Immunology and Cell Biology (1992) 70, 379-390

Generation and cytotoxic profile of human peripheral blood CD4 ^ T lymphocytes MARK J.SMYTH Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia Summary The effects of a variety of metabolic and anti-tumour necrosis factor (TNF) antibodies were utilized to distinguish several different mechanisms of cytotoxicity employed by CD4* effectors isolated from human peripheral blood lymphocytes (PBL). PBL, unseparated high buoyant density T cells and their CD4* T ceil subsets were activated with anti-CD3 monoclonal antibody (MoAb) and interleukin-2 (IL-2) for 1-5 days. CD4* T cells activated with IL-2/anti-CD3 MoAb were cytotoxic when directed by a bispecific anti-nitrophenyl (NP)-anti-CD3 MoAb heteroconjugate against both NP-modified nucleated target cells (TC) and non-nucleated sheep red blood cells (SPLBC). This CD4* T population also lysed L929 in a TNF-a dependent manner. Interestingly, different mechanisms of nucleated and non-nucleated TC directed lysis by CD4* effectors were implied by distinct patterns of sensitivity to cholera toxin (CT) and cyclosporin A (CsA). Cyclosporin A and CT inhibited CD4^ T cell directed lysis of SRBC, but not EL4. Cholera toxin, CsA or EGTA pretreatment also significantly inhibited the release of a-N-benzyloxycarbonyl-L-lysinethiobenzylester (BLT)-esterase activity suggesting that degranulation of CD4"^ effectors may be a critical step in their redirected lysis of SRBC. Overall, these findings suggested that activated human peripheral blood (PB) CD4* effectors can lyse TC by at least three distinct mechanisms: (i) a CsA-sensitive directed lysis of SRBC which correlates with exocytosis and presumably occurs via membrane lesions; (ii) a CsA-insensitive directed lysis of NP-modified nucleated TC that does not appear to involve exocytosis and is metabolically distinct; and (iii) a direct TNF-dependent lysis of TNF-sensitive TC. The highly prolifetative CD4* T cell population could be propagated for at least 35 days while retaining cytotoxicity and secreting up to 80 U/mL of IL-2. These data raise the possibility that anti-CD3 MoAb plus IL-2 activated CD4* T cells may prove effective in adoptive tumour immunotherapy. Key words: cholera toxin, cyclosporin, cytotoxicity, interleukin receptor, lymphokine activated killer, monoclonal antibody, serine protease, tumour necrosis factor.

Introduction Cytotoxic T lymphocytes (CTL) endow the immune system with an effective defence against virus infected and tumour cells.' The mechanisms by which CTL kill tumour cells are fundamental to understanding cellmediated immunity against cancer. Targeted cytotoxicity using bispecific antibodies (BA) can be used to increase understanding of the

general mechanisms of cellular cytotoxicity and clinically in the treatment of cancer. Bispecific antibodies, the main mediators of targeted cellular cytotoxicity, bind to tumour cells via one V region and trigger molecules such as T cell receptors (TCR) on cytotoxic cells via their other V region. This linking of triggering structures to tumour cells induces tumour cell lysis and provides important clues to our understanding of the lytic process. The

Correspondence: M. J. Smyth, Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Vic. 3084. Australia. Accepted for publication 6 August 1992.

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M.J. Smyth

mechanism by which CTL kill their target cells (TC) remains controversial, however killing by lymphocytes involves, in most instances, the vectorial secretion of specialized cytoplasmic granules.^ In particular, these organelles contain a lytic pore-forming protein (PFP)^ and a family of serine proteases (SP)"* which may constitute an intracellular activation pathway for other mediators of lysis. Tumour cell lysis can also occur independently of granule exocytosis.^'^ In general, CD4* T cells have been mainly associated with immune regulation, however subsets of CD4^ T cells have been implicated in mediating delayed type hypersensitivity and TC lysis.^ CD4* T cells have been segregated into T h l and Th2 subsets based on their lymphokine expression.^ Generally CD4* T cell clones with cytotoxic activity belong to the T h l subset, whereas non-lytic clones are of the Th2 type.* In most,^ but not all studies,'** CD4* T cell-mediated cytotoxicity has been attributed to soluble mediators including tumour necrosis factor (TNF)-a and TNF-p. Whereas many CD4* CTL clones have been demonstrated, the generation of lytic activity in human peripheral blood lymphocytes (PBL) CD4* T cell populations is not well characterized. Two categories of T cells mediating BA targeted cytotoxicity have been isolated including; a CD8* CD56* low-density T cell fraction, containing all of the targeted T cell cytotoxicity in unstimulated PBL and mediating non-major histocompatibility complex (MHC) restricted lysis after culture with IL-2, and a CD3* CD56" high-density T cell fraction that had no targeted activity unless stimulated by TCR crosslinking and IL-2." Additionally, unlike the low-density T cells, activated high-density effectors consisted of both CD4* and CD8* T cells. We have recently demonstrated that IL-2/anti-CD3 MoAb activated CD4 ^ peripheral blood (PB) T cells utilize metaboUcally distinct mechanisms of BA-directed iysis when compared with activated CD8 * T cells.'^ These same activated CD4'^ T cell subsets also expressed PFP and SP. In this study, the BA-directed lytic potential and direct cytotoxicity of CD4* T cell subsets from human PBL were characterized against a panel of different TC. The inhibitory effects

of a variety of metabolic inhibitors and antiTNF antibodies were examined and several mechanisms of cytotoxicity employed by CD4* T cell effectors were distinguished.

Materials and methods Materials Recombinant human IL-2 was generously supplied by Cetus Corp. (Emeryville, CA, USA). Bispecific OKT-3-anti-nitrophenyl (NP) heteroconjugated MoAb (composed of Fab' fragments) and NP-O-succinamide were kindly provided by Dr Hideo Yagita, Juntendo University, Tokyo, Japan. Recomhinant human TNF-a was obtained from Genentech Inc. (South San Francisco, CA, USA). Cyclosporin A (CsA) was a generous gift from Sandoz (Basel, Switzerland). Cycloheximide (CHX), ethylene-fcis (oxyethylenenitrilo) tetraacetic acid (EGTA), N-Cbz-Lys-thiobenzylester (BLT), dithiobis (2-nitrobenzoic acid) (DTNB), phorbol myristate acetate (PMA) and a rabbit anti-mouse immunoglohulin (Ig) antibody were all purchased from Sigma Chemical Co. (St Louis, MO, USA). Actinomycin D (Act D)(Calbiochem, La Jolla, CA, USA), cholera toxin (CT) (List Biological Laboratories, Campbell, CA, USA) and anti-human TNF-a and anti-human TNF-P antibodies (polyvalent) (Endogen Inc., Boston, MA, USA) were all purchased. OKT-3 monoclonal antibody (MoAb) was produced from cells obtained from the American Type Culture Collection (Bethesda, MD, USA). OKT-3 MoAb was immobilized by cross linking on Falcon culture dishes (Becton Dickinson Labware, Lincoln Park, NJ, USA) using a rabbit anti-mouse Ig antibody. Target cells Raji, a human EBV^ Burkitt's lymphoma cell line; K562, a human NK-sensitive chronic myelogenous proerythroblastic leukaemia cell line; L929, a murine fibroblast cell line; and EL-4, a murine thymoma eel! line were maintained in culture in RPMI 1640 with 10% heat-inactivated fetal calf serum (FCS), 2 mmol/L L-glutamine, 100 U/mL penicillin and 100 |ig/mL streptomycin (Gibco Laboratories, Grand Island. NY, USA).

Human CD4 * killer lymphocytes Effector populations Peripheral blood (PB) mononuclear cells from normal subjects were separated on FicollHypaque as described.'^ Leucocyte suspensions were washed in Hank's balanced salt solution and were resuspended in RPMI 1640 containing 10% heat-inactivtated FCS. Adherent cells (monocytes and B cells) were removed by incubation on plastic dishes and nylon wool. Highly enriched populations of CD3" CD56- T cells (> 95%) from PB mononuclear cells were obtained by centrifugation of nylon-wool passed cells on discontinuous density gradients of Percoll (Pharmacia Fine Chemicals. Uppsala, Sweden). CD4* T cells were obtained by positively sorting (FACStar; Becton Dickinson, Immunocytometry Systems, Mountain View, CA, USA) CD4^ T cells that were stained with FITC-conjugated anti-CD4. Upon purification, the populations were analysed by flow cytometry to determine the extent of contamination. Cells were phcnotyped by using phycoerythrin- or FITC-conjugated MoAb; anti-CD2 (anti-Leu 5b); anti-CD3 (anti-Leu 4); anti-CD4 (anti-Leu 3a); antiCD8 (anti-Leu 2b); anti-CD16 (anti-Leu U); anti-CD25 (anti-IL-2Ra); and anti-CD56 (anti-Leu 19). Two colour fluorescence measurements were performed on a FACScan IV flow cytometer (Becton Dickinson, Immunocytometry Systems, Mountain View, CA, USA). Lymphocyte

stimulation

PBL, T cells or CD4^ subset were cultured in RPMI 1640 supplemented with 2 mmol/L L-glutamine, 2% heat-inactivated FCS, 100 units/mL penicillin, 100 (xg/mL streptomycin and 6 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepes) buffer, pH 7.3 (Gibco Laboratories, Grand Island, NY, USA). Cultures (1-2 X 10^/mL) were stimulated with the agents above for the indicated times before assaying cytotoxicity, BLT-esterase activity and lymphokine release. Cytotoxicity assays Cytotoxicity was measured in both 4 and 18 h Cr release assays. These and the hetero-

381

conjugated antibody-dependent cytotoxicity assay against the NP-modified TC lines and NP-SRBC were performed as previously described.'^ Target cell lysis was assessed at an E : T ratio of 20 : 1 that yielded maximum or near maximum ^*Cr release from the TC. Similar data were obtained in all experiments at an E : T ratio of 5 : 1 (data not shown). The sensitivity of L929 to TNF-a atid TNF-P was pre-examined in an 18 h ^^Cr release assay and a 1000 IU dose of anti-TNF antibodies determined to inhibit up to 10 ng/mL TNF-a or TNF-P lysis of L929 by more than 80%. In an 18 h ^^Cr release assay, 50% L929 cytotoxicity was observed at a 900 pg/mL dose of TNF-a and a 1800 pg/mL dose of TNF-p. Proliferation assay Triplicate cultures of 2 x 10^ CD4'^ T cells in 200 |j.L of media were seeded in a 96microwell plate and were stimulated with IL-2 (1000 units/mL) and immobilized anri-CD3 MoAb (100 ng/mL). Cultures were incubated for 0-5 days. During the last 4 h of culture (1 jiCi/weil), [^H] TdR (New England Nuclear, Boston, MA, USA) (6.7 Ci/ mmol) incorporation was determinned. Lymphokine release Culture supernatants obtained after treatment of lymphocytes were assayed for human TNF-a (ELISA, Endogen Inc., Boston, MA, USA) and human IL-2 (ELISA, Clinical Immunology Services, Program Resources Incorporated, Frederick Cancer Research and Development Center, Frederick, MD, USA). BLT-esterase assay Activity of BLT-esterase was estimated using a microtitre assay.'^ Two million effectors were incubated with NP-modified TC in the presence of a bispecific anti-CD3-anti-NP heteroconjugate at an E : T ratio of 2 0 : 1. Fifty microlitres of cell-free supernatant were removed and added to 10(J |iL of 1 mmol/L DTNB made up in phosphate buffered saline (PBS), 1 mmol/L CaCls 1 mmol/L MgCl2, pH 7.2. The reaction was initiated by the addition of 50 (JL of 2 mmol/L BLT. The rate of colour development which was measured

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(o.d. 410 iim) on a microplate reader indicated that the ideal duration of the assay was 30 mill. Controls of sample and DTNB alone or DTNB and BLT alone were performed. Total releasable activity was determined by the addition of 0.1% Triton X-100 to the E : T assay wells. All T C used were negative for serine esterase. Activity was expressed as the change in o.d. and % BLT-esterase release was calculated as: Release {%) = supernatant activity xlOO. [total releasable] activity

Results Bispecific antibody-redirected and direct cytotoxicity of human peripheral hlood CD4*T cells PBL were fractionated on Percoll density gradients and the highest buoyant density fraction of T cells (95% C D 3 \ < 2% CD56*) were further sorted into CD4* T cells (> 95%). To find the optimal conditions for generating cytotoxic lymphocytes, PBL, unseparated high-buoyant density T cells and their CD4* subsets were activated with antiCD3 MoAb (100 ng/mL) and IL-2 (1000 units/mL) for 1-5 days. When CD4* T cells were activated the phenotype of the CD4* population was essentially unchanged except for an increase in p55 and p75 IL-2 receptor (IL-2R) expression, as previously demonstrated (data not shown).'^ The majority of CD56^ cells (< 8%) in the CD4^ population were T cells by virtue of their expression of CD3 (> 99%). Isolated CD4" T cells showed no significant proliferative responses to IL-2 or immobilized anti-CD3 MoAb alone. However, culture of CD4'' T cells in the presence of immobilized anti-CD3 MoAb plus IL-2 resulted in their marked proliferation (Fig. 1). CD4* T cell effectors were tested for lytic activity against a panel of unmodified- and NP-modified-TC at an E : T ratio of 20 ; 1. Bispecific antibody-directed lytic potential against NP-modified EL4 and NP-SRBC in the presence of a bispecific anti-CD3-anti-NP heteroconjugate was measured in a 4 h ^'Cr release assay. Direct non-MHC restricted lysis

media IL-2 Bntl-CD3 mAb IL-2/«ntl-CD3 mAb •=•

1 0 '

10' 1

2

3

4

Time (days)

Fig. 1. Proliferation of freshly isolated luimdn CD4* T ceils by immobilized anti-CD3 MoAb and IL-2. Human CD4'' T cells were isolated by positive sorting and cultured for various periods of time in 96-microwell plates with media alone, IL-2 alone (1000 units/mL), anti-CD3 MoAb alone (100 ng/mL) and anti-CD3 MoAb (100 ng/mL) and IL-2 (1000 units/mL). The cells were pulsed with [^H]-TdR for 4 h before harvest.

against K562, the human NK-sensitive target, and Raji, the human NK-insensitive, lymphokine activated killer (LAK) target, was also measured in a 4 h assay. Direct TNFdependent lysis against L929. the TNFsensitive target, was measured in an 18 h ^'Cr release assay. Whole PBL, but not highbuoyant density T cells or CD4* T cells, displayed consistent and significant NK activity against K562 in the absence of IL-2/ anti-CD3 MoAb stimulation. NK activity was enhanced and LAK activity developed in PBL, but not T cells, within 24 h of activation and maximal levels of NK and LAK activity in PBL were obtained after 3 days (data not shown). By contrast, whole PBL, high-buoyant density T cells and CD4* T cells activated with IL-2/anti-CD3 MoAb for 3 days directly lysed the TNF-sensitive L929 T C (Fig. 2a). The development of lytic activity against L929 correlated with TNF-a production by the activated CD4 * T cell population (Table 1). Figure 2b, c demonstrated that both PBL and CD4 * T cell populations acquired cytotoxic potential against NP-EL4 and NPSR^C in the presence of a bispecfic anti-CD3anti-NP heteroconjugate. PBL and T cells

Human CD4* killer lymphocytes PBL 0 T + IL-2 /anIi-CD3 PBL + IL-2/anti-CD3 D CD4+ CD4^-1 IL-2/anti-CD3 60 -1

(a)

S 30 o

IIJIJ II

o 20 O 10 0

Table 1. Effect of pretreatment with metabolic inhibitors on the TNF-a production by IL-2/antiCD3 MoAb-activated human PB CD4* T cells.

Pretrcatment**

5- 40

383

TNF-a

850 ±35* 15±7 15±4 20±0 0±0 18±6

Media ActD CHX CT EGTA CsA Unactivated

60^ 50>^ 40 u X 30 o

ii

o 20 O 100

80 — 70 ^60-

0±0

(b)

(c)

*TNF-a (pg/mL) as measured by ELISA. ^High buoyant density CD4* T cells were activated with IL-2 (1000 units/mL) and immobilized anti-CD3 MoAb {100 ng/mL) for 66 h. These were then washed and resuspended in media for 6 b. Two million effectors were pretreated with metabolic inhibitors for 2 h and then in the presence of these agents IL-2 (1000 units/mL) and anti-CD3 MoAb were added. Supernatants were assayed for TNF-a production 24 h later. *Represents the mean ± s.e.m. for two experiments.

D {10|ig/mL); CHX {lO^g/mL); CT (5 Mg/mL); EGTA (5 mmol/L); CsA (

30 10 0

Time (days)

Fig. 2. Cytotoxicity of PBL, high-buoyant density T (unseparated) and CD4* T cells. PBL and T cells (unseparated/CD4*) were treated for 1-5 days with IL-2 {1000 units/niL) and anti-CD3 MoAb (100 ng/mL) as indicated. Effector cells were washed and resuspended in media 6 h before cytotoxicity assay, (a) TNF-dependent direct cytotoxicity against L929 was measured in an 18 h ^'Cr release assay. BA-directed lytic potential in the presence of an anti-CD3-anti-NP heteroconjugate against NP-modified-EL4 (b) and NP-modifiedSRBC (c) was measured in a 4 h ^'Cr release assay. All assays were performed at an E : T ratio of 20 : 1 . Results expressed as the mean ± s.e.m. of triplicate samples are representative of 3 experiments (3 donors). T cell effector populations did not display redirected lytic potential in the absence of heteroconjugate or against unmodified EL4 or SRBC (< 3%). T/CD4* effectors displayed less than 3% cytotoxicity against unmodified TC.

developed maximal levels of BA-directed lytic potential against NP-SRBC within 24 h. As SRBC are non-nucleated, CD4^ CTL were capable of lysing TC by a mechanism independent of DNA degradation. Kinetically, the induction of lytic activity of CD4* andT cells against either NP-SRBC or L929 was concurrent, however the development of cytotoxic potential of CD4^ T cells against NP-EL4 was delayed compared with that of T cells. These data suggested that the mechanism used by CD4* effectors to lyse NP-SRBC may be more rapidly activated than their mechanism to lyse nucleated NP-EL4 TC and raised the possibility that these two mechanisms may indeed be distinct. T/CD4* effectors displayed less than 3% cytotoxicity against unmodified TC whereas activated PBL were also lytic {15-25%) against unmodified SRBC {data not shown). Over short-term culture, lytic activity against all susceptible targets using CD4* effectors was maximal at 3 days and therefore in following experiments a 72 h activation period was standardly employed.

M.J. Smyth

384

Distinct mechanismsojl of lysis used by activatedCD4^ Tee Us

modes of lysis used by activated CD4* T cells. The doses of metabolic inhibitors and anti-TNF antibodies chosen were based on our previous With the optimal conditions for generating study using this same T cell effector subset and lytic activity in purified CD4* T cells estabTC.'2 Unactivated CD4'^ T cells did not lyse lished, the different mechanisms of cytotoxicity L929, NP-EL4 or NP-SRBC targets (Fig. 3). used by these effectors were examined. The Pretreatment of activated CD4* effectors with effects of a panel of metabolic inhibitors upon each of the metabolic inhibitors almost comlysis of NP-EL4 (a nucleated, NP-modified pletely abrogated their lytic activity against and TNF-resistant TC), NP-SRBC (a nonL929. Each of these metabolic inhibitors was nucleated and NP-modified TC) and L929 (a demonstrated to inhibit TNF-a production by nucleated, unmodified and TNF-sensitive TC) were compared. This panel included the fol- these cells (Table 1).'^ In addition, both the anti-TNF-a and anti-TNF-P antibodies signiflowing: Act D for inhibiting RNA synthesis; icantly reduced the lytic activity of activated CsA for selectively inhibiting lymphokine CD4* effectors. Clearly CD4^ T cell effectors mRNA and protein synthesis; CHX for inhiblysed L929 in a TNF-dependent manner. iting protein synthesis; CT for increasing intracellular cAMP; and EGTA for chelating diPretreatment of activated CD4* T cell valent cations. Anti-TNF antibodies were also effectors with Act D, CHX or EGTA abrogemployed to assess the role of TNF in the various ated their BA-directed cytolytic potential

40 n media Cholera Toxin Cyclosporin A Actinomycin D Cycloheximlde

30 -

o 'x o o

o

20 -

10 -

0 NP-EL4

L929

EGTA anti-TNF- a anti-TNF- p no lL-2/anti-CD3

NP-SRBC

Target Pig. 3. Effect of pretreatment with metahoUc inhibitors or anti-TNF antibodies on the cytotoxicity of CD4* T cell subsets. Separated CD4" T cells activated with IL-2 (1000 units/mL) and anti-CD3 MoAb (100 ng/mL) for 66 h were washed and resuspended in media for 6 h. These effectors were pretrcated with metabolic inhibitors: C T (5 ^g/mL); CsA (lO^imol/L); Act D (10 Hg/mL); CHX (10 Mg/mL): or EGTA (5 mmol/L); and anti-TNF antibodies. anti-TNF-a (1000 iu) or anti-TNF-p (1000 iu). for 2 h. These metabolic inhibitors, in the presence of these agents, were tested for BA-directed cytolytic potential (with and-CD3-anti-NP heteroconjugate) against NP-modified-EL4 or NP-modified-SRBC in a 4 h ^'Cr release assay and direct cytotoxicity against L929 in an 18 h ^'Cr release assay. Results expressed as the mean ± s.e.m. of triplicate samples are representative of 2 experiments (2 donors) at an E : T ratio of 20 : 1. Unactivated CD4^ T cells cultured in media (no !L-2/anti-CD3) did not significantly lyse any T C (< 5%). Effector populations did not display redirected cytolytic potential in the absence of heteroconjugate (< 5%). None of the treatments with metabolic inhibitors were toxic to the effectors or targets as determined by trypan blue exclusion.

Human 004*^ killer lymphocytes against NP-EL4 (Fig. 3). Neither an antiTNF-a nor an anti-TNF-p antibody inhibited CD4* effector lysis of NP-EL4. This mechanism of BA-directed lysis by activated human PB CD4* T cells has been consistent against a number of NP-modified, nucleated TC including TNF-sensitive {L929, WEHI 164) and TNF-insensitive (EL4, YAC-1) cell lines (Smyth, unpubl. data).'"^ Although not demonstrated here, redirected lysis of nucleated TC appears to involve the induction of DNA degradation as well as the release of ^'Cr from labelled TC.^'* In order to better understand the mechanisms of lysis used by CD4^ T cells and to assess their capacity to cause membrane damage in TC, the BA-redirected lytic activity of these effectors against non-nucleated NPSRBC was also assessed {Fig. 3). Activated CD4" T cells iysed NP-SRBC in a lytic assay only in the presence of the bispecific antiCD3-anti-NP heteroconjugate. Interestingly, lysis by CD4* effectors was inhibited by CT, CsA and EGTA. Pretreatment of activated CD4* T cells with Act D or CHX or co-culture with anti-TNF antibodies did not inhibit their directed lysis of NP-SRBC. First, these data suggested that CD4^ T effectors Iysed NPSRBC by a TNF-independent mechanism. Second, since Act D and CHX both inhibited CD4* T cell-mediated lysis of NP-EL4, a clear distinction between CD4'^ T cellmediated lysis of nucleated (i.e. EL4) and non-nucleated {i.e. SRBC) TC had been demonstrated. Finally, CsA inhibited CD4* T cell directed lysis of NP-SRBC, but not, NP-EL4, implying that CD4^ T cells Iysed nucleated and non-nucleated TC by different mechanisms. The expression of BLT-esterase activity in T cells is thought to primarily be a measure of SP A activity. As such, the level of BLTesterase activity in the supernatants of cultures from lytic assays are a measure of the degranulation and exocytosis of T cell effectors. The release of BLT-esterase from activated CD4* T cells seldom rose above 25% at an E : T ratio of 20 : 1 {Fig. 4), however, BLT-esterase release from activated CD4'' T cells was less than 5% in the absence of the bispecific anti-CD3-anti-NP heteroconjugate. Comparatively, unactivated CD4^ T cells contained

385

little BLT-esterase activity {five- to 10-fold less, data not shown) and did not release BLT-esterase activity in lytic assays {Fig. 4). Interestingly, in the presence of either NPEL4 or NP-SRBC TC, only CT, CsA or EGTA pretreatment of CD4'^ T cell effectors significantly inhibited the release of BLTesterase activity {Fig. 4). This inhibition of BLT-esterase release paralleled the effect of CT, CsA or EGTA on the BA-directed lytic potential of CD4'^ effectors upon NP-SRBC TC. This correlation suggested that degranulation of CD4* effectors may be a critical step in their directed lysis of non-nucleated SRBC. By contrast. Act D and CHX pretreatment of CD4^ effectors, which inhibited their BAdirected lysis of NP-EL4, had no effect on their release of BLT-esterase activity. In addition, CsA had no effect on CD4'^ T cell directed lysis of NP-EL4, but did inhibit their BLT-esterase release. Therefore it is doubtful that activated C D 4 ' T cells require granule exocytosis to lyse NP-EL4 in a BA-directed manner. Long-term generation ofCD4* populations

helper/killer

The IL-2 secretion of positively sorted CD4* T cells expanded for 14 days with immobilized anti-CD3 MoAb plus IL-2 was examined. The CD4^ T cells (washed to remove IL-2 from the media) produced 77 ± 4 units/mL of IL-2 {over a 24 h period) by stimulation with PMA {10 ng/mL) and ionomycin (1 jig/mL), but no IL-2 in the absence of stimulation or in the presence of PMA or ionomycin alone. Immobilized anti-CD3 MoAb {100 ng/mL) could also stimulate CD4^ T cells to produce IL-2 {18 ± 1 units/ mL). Thus, CD4^ T cells expanded in vitro by immobilized anti-CD3 MoAb plus IL-2 demonstrated an ability to produce IL-2 that was triggered through TCR-CD3 complex binding and subsequent intracellular signals. The CD4* T cells demonstrated growth by restimulation with immobilized anti-CD3 MoAb {days 0,14 and 28} plus lL-2 (days 7,14,21,28 and 35). The growth rate of CD4* helper/killer populations became constant after a 7 day culture and cells were increased in numbers approximately 15-fold

386

M.J Smyth 30 media Cholera

Toxin

Cyclosporin A Actlnomycln

D

Cycioheximide

o

EGTA no IL-2/antl-CD3

I I-

_J CQ

NP-SRBC

NP-EL4 Target

i

Fig. 4. Effect of pretreatment with metabolic inhibitors on tbe BLT-esterase release of CD4* T cell subsets. Separated CD4 ' T cells were activated with IL-2 {1000 units/mL) and anti-CD3 MoAb {too ng/mL) for 66 h. These effectors were then washed and resuspended in media for 6 h. Effecton were pretreated with metabolic inhibitors CT (5 ^ig/mL); CsA {lO^mol/L); Act D {lO^Jg/mL); CHX {10 (ig/niL); or EGTA {5 mmol/L) for 2 h. Two million of all of these effectors were washed and added to 10^ NP-modified-EL4 or NP-modified-SRBC TC {E : T = 20 : 1) m the presence of bispecific anti-CD3anti-NP heteroconjugate. Tbese samples were tben assayed for total BLT-esterase content and BLT-esterase release. Tbe results are expressed as the mean % BLT-esterase release ± s.e.m. of duplicate samples representative of 2 experiments {2 donors). Total BLT-esterase content (o.d.) was in the range 0.112-0.215 {untreated CD4* T cells) and 1.053-1.411 {IL-2/anti-CD3 MoAb activated CD4* T cells ± metabolic inhibitors). No BLT-esterase release was detected in the absence of beteroconjugate.

by a 7 day culture {Fig. 5). During the culture, C D 4 * T cells maititained their helper function to produce IL-2 and BA-directed lysis.

Discussion Data on the subject of T cell-mediated cytotoxicity suggest that no single mechanism is likely to provide a satisfactory explanation of this process. In a previous report we presented data suggesting that at least two distinct effector mechanisms were used by human PB CD4" and CD8* T cells to lyse nucleated T C ' The contribution of exocytosis and lymphokine production in human PB CD4^ I ' cell lysis of nucleated and non-nucleated TC was assessed. Although these data show that exocytosis, lymphokine production and cytolysis are linked with respect to their requirements for activation, functionally these have been separated by using metabolic inhib-

itors and various TC. Lymphokine production, exocytosis and cytolysis could be selectively inhibited in CD4* T cells. In CD4* T cells. Act D and CHX inhibited TNF-a production, direct TNFdependent lysis of L929 and BA-directed lysis of nucleated TC, but did not inhibit exocytosis of BLT-esterase or BA-directed lysis of non-nucleated SRBC. This finding is in accord with Ju et al. who demonstrated that CD4* T cell clones can mediate lysis through a TNF-independent pathway requiring effector protein synthesis."' Other studies have suggested that TNF-a or TNF-p of CD4 * T cells'^ are soluble mediators of nucleated TC lysis. However, the following data argue strongly against a role for TNF in CD4^ clones^ and leukalexin in CD8* T cellmediated BA-directed lysis of nucleated TC. First, the rate of DNA fragmentation (> 620 b) by these mediators is slow in comparison with effector-mediated lysis of TC. Second,

Human CD4^ killer lymphocytes

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lysis by CD4'^ T cell clones but did inhibit CTL SP release in response to antigen.'**'"' These findings strongly implicated that 80 CD4^ CTL can lyse nucleated TC by a mechanism other than exocytosis of granule 60 PFP and SP. In general, these data suggested that CD4* T cells possess distinct mecha40 nisms of BA-directed lysis that do not conform with a granule exocytosis or TNFmediated model of lysis. This novel 20mechanism is Ca^ ^ dependent. Future effort will be directed towards establishing whether the protein synthesis requirement of C D 4 ' T 14 2 1 2 8 35 effectors involves a unique triggering moleTime (days) cule or intracellular metabolic pathway. T cell receptor-CD 3-mediated exocytosis, Fig. 5. Long-term culture of CD4* helper/killer populations by immobilized anti-CD3 MoAb plus as measured by the release of BLT-esterase IL-2. Isolated CD4* T cells (10^) were cultured activity, appears to be the mechanism of long-term with immobilized anti-CD3 MoAb release of lysis-relevant molecules including (100 ng/mL) plus IL-2 (1000 units/mL) using cytotoxic Iymphokines and PFP. PFP and SP 96-microweIl plates (2xlOVweIl). When cell are colocalized in the same individual CTL growth reached a peak (at 7 day intervals), the cells granules and thus inhibition of BLT-esterase were passaged into several wells with fresh IL-2. release implies an inhibition of PFP release. The cells were diluted back to 10*^ cells/well in Moreover, it has been shown that at least with 96-microwell plates after counting the accumulaCD4^ CTL, the ability to lyse modified tive number of cells. Stimulation of the cells with immobilized anti-CD3 MoAb was carried out at 0, SRBC correlates with the presence or absence 14 and 28 days of culture. The data are expressed as of PFP mRNA in the CTL.''' Sheep red blood the fold increase in cell number (•) for every 7 day cell lysis by CTL might be considered a period. The helper and killer functions of the measure of the PFP-degranulation pathway of CD4^ T cells were assessed. The ability of CD4* CTL. The capacity of CD4* T cell effectors helper/killer populations to produce IL-2 and to to lyse SRBC excludes the destruction of the lyse NP-modified-EL4 TC were measured at 7 day TC nucleus as a mechanism of lysis. It has intervals during long-term culture. The lysis of been noted that CD4^ T cells activated with NP-EL4 (% cytotoxicity; M) was performed at an IL-2/anti-CD3 MoAb express PFP, SP A and E : T ratio of 20 : 1 and the IL-2 activity (units/ mL; Vm) of culture supernatants from PMA and B, and a variety of cytokines.^^'"^ Clearly the ionomycin stimulated CD4 * T cells was measured. panel of metabolic inhibitors used in this study had a very similar pattern of activity upon CD4^ T cell BLT-esterase release and SRBC lysis. Since the release of BLT-esterase is EL4 is resistant to high concentrations of believed to be a marker for the release of PFP, TNF-a and TNF-p. Third, CsA efficiently these data suggested that granule proteins may abrogates CD4* T cell production of TNF-a be sufficient to cause lysis of SRBC by CD4* but has no effect on CD4* T cell-mediated CTL. Bispecific antibody-directed lysis of lysis of nucleated TC. Finally, neutralizing SRBC by CD4* effectors in the absence oi de anti-TNF antibodies did not inhibit CD4* novo RNA or protein synthesis suggested that T cell BA-directed lysis of nucleated TC. their cytolytic machinery is preformed and Cyclosporin A, a strong inhibitor of granule rapidly expressed. However, it is also quite exocytosis, profoundly inhibited BA-directed conceivable that the inhibitory effects of lysis of SRBC and exocytosis of BLT-esterase, EGTA, CsA and CT on SEIBC lysis by human but had little or no effect on CD4* T cell PB CD4* T cells involve cellular loci other BA-directed lysis of nucleated TC. Interestthan secretory pathways. Although not examingly, the studies of Ju et al. and Lancki et al. ined herein, pretreatment of T cell effectors also indicated that'CsA did not inhibit cyto100 n

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for 2 h is sufficient to demonstrate the inhibitory activity of Act D and CT, whereas the effects of CHX and EGTA are transient with washing (data not shown).^^ In addition, de novo synthesis of TC RNA and protein, and TC cAMP levels have previously been demonstrated not to be critical, suggesting that in these forms of T cell-mediated lysis,^^ the TC plays a relatively passive role. The nature of the trigger in CD4^ T effectors that mediates their lysis of nucleated TC in BA-directed cytoxicity remains to be elucidated. It is only clear that receptor-mediated contact of the effector and its TC is necessary. Clonal populations of CD4* CTL or Th have previously been analysed for cytolytic potential.'^ Three patterns of lysis were observed among the T cell clones. Some CD4* Th2 effectors efficiently lysed nucleated TC or SRBC in an antibody-directed manner. CD4^ T h l and some Th2 T cells only lysed nucleated TC and other Th2 clones failed to lyse nucleated or SRBC TC at all. In addition, CD4* T h l failed to express detectable levels of PFP or SP B mRNA. Overall, this study postulated that some CD4* Th2 effectors, but not Thl effectors, employed a membraneattack mechanism of lysis involving PFP and SP. Herein, the utilization of heterogeneous populations of T cells, such as PB CD4'' T cells, has not permitted the identification of the relative contributions of individual phenotypes to a particular lytic mechanism. However, CD4* T cells were shown to lyse TC by at least three different mechanisms, thereby suggesting that there were the same functionally distinct effectors within CD4* subsets of human PB T cells. Within CD4^ T cells the Leu8" subset contains nearly all CD4* precursors of alloantigen specific CTL.'^ Preexisting CTL in PB have been shown to be preferentially present within the CD45RO* memory population of both CD4* and CD8^ subsetSj^o however these T cells are distinct in that they require co-stimulation via CD28/B7 interactions in the absence of an overriding TCR/CD3-mediated activation. Further fractionation of CD4^ T cells will be necessary to dissect the populations expressing distinct mechanisms of lysis. In addition, the expression of helper {IL-2 production) and killer function in the CD4^ population must be

confirmed at the clonal level using clones derived from culture of CD4* T cells in the presence of IL-2 and anti-CD3 MoAb. CD4* populations were able to expand in vitro for a long time without losing their functions. PBL in the presence of IL-2 alone yield about a 10- to 20-fold increase in cell number after 14 day cultures.^' Our culture system, however, yields about a 300-fold {15 X 20) increase in cell number in the initial 14 day period. This compares favourably with the culture system of Nishimura et al., who report a 500- to 2000-fold increase in CD4* T cells cultured in IL-2 and anti-CD3 MoAb.'^ Isolation of CD4^ T cells may be complicated, but 20-50 mL of blood is enough to obtain sufficient numbers of cytotoxic CD4^ T cells for tumour therapy. It remains to be determined whether efficient antitumour CD4^ effectors can be generated from the PBL or tumour infiltrating lymphocytes from a wide variety of cancer patients. While the adoptive transfer of large numbers of syngeneic LAK cells in combination with IL-2 reduces metastatic tumour growth in mouse tumour models and causes some regression of established tumours in humans,^ ongoing trials in humans indicate that LAK therapy may be thwarted by limited LAK cell tumour localization and extreme IL-2 toxicities. Therefore, a more specific subset of targeted lymphocytes may improve adoptive tumour immunotherapy in humans. Furthermore, the failure of an effective anti-tumour immune response in the host might primarily be caused by a helper deficiency as poorly immunogenic tumour cells can elicit a powerful host effector response when the tumour produces IL-2.^"* These data coupled with limited range/half-life of systemic IL-2/LAK administration have suggested that helper functions at the local site of tumours may be beneficial. In this light, we are encouraged to develop an efficient strategy for isolating and targeting CD4* T helper/killer cells to tumours. Future efforts will be made to examine the ability of CD4* populations from healthy donors and cancer patients to inhibit the growth of anti-CD3 MoAb hybridomas in nude mice. In addition, the development of anti-CD3-anti-colon tumour/melanoma BA will enable assessment of the activity of CD4 *

Human CD4* killer lymphocytes

T cell populations against human tumours in vitro and in vivo.

Acknowledgements I thank Dr Yoko Norihisa and Flow Cytometry. Clinical Immunology Services (CIS), Program Resources Inc. (PRI), FCRDC, Frederick, MD for T cell sorting and cytokine assays, Mr William Bere for expert technical assistance and for providing SRBC targets, Dr John Ortaldo for his support and helpful discussion and Ms Toula Athanasiadis for her secretarial assistance. This work was supported by a C. J. Martin Travelling Fellowship, NH &MRC.

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