FACS analysis (13). The experiments contained in Table 1 examined the CD4/ CD8 phenotype of these killer cells. As shown by the results of Experiments 1 and 2, the T4( -), T8-enriched T cell populations were activated to kill Cur, Daudi, or K562 targets with greater efficiency than unseparated T cells or T8( -), T4enriched T cells. However, some cytotoxic activity directed at each of these tumor targets could also be generated from T8-depleted T cell populations. These results suggested that the precursors of these cytotoxic cells were heterogeneous with respect to CD4/CD8 phenotype or that they were derived from CD4( -), CD8( -) precursors that were activated with greater efficiency in the presence of CD8( +) T cells. To address these possibilities, experiments were carried out using highly purified T8 or T4 cells positively selected on a fluorescence-activated cell sorter. As shown by the results of Experiments 3 and 4 in Table 1, the majority of cytotoxic effecters capable of lysing Cur, Daudi, or K562 targets arose from CD8( +) precursors that could be activated in the absence of CD4( +) helper T cells. However, cytotoxic T cells could also be generated from highly purified CD4( +) T cell precursors. The cytolytic activity of cells derived from CD4enriched precursors was always less than that of CD8-enriched or unseparated T cells, but was mediated predominantly by CD4( + ) effector cells and not the small percentage of contaminating CD4( - ) cells. Thus, CD4( + ) cells selected with the FACS after activation of CD8( - ) cells manifested lytic activity (Table 1, Experiment 5). These results indicate that the killer cells generated in this system were heterogeneous, expressing either CD4 or CD8. Of interest, the relative sensitivity of a panel of tumor targets to lysis by CD8( +) cells was indistinguishable from that mediated by CD4( + ) cells. Generation of Cytotoxic Effector Cells from Leu-Leu-OMe-Resistant Lymphocytes Is Not Exclusively Lectin Dependent As PHA was used to activate the cytotoxic cells responsible for tumor lysis i’f these studies, it was possible that the small amount of lectin carried into cytotoxicity assays with the effector cells was playing a role in target cell binding and lysis. To examine this possibility, Leu-Leu-OMe-treated lymphocytes were cultured with immobilized Mab to CD3 (64.1). This provided a means to activate the T cells in the absence of lectin and accessory cells (35). As demonstrated by the

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TABLE 1 PHA-ACTIVATEDTCELLSCYTOTOXIC FORTUMORCELLSAREGENERATED AND OKT8 POSITIVE PRECURSORS

Expt 1

T cell precursor population” ~__ Unseparated T cells T8 negative T4 negative

Purification of effector cells after cultureh

FROM BOTH OKT4 Specific lysis

Cur

Daudi

41 -

29

KS62 14 I’i 19

52

1 -_

2

T8 negative T4 negative

-

IS 112

3 40

ND ND

3

Unseparated T cells T8 negative T8 positive

-

56 21 68

22 c 29

II8 68 ND

Unseparated T cells T4 positive T4 negative

-

40 12 57

18 6 24

67 17 ND

75

-

74,

II 12

-

15 Ih

4

5

Unseparated T cells T8 negative T8 negative

T4 positive

0 In Experiments 1 and 2, cells were purified by panning followed by antibody and complement treatment as described under Materials and Methods. In Experiments 3 and 4, cells were incubated with OKT8 (Experiment 4) or OKT4 (Experiment 5) and then separated by FACS. All T cell populations were treated with 250 p,M Leu-Leu-OMe to deplete LGL before culture with PHA for 5 days and assay of cytotoxic activity. In Experiment 3, the positively selected T8-enriched cells were 99.5% OKT8( + 1; in Experiment 4. the positively selected T4-enriched cells were 98.6% OKT4( + 1. b In Experiment 5, T4( +) cells were initially purified by panning and antibody and complement treatment. After Leu-Leu-OMe treatment and culture with PHA for 4 days, activated T cells derived from T4-enriched precursors were 98.3% OKT4( +) and 1.3% OKT8( + 1. A fraction of these cells was then positively selected by FACS (postsort analysis 99.7% OKT4(+)) and assayed in parallel with unsorted cells.

experiments detailed in Table 2, Leu-Leu-OMe-treated peripheral blood lymphocytes cultured for 4 days with the immobilized anti-CD3 Mab, 64.1, were comparable to PHA-activated cells with respect to the capacity to lyse Cur and K562. Since the immobilized anti-CD3 Mab is unavailable for effector-target cell bridging, these findings suggest that Leu-Leu-OMe-treated peripheral blood lymphocytes can be activated to kill tumor targets by mechanisms that are independent of lectin or Mab bridging between effector and target cell. Lysis of Phenotypically Disparate Tumor Targets Appears to be Mediated by a Common Population of Cytotoxic T Cells

The studies detailed in Fig. 2 were designed to assess whether lysis of various tumor targets was mediated by a common population of cytotoxic effector cells or perhaps by multiple distinct populations of cytotoxic ceils, each with the potential to lyse discrete subsets of tumor cells. The targets chosen for this study included Cur, a renal cell carcinoma line, which expresses class I but not class II MHC

MECHANISMS

OF

TUMOR

TABLE GENERATION

Expt 1

2

OF TUMORICIDAL

CELL

LYSIS

2

ACTIVITY FROM LEU-LEU-OME REQUIREMENT FOR LECTIN

T cell activation stimulus

411

BY T CELLS

TREATED

T CELLS:

LACK

OF

Specific Lysis” cur

K-562

PHA Immobilized

64.1

67 58

52 82

PHA Immobilized

64.1

52 38

54 19

a In these experiments, where indicated, Leu-Leu-OMe-treated NAC were cultured for 4 days with immobilized 64.1. Parallel cultures were established in which 25,000 Leu-Leu-OMe-treated NAC were cocultured with 25,000 radiated adherent cells and 0.5 &ml PHA. At the end of 4 days, ‘iCr-labeled targets were added directly to culture wells and the percentage of specific lysis over 18 hr was determined. Enumeration of effector cell numbers present in control culture wells indicated that E:T ratios present in such assays were: PHA-stimulated cells, Experiment 1: 49:l; immobilized 64.l, Experiment 1: 80: 1; PHA-stimulated cells, Experiment 2: 100: 1; immobilized 64. l-stimulated cells, Experiment 2: 20: 1. Supematants harvested from 64. I- or PHA-stimulated cultures uniformly caused <:5% lysis of Cur or K562 in 18-hr assays.

antigens (personal observation); Daudi, a B lymphoblastoid cell line which expresses class II but not class I MHC antigens (36); and K562, an erythroleukemia cell line that does not express MHC antigens (37). As shown by the data contained in Fig. 2, PHA-activated Leu-Leu-OMe-resistant effector cells killed each of these targets. Moreover, killing of each of these phenotypically disparate tumor cell lines was competitively inhibited by the presence of each of the other tumor cell lines (Fig. 2) but not by Leu-Leu-OMe-treated PBM (data not shown). Furthermore, the efficiency of cold target inhibition by each cell type was proportional to the ease with which it was lysed by PHA-activated T cells. These data suggest that a common population of effector cells is responsible for the lysis of all three targets. Kinetics of Tumor Cell Lysis by T Cells Is Dgferent from that Mediated CD3 Negative LGL

by

The studies detailed in Fig. 3 were carried out to assess in greater detail the kinetics of target cell lysis by the CD3( +) killer cells generated from LeuLeu-OMe-treated lymphocytes and to compare their cytotoxic potential to that of CD3( -) LGL. After PHA activation of Leu-Leu-OMe-treated lymphocytes, the cumulative K562 lysis caused by these cells over 20 hr at an E:T ratio of 10: 1 was equivalent to that caused by rIL-2 activated CD3( -) LGL at a 5: 1 E:T ratio. However, the rate at which such target cell killing was accomplished by the two populations of cytotoxic cells was quite different. Thus, >70% of total (20 hr) target cell lysis of either K562 (top) or Cur (bottom) by CD3( -> LGL was achieved during the first 8 hr of assay. In contrast, the killing generated from Leu-Leu-OMe-treated lymphocytes proceeded more slowly with <45% of the maximal killing of either target occurring during the first 8 hr of assay. When kinetics of target cell lysis were examined using E:T ratios ranging from 2.5:1 to

412

THIELE

Cold

AND

LIPSKY

Target Inhibitron

Cold Target Inhibition

Ratlo

of Unlabeled

to 5’Cr

of Daudi

Lysis

of K562

Ly%

- Labeled

Targets

FIG. 2. Tumor cell fysis by PHA-activated T cells is susceptible to cold target inhibition by phenotypically disparate tumor cells. PHA-activated T cells generated from Leu-Leu-OMe-treated precursors were assessed for ability to lyse 51Cr-labeled Cur (top), Daudi (middle), or KS62 (bottom) targets in the presence of varying numbers of unlabeled tumor cells. “Cr release was measured in 8-hr assays.

40: 1, it was uniformly noted that ~30% of total K562 or Cur lysis by CD3( - ) LGL occurred during the final 12 hr of a 20-hr assay, whereas >50% of total killing of either target by PHA-activated T cells routinely occurred during this portion of the assay (data not shown). Furthermore, addition of 1000 U/ml rIL-2 to PHAstimulated cultures of Leu-Leu-OMe-treated lymphocytes did not generate effectors with accelerated kinetics of target cell lysis. Moreover, addition of PHA to rIL-2-stimulated LGL cultures did not result in the generation of effecters with retarded killing of tumor targets. Thus, the kinetics of tumor cell lysis by PHAactivated, CD3( +), NK antigem -) lymphocytes were distinctly different from that of CD3( -) LGL, even when identical activation stimuli were used. Furthermore, this difference in rate of target cell lysis was a characteristic of the effector lymphocytes and was relativeIy independent of the tumor cell target which was used to assay cytotoxicity. Soluble cytolytic factors have been shown to be released after PHA stimulation of T cells and to require longer assays to assess cytotoxic activity (32-34). How-

MECHANISMS

OF

TUMOR

CELL

LYSIS

BY

T CELLS

413

K562

Durationof

Assay

(hours)

FIG. 3. Kinetics of tumor cell lysis by PHA-activated Leu-Leu-OMe-treated T cells and CD3( -) NK cells. CD3( -) lymphocytes cultured for 4 days with 1000 U/ml IL-2 alone (m) or PHA and 1000 U/ml IL-2 (A) or Leu-Leu-OMe-treated lymphocytes cultured for 4 days with PHA alone (0) or PHA and 1000 U/ml rIL-2 (El) were assayed for ability to lyse K562 and Cur targets. Data are given for lysis mediated by CD3( -) effector cells at E:T ratios of 5: 1 and CD3( +) effector cells at E:T ratios of 10: 1.

ever, in additional experiments (data not shown) no evidence could be found that Cur or K562 lysis was mediated by soluble factors present in the culture supernatant of tumor cell or PHA-stimulated cytotoxic T cells generated from PHAactivated Leu-Leu-OMe-treated lymphocytes. T Cell Surface Determinants

Involved

in Cytolysis by PHA-Activated

T Cells

To examine the role of class I MHC antigens and the CD8 molecule in the killing of targets by PHA-activated T cells, purified T8 cells were obtained by depletion of both OKT4( +) T cells and OKMl, Leu-lib, or HNK-l(+) LGL. These T8 cells were then stimulated either with allogeneic M$ or with PHA and syngeneic M+ for 5 days and assayed for the ability to lyse stimulator lymphoblasts or Cur targets, respectively. As shown in Fig. 4, both lysis of allogeneic lymphocytes by specific CTL and Cur lysis by PHA-activated T8 cells were comparably inhibited by OKT3. However, killing of stimulator lymphoblasts by MLC-activated T8 cells was profoundly inhibited in the presence of antibodies to HLA-A,B,C antigens (Table 3, Experiments 1 and 2), whereas no inhibition of Cur lysis by these antibodies was noted. Furthermore, OKT8 inhibited killing by allospecific T8 cells over a broad concentration range (I-100 p,g/ml), but killing of Cur targets by PHA-activated T8 cells was not inhibited by this antibody. In fact, these concentrations of OKT8 augmented Cur lysis. In the experiments detailed in Fig. 5, antibody 60.3 directed against the LFA-1, CR,, p150,95 family of leukocyte antigens and the anti-CD2 antibodies, 9.6 and OKTll, were assessed for the ability to inhibit lysis of Cur and K562 targets by PHA-activated Leu-Leu-OMe-treated lymphocytes. As shown in the top panel of the figure, K562 lysis by these cells was profoundly inhibited by 60.3, whereas

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I J Log Antibody

Concentration

(pg/ml)

4. Lysis of tumor cells by PHA-activated T cells and lysis of allolymphoblasts by mixed lymphocyte culture (MLC)-activated CTL are inhibited by OKT3. Similar concentrations of NK cell-depleted T8 cells were cultured with PHA and autologous mitomycin C-treated M+ or with allogeneic M$ for 5 days and then assessed in 8-hr cytotoxicity assays for the ability to lyse Cur or allogeneic lymphoblasts, respectively, in the presence of the indicated concentrations of SPA columnpurified OKT3. The percentage inhibition of specific target lysis was calculated from the formula: % inhibition = 100 X (control specific lysis - experimentaf specific lysis)/control specifK lysis. Data are given for the mean C SEM of three experiments. FIG.

OKTll had no effect on K562 lysis, and 9.6 was only modestly inhibitory at the highest concentration tested. In contrast, as shown in the lower panel, 60.3 had no effect on Cur lysis, whereas both 9.6 and OKTI 1 inhibited Cur lysis more effectively than K562 lysis. As shown in Fig. 6, the effect of 60.3 on tumor lysis TABLE CUR Lvsrs __~

Expt -___

-~~~

BY PHA-ACTIVATED ~~-_

.-__

CD8

POSITIVE

ANTI-HLA-A,B,C,

~~_ .._.

Antibody addition” to cytotoxicity assay

3

T CELLS CANNOT ANTIBODIES

BE INHIBITED

MLC-activated cytotoxicity vs allolymphoblastsb

BY OKT8

OR

PHA-activated cytotoxicity vs Cur”

1

Nil OKT8. 1 t&ml OKT8, 10 &ml OKT8, 100 kg/ml Anti-HLA-A,B,C, IO t&ml Anti-HLA-A,B,C, 100 kg/ml

Percentage specific lysis’ 61 46 40 37 32 18

20 7.5 2? 2: 26 22

2

Nil OKT8, 10 pg/ml Anti-HLA-A,B,C, 10 pglml

30 16 17

II 31 2x

3

Nil OKT8, 1 kg/ml OKT8. 10 pg/ml

59 39 34

34 55 hi

u The indicated concentrations of Sephacryl 300-purified OKT8 (Experiments I and 3) or SPApuritied OKT8 (Experiment 3) or a mixture of anti-HLA-A,B.C, Mab MB40.5, and PA2.6 (I:1 by protein concentration) were added to 8-hr cytotoxicity assays. ’ NK cell-depleted T8 cells were prepared and cultured as described in Fig. 4. ( Data are given for 50: 1 effector:target ratios.

MECHANISMS

OF

TUMOR

CELL

LYSIS

BY

T CELLS

415

0

20

60 80

4

-;

Log Antibody

0 1 Concentration

2

I

(pghl)

FIG. 5. Target cell differences in ability of anti-CD2 or anti-LFA-1 antibodies to modulate cytotoxic activity of PHA-activated killer cells generated from Leu-Leu-OMe-treated precursors. LeuLeu-OMe-treated lymphocytes were cultured with PHA for 4 or 5 days and were then assessed for the ability to lyse K562 and Cur targets in 18-hr assays in the presence of indicated concentrations of SPA column-purified Mab OKTI 1, 9.6, and 60.3. Data are expressed as mean f SEM of five experiments with the percentage of inhibition calculated as in Fig. 4.

appears to be a function of the target rather than the effector cell which is used. Thus, lysis of Cur by either CD3( +) T cells derived from Leu-Leu-OMe-treated precursors or by CD3( -) LGL was unaffected by addition of 60.3 to the assay. Lysis of another renal cell carcinoma line, Caki, by either effector was only minimally inhibited by 60.3. By contrast, lysis of the myeloid targets K562 and U937 by either class of effector cells was profoundly inhibited in the presence of 60.3. In additional experiments not shown, effector cells and targets were separately preincubated with 60.3. K562 lysis was only inhibited when 60.3 was bound to cytotoxic effector cells and not to K562 targets, whereas Cur lysis was not inhibited by preincubation of either effector or target cells with 60.3.

CW

Caki

K562

u937

6. CD3( -)-activated killer cells and CD3( +) PHA-activated killer cells have similar target cell specific susceptibility to anti-LFA-1 inhibition of tumor cell lysis. CD3( +) killer cells were generated by PHA activation of Leu-Leu-OMe-treated lymphocytes and CD3( -) killer cells by rIL-2 activation (100 U/ml for 3 days) of CD3(-) lymphocytes. Lysis of the indicated targets in 18-hr assays was assessed in the presence or absence of 1 t&ml of SPA column-purified Mab 60.3. Data are expressed as mean ? SEM of three experiments where the percentage of control cytotoxicity was calculated from the formula: Percent of control = 100 x experimental % specific lysis/control % specific lysis. FIG.

416

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Whereas the target-specific pattern of 60.3-mediated inhibition of tumor cell lysis was comparable for CD3( +) and CD3( - ) effecters, this was not the case for the inhibitory effects of 9.6. As previously noted by other investigators (38, 39), lysis of K562 cells by freshly isolated NK cells was only modestly inhibited by 9.6 (~25% inhibition by 10 pg of 9.6/ml over a broad range of E:T ratios, data not shown). However, as shown in Fig. 7, lysis of Cur by PHA-activated CD3( + ) effecters was significantly inhibited by I-10 pg 9.6/ml, whereas lysis by t-IL2-activated CD3( - ) LGL was not. Indeed, the level of 9.6-mediated inhibition of Cur lysis by PHA-activated T cells was comparable to the degree of 9.6-mediated inhibition of alloantigen-specific CTL (38 i_ 6% inhibition at 50: 1 E:T by 10 pg 9.6/ml, n = 3). DISCUSSION

The role of various effector cell surface antigens in target cell binding and lysis by CD3( +) clones cytotoxic for tumor cell lines has been studied in detail (2, 40, 41). Such CD3( +) clones have generally been functionally similar to freshly isolated peripheral blood NK cells. However, the phenotype of the peripheral blood precursors of such clones remains unclear. Furthermore, as functional studies with such clonal populations can only be performed after they have been expanded under long term culture conditions, the functional similarity of such effector populations to MHC-unrestricted cytotoxic T cells, recently demonstrated to be spontaneously activated in vivo (8) or after brief in vitro cultures ( 13. 42), remains unclear. The purpose of the present studies was to functionally characterize a well-defined population of freshly activated tumoricidal T cells. A number of investigators have recently described subsets of human peripheral blood T cells which either spontaneously lyse tumor targets or, after brief activation with IL-2 or in the presence of lectin, can be shown to exert such activity (8,42,43). Such T cell subsets have been shown to share certain antigens with NK cells such as NKHl (8) or HNK-1 (Leu-7, (42)). or to be both CD4( - ) and CDE( -) (43) and have been hypothesized to be the precursors of previously described T cell clones with NK-like activity (8). Alternatively, alloantigenspecific cytotoxic T cells have been observed to acquire MHC-unrestricted cyto-

FIG. 7. Mab 9.6 inhibits Cur lysis by CD3( +) killer cells but not by CD3( ~ ) killer cells. Activated killer cells and cytotoxicity assays were performed as in Fig. 5. Results are given for the mean -+ SEM of three experiments.

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toxic activity after long intervals of in vitro culture (44) and may therefore give rise to such NK-like CD3( +) clones. In contrast, the cytotoxic T cells characterized in the current studies clearly arise from a different precursor population. The removal of NK cells and allospecific CTL precursors by Leu-Leu-OMe treatment of peripheral blood lymphocytes (12, 13) produces a population of initially noncytolytic T cells, which after 2-6 days of activation by PHA or immobilized anti-CD3 develops MHC-unrestricted cytolytic activity against a broad spectrum of tumor targets (13). The activated cells responsible for this lytic activity are generated from CD3(+), CD4, or CD8( +), but CD16 (Leu-ll)(-), CDllb (OKMl, Leu-15)( -), HNK-1 (Leu7)( -), and NKH- I( - ) precursors and remain CD3( +), and CD16 (Leu-ll)(-), CDllb (OKMl, Leu-15)( -), and NKH-1 (N901)( -)((13) present studies). The use of Leu-Leu-OMe, therefore, permitted the functional characterization of activated T cells that were free of contaminating NK cells or allospecific CTL. In addition, these cells would appear to be distinct from the majority of lymphokine-activated killer (LAK) cells which derive from predominantly CD3( -), CD16( +), or NKHl( +) precursors (45-47). After peripheral blood lymphocytes are preincubated with 250 FM Leu-Leu-OMe, no tumoricidal activity can be detected after brief cultures (12-24 hr) with rIL-2 or PHA-stimulated T cell lymphokines. After 3-5 days of culture with high concentrations of rIL-2, minimal cytotoxicity against tumor cells can be generated from Leu-Leu-OMe-treated T cells, but the number of lytic units of cytotoxic activity generated in rIL-2-stimulated cultures is always ~5% of that activated by PHA stimulation (data not shown). Thus, stimuli other than IL-2 appear to be essential for optimal activation of the majority of this population of tumoricidal T cells. Of interest, immobilized anti-CD3 Mab was comparable to PHA in the capacity to activate the cytolytic function of this population of T cells. Thus, lectin attached to effector cells was not necessary for target cell lysis. Rather, the role of PHA in the culture systems used in these studies appeared to involve stimulation of T cell activation. This conclusion has been further supported by recent studies carried out in our laboratory with a variety of PHA-activated CD4( +) or CD8( +) T cell clones with tumoricidal activity (48). Whereas such clones often failed to maintain cytotoxic activity when cultured with rIL-2 alone, in all cases after prolonged culture in the absence of PHA these cloned T cells could be activated to kill both Cur and K562 targets when stimulated with immobilized anti-CD3 Mab (48). The present studies demonstrate that PHA-activated killer cells derived from Leu-Leu-OMe-treated lymphocytes are capable of cell-mediated tumor cell lysis by mechanisms that are similar but not identical to those employed by NK cells or by antigen-specific T cells, As has been previously reported (13), lysis of tumor targets by these cells proceeds more slowly than does lysis of the same target by CD3( -) LGL. The present studies demonstrate that this delayed rate of target cell lysis is indeed a characteristic of this class of CD3( +) cytotoxic cells and is not a feature of the culture conditions used to activate these cells, or of the tumor targets used in the assay. Lysis of tumor targets by PHA-activated CD3( +) cytotoxic cells could not be attributed to soluble factors alone, but rather appeared to be dependent on cell to cell contact involving certain leukocyte function-associated antigens previously

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shown to be of importance in cellular adhesion (37, 49, 50). Thus, killing of K562 and U937 cells by these cells was modulated by antibody 60.3 directed against the LFA-1, CR,, p150,95 family of antigens. The present studies demonstrate that 60.3 inhibits cytotoxicity mediated by PHA-activated CD3( +), NK antigem - ) cells in a target cell-dependent fashion. Moreover, preincubation studies indicate that such inhibition is caused by 60.3 binding to an effector cell antigen and not mediated by binding to target cells and thus is similar to its mode of action in other cytotoxicity assays (38, 41). When tumor targets of nonhemopoietic origin were used, a relative lack of 60.3 inhibition of killing by both CD3( + ) non-NK cells and CD3( -) NK-like effector cells was observed. Such findings indicate that 60.3 is not directly inhibitory of all cytolytic activity of either cell type. Moreover, target cell lysis that is not inhibited by 60.3 was found to be susceptible to cold target competition by targets whose lysis was inhibited by 60.3. These results suggest that the same CD3( + ) PHA-activated killer cell can utilize LFA-l-like antigens as well as other molecules to establish interactions with target cells, although it would appear likely that recognition of an additional structure expressed by the target cell is also necessary for killing. An additional interaction molecule utilized by PHA-activated killers to lyse targets is the CD2/LFA-2 antigen recognized by Mab 9.6. The capacity of Mab 9.6 to inhibit killing by these killer cells could have been explained by a number of previously observed effects of this antibody on T cell function. Thus, antibodies directed against the 50-kDa protein recognized by 9.6 have been shown to inhibit binding of T cells to sheep erythrocytes or a variety of other targets (38, 51). In addition, 9.6 and other anti-CD2 antibodies have also been shown to generate a transmembrane signal which modulates various T cell functions (52). Previous reports have demonstrated the profound inhibitory effects of Mab 9.6 on antigenspecific CTL function, whereas varying degrees of inhibition of the function of freshly isolated NK cell have been reported (38, 39). In the current studies, 9.6 caused consistent inhibition of antigen-specific CTL, but only modest inhibition of K562 lysis by freshly isolated NK cells and no inhibition of the lysis of Cur or K562 targets by rIL-2-activated CD3( -) LGL. The effects of 9.6 on tumor cell lysis by the PHA-activated CD3( +), NK antigem - ) cytotoxic cells were target cell dependent, with much more profound inhibition of the killing of nonhemopoietic target cells observed compared to that of tumor cell targets of hemopoietic origin. Of interest, the ability of 9.6 to inhibit lysis of specific targets was found to be inversely related to the degree of 60.3 inhibition. Thus, LFA-1 and LFA-2 may serve a similar role in effector-target contact, with LFA-Zmediated interactions playing a greater role in situations where target cells do not possess epitopes capable of binding LFA-1. As has been noted with selected T cell clones, tumor cell lysis by the cytotoxic effector cells characterized in these studies was modulated by anti-CD3 antibodies and thus the effector cells were clearly T cells. It was, therefore, of interest to determine whether CD4 or CD8 antigens on the effector cell or class I or class II MHC antigens on the tumor targets played a role in target cell recognition or lysis. The finding that lysis of class I MHC antigem +) Cur targets or class II MHC antigen( + ) Daudi could be efficiently inhibited by MHC antigen( - ) K562 cells

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suggested that at least a substantial fraction of cytotoxicity was mediated in an MHC-unrestricted fashion. The lack of correlation between CD8 or CD4 phenotype of cytotoxic cell precursors and the class I or class II MHC antigen expression of effector cells further suggested that these effecters were quite different from antigen-specific T cells with respect to mechanisms of tumor cell recognition. When purified CD3( +), CD8( +) T cells were used to generate alloantigenspecific CTL and PHA-activated tumoricidal cells, it became clear that unlike antigen-specific CTL, there was no essential role for the CD8 antigen or class I MHC antigens in the killing of Cur cells by these PHA-activated killer cells. Indeed, the presence of OKT8 antibody augmented Cur lysis by PHA-activated T cells. This finding was reproducibly seen with unpurified OKT8-containing ascites as well as with purified OKT8 antibody (data not shown). Whereas the nature of such augmentation is unclear, it is distinctly different from the effects of OKT8 on antigen-specific CTL killing. The cytotoxic T cells examined in this report would appear to be a component of a proposed third class of cytotoxic lymphocytes designated MHC-unrestricted CTL (53). Of interest, the cytotoxic cells characterized in these present studies appear to be derived from precursors which are in large part phenotypically distinct from the CD3( +), NKHl (Leu-19)( +), or CD3( +)/CD4( -)/CD8( -) subsets of freshly isolated peripheral blood T cells (8,43) or the CD3( +), NKHl( +) T cell clones (1, 2, 10) that have previously been noted to possess MHC-unrestricted CTL function (8,43). It is not clear, however, that any functional differences exist between such subsets of MHC-unrestricted cytolytically active T cells. Furthermore, we (48) and other investigators (54) have noted that after longer term culture (~4 weeks), essentially all T cell clones are induced to express antigenic markers such as NKHI which have previously been associated only with cells with NK-like function. Indeed, when a large panel of PHA-stimulated clones has been examined for MHC-unrestricted tumoricidal activity, after prolonged in vitro culture, significant cytotoxicity was exhibited by all (48). Such observations suggest that MHC-unrestricted CTL function is an activation-dependent property common to all subsets of T cells. It remains to be determined whether all T cells mediate such tumoricidal activity by similar mechanisms. The functional relevance of MHC-unrestricted T cell cytotoxicity is at present unclear. However, Lanier et al. (8) and Goto and Zvaifler (55) have recently identified MHC-unrestricted cytotoxic T cells in freshly isolated peripheral blood of normal individuals and in the synovial fluid of patients with inflammatory arthritis. The present observations that such activity is mediated not only by cells with NK-like phenotype or by CTL after prolonged in vitro activation but can also be mediated by CD4( + ) or CD8( + ) T cells stimulated by relatively short term activation regimens suggest that MHC-unrestricted cytotoxicity might be generated in vivo from a variety of T cell subsets. Recent clinical experience with cancer immunotherapy regimens suggests that generation of such MHCunrestricted cytotoxic function, while of some value in eliminating tumor cells, may also cause adverse effects on normal cell or organ function (56). Increased understanding of the nature of cytolytic mechanisms employed by these cells may be of value in devising improved strategies for the use of lymphokineor lectin-

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activated killer cells in the immunotherapy of human malignancies. Moreover, increased insight into the function of these cells may promote a better understanding of antigen-nonspecific effects of immune responses generated as part of a variety of inflammatory diseases. ACKNOWLEDGMENTS The authors thank Melanie Owen and Ruth Pettigrew for excellent technical assistance, and Mrs. Renate Davis and Mrs. Elsa Abraha for preparing the manuscript. This work was supported by Public Health Services Grants AM19329 and AM09989, and by Grant IN-142 from the American Cancer Society. Dr. Thiele is the recipient of NIH Clinical Investigator Award AM01251. and an award from the Clay Weed Memorial Cancer Fund Trust.

REFERENCES 1. Hercend, T.. Reinherz, E. L.. Meuer, S., Schlossman. S. F., and Ritz, J., Phenotypic and functional heterogeneity of human cloned natural killer cell lines. Nuture (London) 301, 158-160, 1983. 2. Hercend, T., Meuer, S., Brennan, A., Edson, M. A.. Acute, 0.. Reinherz. E. L.. Schlossman. S. F., and Ritz, J., Identification of a clonally restricted 90 dK heterodimer on two human cloned natural killer cell lines: Its role in cytotoxic effector function. J. Exp. Med. 158, 1547-1560, 1983. 3. Acha-Orbea, H., Groscurth, P., Lang, R.. Stitz, L.. and Hengartner, H., Characterization of cloned cytotoxic lymphocytes with NK-like activity. 1. Immunol. 130, 2952-2959, 1983. 4. Brooks, C. G.. Urdal, D. L., and Henney, C. S., Lymphokine-driven “differentiation” of cytotoxic T-cell clones into cells with NK-like specificity: Correlation with display of membrane macromolecules. Immunol. Rev. 72, 43-72. 1983. 5. Shortman, K., Wilson, A., and Scollay, R., Loss of specificity in cytolytic T lymphocyte ctones obtained by limited dilution culture of Lyt2+ T cells. J. Immunol. 132, 584-593, 1984. 6. Roberts, K., and Moore, M., A clonal analysis of human peripheral blood lymphocytes displaying natural killer-like activity. Eur. J. lmmunol. 15, 448-456. 1985. 7. Lanier, L. L., Kipps, T. J., and Phillips, J. H., Functional properties of a unique subset of cytotoxic CD3 + T lymphocytes that express Fc receptors for IgG (CD16/Leu- I 1 antigen). J. &p. Med.

162, 2089-2106,

1985.

8. Lanier, L. L., Le, A. M., Civin, C. I., Loken, M. R., and Phillips. J. H.. The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J. Immunol. 136, 4480-4486. 1986. 9. Allavena, P., and Ortaldo, J. R., Characteristics of human NK clones: Target specificity and phenotype. .I. Immunol. 132, 2363-2369, 1984. 10. Hercend. T., Griffin, J. D., Bensussan, A., Schmidt, R. E., Edson, M. A., Brennan, A., Murray, C., Daley, J. F., Schlossman, S. F.. and Ritz. J., Generation of monoclonal antibodies to a human natural killer clone: Characterization of two natural killer-associated antigens, NKHl, and NKHZ, expressed on subsets of large granular lymphocytes. J. C/in. Invest. 75, 932-943. 1985. 11. Lanier, L. L., Cwirla, S.. Federspiel, N., and Phillips, J. H., Human natural killer ceils isolated from peripheral blood do not rearrange T cell antigen receptor B chain genes. J. Exp. Med. 163, 209-214, 1986. 12. Thiele, D. L., and Lipsky, P. E., The immunosuppressive activity of L-leucyl-L-leucine methyl ester: Selective ablation of cytotoxic lymphocytes and monocytes. J. Immunol. 136, 1038-1048, 1986.

Thiele. D. L., and Lipsky, P. E., Leu-Leu-OMe sensitivity of human activated killer cells: Delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets. J. Irnmuml. 137, 1399-1406, 1986. 14. Thiele, D. L., and Lipsky, P. E., Regulation of cellular function by products of lysosomal enryme activity: Elimination of human natural killer cells by a dipeptide methyl ester generated from r-leucine methyl ester by monocytes or polymorphonuclear leukocytes. Proc. Nnrl. Acrrd. Sci. 13.

USA

82, 2458-2472.

1985.

MECHANISMS

OF TUMOR

CELL LYSIS BY T CELLS

421

15. Kung, P. C., Goldstein, G., Reinherz, E. L., and Schlossman, S. F., Monoclonal antibodies defining distinctive cell surface antigens. Science 206, 347-349, 1979. 16. Breard, J., Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. F., A monoclonal antibody reactive with human peripheral blood monocytes. J. Immunol. 124, 1943-1948, 1980. 17. Abo, T., and Balch, C. M., A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J. Zmmunol. 127, 102&1029. 1981. 18. Parham, P., Monoclonal antibodies against two separate alloantigenic sites of HLA-B40. Immunogenetics 13, 509-527, 1981. 19. Parham, P.. and Bodmer, W. F., Monoclonal antibody to a human histocompatibility alloantigen. HLA-AZ. Nature (London) 276, 397-399, 1978. 20. Beatty. P. G., Ledbetter, J. A., Martin, P. J., Price, T. H., and Hansen, J. A., Definition of a common leukocyte cell-surface antigen (Lp95-150) associated with diverse cell-mediated immune functions. J. fmmunol. 131, 2913-2918. 21. Hansen, J. A., Martin, P. J., Beatty, P. L., Clark, E. A.. and Ledbetter. J. A., Human T lymphocyte cell surface molecules defined by the Workshop monoclonal antibodies (“T cell protocol") In “Leukocyte Typing” (A. Bernard, L. Boumsell, J. Dausset. C. Milstein, and S. F. Schlossman, Eds.), p. 195, Springer-Verlag, Berlin, 1984. 22. Tadmori, W., Reed, J. C., Nowell, P. C.. and Kamoun, M., Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: Suppression of IL2 production by anti-p50 monoclonal antibodies. J. Zmmunol. 134, 1716, 1985. 23. Lanier, L. L., Le, A. M., Phillips, J. H., Warner, N. L., and Babcock, G. F., Subpopulations of human natural killer cells defined by expression of the Leu 7 (HNK-1) and Leu 11 (NK-15) antigens. J. fmmunol. 131, 1789-1796, 1983. 24. Perussia, B., Starr, S., Abraham, S., Fanning, V., and Trinchieri, G., Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. I. Characterization of the lymphocyte subset reactive with B73.1. J. Immunol. 130, 2133-2141. 1983. 25. Griffin, J. D., Hercend, T., Beveridge, R. P., and Schlossman, S. F.. Characterization of antigen expressed by human natural killer cells. J. Immunol. 130, 2947-2951, 1983. 26. Lampson, L. A., and Levy, R., Two populations of Ia-like molecules on a human B cell line. J. Immunol. 125, 293-299, 1980. 27. Lozzio, C. B., and Lozzio, B. B., Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45, 321-334, 1975. 28. Prager, M. D., and Kanar, M. C., Effect of dimethylsulfoxide and butyrate on 5’-nucleotidase of human renal carcinoma cells. Cancer Let?. 24, 81-88, 1984. 29.. Klein, E.. Klein, G., Nadkarni, J. S., Nadkami, J. J., Wiqzell, H., and Clifford, P.. Surface IgM-kappa specificity on a burkitt lymphoma cell in vivo and in derived culture lines. Cancer Res. 28, 130~1310,

1968.

30.

Larrick, J. W., Fischer, D. G., Anderson, S. J., and Koren, H. S., Characterization of a human macrophage-like cell line stimulated in vitro: A model of macrophage functions. J. Immunol. 125,

31,

Thiele, D. L., and Lipsky, P. E., Modulation of human natural killer cell function by L-leucine methyl ester: Monocyte-dependent depletion from human peripheral blood mononuclear cells. J.

6-12,

Immunol.

1980.

134,

786-793,

1985.

Spofford, B. T., Dayness, R. A., and Granger, G. A., Cell-mediated immunity in vitro: A highly sensitive assay for human lymphotoxin. J. Immunol. 112, 211 l-21 16, 1974. 33. Ortaldo, J. R., Mason, L. H., Mathieson, B. J., Liang, S.-M., Flick, D. A., and Herberman, R. B., Mediation of mouse natural cytotoxic activity by tumor necrosis factor. Nature (London)

32.

321, 700-702,

1986.

Degliantoni, G., Murphy, M., Kobayash, M., Francis, M. K., Perussia, B., and Trinchieri, G., Natural killer (NK) cell-derived hematopoietic colony-inhibiting activity and NK cytotoxic factor. Relationship with tumor necrosis factor and synergism with immune interferon. J. Exp. Med. 162, 1512-1530. 35. Geppert, T. D., and Lipsky, P. E., Accessory cell independent proliferation of human T4 cells stimulated by immobilized monoclonal antibodies to CD3. J. Zmmunol. 138, 1660-1666, 1987. 36. Arce-Gomez. B.. Jones, E. A., Barnstable, C. J., Solomon, E.. and Bodmer, W. F., The genetic 34.

422

THIELE

AND LIPSKY

control of HLA-A and B antigens in somatic cell hybrids: Requirements for p2 microglobulin. Tissue Anrigens 11, 96-l 12. 1978. 37. Kombluth, J., Spear, B., Raab, S. S., Wilson, D. B., Evidence for the role of class 1 and class II HLA antigens in the lytic function of a cloned line of human natural killer cells. J. Immune/. 134, 728-735, 1985. 38. Krensky, A. M., Sanchez-Madrid, F., Robbins, E., Nagy. J. A., Springer, T. A.. and Burakoff, S. J., The functional significance, distribution, and structure of LFA-1, LFA-2, and LFA-3: Cell surface antigens associated with CTL-target interactions. J. Immunol. 131, 611-616, 1983. 39. Fast, L. D., Hansen, J. A.. and Newman, W., Evidence forT cell nature and heterogeneity within natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) effectors: A comparison with cytolytic T lymphocytes (CTL). J. Immunol. 127, 448-452. 1981. 40. Moretta, A., Pantaleo, G., Mingari, M. C.. Melioli, G.. Moretta, L., and Cerottini, J.-C., Asxignment of human natural killer (NK)-like cells to the T cell lineage: Single allospecific T celi clones lyse specific or NK-sensitive target cells via distinct recognition structures. Eur. J. Immunol. 14, 121-125, 1984. 41. Schmidt, R. E.. Bartley, G., Levine. H.. Schlossman, S. F.. and Ritz. J., Functional characterization of LFA-1 antigens in the interaction of human NK clones and target cells. J. Itnmunnl. 135, 1020-1025, 1985. 42. Phillips, J. H., and Lanier, L. L.. Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen. J. Immunol. 136, 1.579-1585. 1986. 43. Lanier, L. L.. Ruitenberg, J. J.. and Phillips, J. H., Human CD3 + T lymphocytes that express neither CD4 nor CD8 antigens. J. Exp. Med. 164, 339-344. 1986. 44. Irle, C., Beatty. P. G., Mickelson, E. M., and Hansen, J. A.. Change in functional phenotype of cloned human alloreactive cytolytic T cells. Humun Imtnunol. 11, 183-191, 1984. 45. Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg. S. A.. Lymphokine-activated killer cell phenomenon: Lysis of natural killer-resistant fresh solid tumor cells by interleukin Z-activated autologous human peripheral blood lymphocytes. J. Exp. Med. 155, 1823-1841. 1982. 46. Itoh, K., Tilden, A. B., Kumagai. K.. and Balch, C. M.. Leu-11 + lymphocytes with natural killer (NK) activity are precursors of recombinant interleukin 2 (rlL-2)-induced activated killer (AK) cells. J. Immunol. 134, 802-806. 1985. 47. Phillips, J. H., and Lanier, L. L.. Discussion of the lymphokine-activated killer phenomenon: Relative contribution of peripheral blood natural killer cells and T lymphocytes to cytolysis. J. Exp. Med. 164, 814-825, 1986. 48. Pate], S. S., Thiele, D. L., and Lipsky, P. E., MHC unrestricted cytolytic activity of human T cells: Analysis of precursor frequency and effector phenotype. J. fmmunol. 139, 3886-3895, 1987. 49. Sanchez-Madrid, F., Nagy, J. A.. Robbins, E.. Simon. P., and Springer. 1‘. A.. A human leukocyte differentiation antigen family with distinct a-subunits and a common P-subunit: The lymphocyte function-associated antigen (LFA-1) and C3bi complement receptor (OKMlIMac-1). and the ~150.95 molecule. J. Exp. Med. 158, 1785-1803, 1983. 50. Patarroyo, M., Beatty, P. G., Fabre. J. W., and Gahmberg. C. G.* Identification of a cell surface protein complex mediating phorbol ester-induced adhesion (binding) among human mononuclear leukocytes. Stand. J. Immunol. 22, 171-182. 1985. 51. Selvaraj, P., Plunkett. M. L., Dustin. M.. Sanders, M. E.. Shaw. S.. and Springer, T. A.. The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3. Native (London) 326, 400-103, 1987. 52. Brottier, P.. Boumsell. L., Gelin, C.. and Bernard. A., T cell activation via CD2[T.gp501 molecules: Accessory ceils are required to trigger T cell activation via CD2-D66 plus CD2-9.6/TI 1 1 epitopes. J. Immunol. 135, 1624-1631, 1985. 53. Lanier, L. L.. Phillips, J. H., Hackett, .I., Jr.. Tutt. M.. and Kumar. V.. Natural killer cells: Definition of a cell type rather than a function. J. Immunol. 137, 2735-2739. 1986. 54. Lanier. L. L.. Le, A. M.. Ding, A.. Evans. E. L., Krensky, A. M.. Clayberger. C.. and Phillips. J. I-1.. Expression of Leu-19 (NKH-I) antigen on IL?-dependent cytotoxic and non-cytotoxic 7 cell lines. J. lmmunoi. 138. 2019-2023. 1987.

MECHANISMS

OF TUMOR

CELL LYSIS BY T CELLS

423

55. Goto, M., and Zvaifler, N. .I., Characterization of the natural killer-like lymphocytes in rheumatoid synovial fluid. J. Immunol. 134, 1483-1486, 1985. 56. Rosenberg, S. A., Lotze, M. T., Muul, L. M., Chang, A. E., Avis, F. P., Leitman, S., MarstonLinehan, W., Robertson, C. N., Lee, R. E., Rubin, J. T., Seipp, C. A., Simpson, C. G., and White, D. E., A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N. Engl. J. Med. 316, 889-897, 1987. Received March 4, 1988: accepted with revision August 16, 1988