Immunology 1990 70 357-364

Generation of alloreactive cytolytic T lymphocytes by immobilized anti-CD3 monoclonal antibodies. Analysis of requirements for human cytolytic T-lymphocyte differentiation R. DE JONG, M. BROUWER, V. I. REBEL, G. A. VAN SEVENTER, F. MIEDEMA & R. A. W. VAN LIER Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, The Netherlands

Acceptedfor publication 13 March 1990

SUMMARY Requirements for the induction of human cytolytic T-lymphocyte (CTL) activity were studied in a monocyte-free T-cell activation system that uses immobilized anti-CD3 monoclonal antibodies (mAb) as a stimulus. Alloreactive CTL with specificity for HLA-A and -B locus antigens could be demonstrated within 2 days after the initiation of activation. CTL induction in purified T cells initiated by an optimal concentration of immobilized anti-CD3 mAb was not enhanced by the addition of monocytes or exogeneous cytokines, whereas addition of anti-CD25 mAb largely blocked the response. Upon suboptimal anti-CD3 mAb stimulation, addition of recombinant interleukin (rIL)-2, rIL-1 and rIL-4, but not recombinant interferon-gamma (IFN-y) or rIL-6, potentiated the development of CTL activity. Finally it was shown that immobilized anti-CD3 mAb induced significant levels of CTL activity in both purified CD4+ and CD8 + cells. This study indicates that the requirement for cytokines in the differentiation of CTL precursors depends on the strength of the activation signal delivered through the T-cell receptor.

Grabstein, 1987; Collins et al., 1988; Pfeifer et al., 1987) and IL6 (Takai et al., 1986; Takai et al., 1988; Okada et al., 1988) may function as CTL differentiation factors. It has been suggested that the regulatory function of IL-6 is dependent on the presence of IL-2 (Takai et al., 1986,1988; Okada et al., 1988), whereas IL4 may act as CTL differentiation factor in an IL-2-independent manner (Pfeifer et al., 1987). Although much interest has been focused on the functional activity of interferon-gamma in the generation of CTL, data are still not conclusive (Takai et al., 1986; Chouaib et al., 1988; Shau, Gray & Golub, 1988; Farrar, Johnson & Farrar, 1981; Chen et al., 1986; Simon et al., 1986). It has been reported that culturing human peripheral blood mononuclear cells (PBMC) with soluble or immobilized antiCD3 monoclonal antibodies (mAb) generates CTL activity in monocyte-dependent T-cell activation systems (Jung et al., 1986; Scott et al., 1988; Jung, Martin & Muller-Eberhard, 1987). Specificity of these anti-CD3 mAb-induced CTL has not been

INTRODUCTION The differentiation of non-cytolytic T-lymphocyte precursors (CTLp) into CTL is thought to be mediated by an activation signal delivered through the T-cell receptor (TcR). Furthermore, antigen-triggered T-helper (Th) cells and antigen-presenting cells are considered to participate in this process by the production of cytokines. Although it has been established that interleukin (IL)-2 plays an important role in CTL generation, it is still controversial whether IL-2 alone is sufficient for the proliferation and differentiation of lectin-stimulated CTLp (Erard et al., 1985; Vohr & Hunig, 1985) or whether additional lymphokines are required (Gromo et al., 1987; Cernetti, Steinman & Granelli-Piperno, 1988; Hardt, Diamantstein & Wagner, 1985; Horohov, Stocks & Siegel, 1988). Recent reports have shown that both IL-4 (Widmer et al., 1987; Widmer & Abbreviations: B-LCL, Epstein-Barr virus-transformed B-lymphoblastoid cell lines; CTLp, cytolytic T-lymphocyte precursor; E: T ratio, effector: target ratio; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; IFN, interferon; LAK, lymphokineactivated killer; mAb, monoclonal antibody (ies); NK, natural killer; PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; rIL,

demonstrated yet. In the present study a T-cell activation system, in which plastic immobilized anti-CD3 mAb (CLB-T3/3) acted as stimulus, was used to generate CTL activity in peripheral blood T cells. Specificity of anti-CD3 mAb-induced CTL was shown by demonstrating the generation of alloreactive CTL. Involvement of lymphokine-activated killer (LAK) and natural killer (NK) cell activity was excluded since CD1 lb+ (Dianzani et al., 1989) and CD16+ (Kabelitz, 1989; Lanier et al., 1989) cells, respectively, were stringently depleted. T-cell proliferation

recombinant interleukin; TcR, T-cell receptor; Th, T-helper. Correspondence: Dr R. A. W. Van Lier, c/o Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, PO Box 9406, 1006 AK Amsterdam, The Netherlands.

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induced by immobilized anti-CD3 mAb is proportional to the density of the immobilized antibody and is independent of the presence of monocytes, monocyte-derived factors or LFA-Imediated intercellular adhesion (Geppert & Lipsky, 1987; Van Lier et al., 1989; Van Noesel et al., 1988). By varying the density of the immobilized anti-CD3 mAb, this system allows one to study the requirements for co-stimulatory factors in CTL induction in relation to the strength of the activation signal delivered through the CD3/TcR complex. The present data indicate that optimal T-cell stimulation via the CD3/TcR complex induces CTL differentiation in purified T cells. In the case of suboptimal CD3/TcR triggering, exogeneous cytokines appear to be required for the generation of optimal levels of CTL activity. MATERIALS AND METHODS Monoclonal antibodies The mAb CLB-T3/3 (IgG2a; Van Lier et al., 1989) and CLB-T3/ 4.1 (IgGI), specific for CD3 (Van Lier et al., 1987a), the antiCD25 mAb (CLB IL-2R/I; Van Lier et al., 1987b), anti-CDl9 mAb (CLB CD 19; De Rie et al., 1989b), anti-HLA class II mAb (El; De Rie et al., 1989a), anti-CD16 mAb (Huizinga et al., 1989) and anti-CD 14 mAb (Van der Schoot, Visser & Tetteroo, 1989) were produced at the Central Laboratory of the Red Cross Blood Transfusion Service, Amsterdam. Anti-CD 1 lb mAb (OKM) was purchased from Ortho Pharmaceutical, Raritan, NJ. Phycoerythrin (PE)-conjugated Leu-2a (CD8) mAb and fluorescein isothiocyanate (FITC)-conjugated Leu-3a (CD4) mAb were purchased from Becton-Dickinson Immunocytometry Systems (Mountain View, CA).

Cytokines Recombinant human cytokines were kindly provided by the

following investigators or laboratories and used at the concentration indicated: IL-I# (50 U/ml), Dr P. T. Lomedico (Hoffmann-La Roche, Nutley, NJ); IL-2 (50 U/ml), Sandoz (Vienna, Austria); IL-4 (50 ng/ml), Dr M. Schreier (Sandoz, Basel, Switzerland); IL-6 (50,000 U/ml), Dr J. P. J. Brakenhoff (the Central Laboratory of the Red Cross Blood Transfusion Service, Amsterdam) (Brakenhoff et al., 1987); IFN-y (1500 U/ ml), Dr P. Van der Meide (TNO, Rijswijk). Cells PBMC were obtained by Ficoll-Isopaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation of anti-coagulated blood from healthy bloodbank donors. Lymphocytes and monocyte fractions were isolated out of PBMC by counterflow centrifugation elutriation. Purified T cells were negatively selected from the lymphocyte preparations using magnetic immunoselection. Lymphocyte suspensions were incubated with a mixture of anti-CD 19, anti-CD 16, anti-CD 11 b, antiHLA class II and anti-CD 14 mAb for 45 min at 4°. Subsequent incubation with anti-mouse IgG-coated immunomagnetic beads (Dynabeads M-450, Dynal A.S., Oslo, Norway) for 1 hr at 40 was followed by removal of the bead/mAb-coated B cells, NK cells, LAK cells, activated (i.e. class II +) T cells and residual monocytes with a Dynal magnetic particle concentrator (Dynal MPC 1, Dynal A.S.). The resulting T-cell fraction was > 99% CD3+ and did not yield detectable levels of CDIl9, CD1l6, CDl lb, CD14 or HLA class Il-positive cells, as determined by

fluorescence-activated cell sorter (FACS) analysis. Removal of monocytes was functionally confirmed by the lack of IL-6 production (i.e. 98%. The depleted populations contained less than 0-5% CD4+ or CD8+ cells, respectively. The human erythro-myeloid leukaemia cell line K562, the Bcell line Daudi and the murine mastocytoma cell line P815 were maintained in RPMI-1640 medium supplemented with 10% FCS and antibiotics. Epstein-Barr virus (EBV)-transformed Blymphoblastoid cell lines (B-LCL) were obtained by infection of B-cells with the B95-8 strain of EBV. Induction of cytotoxicity Culture wells (Nuclon, Nunc, Denmark) were coated overnight with anti-CD3 mAb (CLB-T3/3) in phosphate-buffered saline (PBS). CLB-T3/3 was used at a concentration of 5 yg/ml for optimal stimulation and 5 ng/ml for suboptimal stimulation; I ml was used to coat wells of 24-well culture plates (cytotoxicity assay) and 100 p1 for the 96-well flat-bottomed microtitre plates (proliferation assay). After incubation, the wells were washed two times with PBS. For cytotoxicity experiments, 0-5 x 106 lymphocytes/well were cultured in anti-CD3 mAb-coated plates at 370 in I ml of IMDM supplemented with 15% pooled human serum and antibiotics in the absence or presence of cytokines, as indicated. Where indicated irradiated (2000 rads) autologous monocytes (50,000/well) were added. After 4 days or at Days 15 in time-course experiments, stimulated lymphocytes (effector cells) were harvested, viable cells counted and cytolytic activity tested in 5'Cr-release assay at the indicated effector: target (E: T) ratios. Cells cultured without stimuli served as negative controls. Anti-CD3-mediated cytotoxicity (Leeuwenberg et al., 1985; Staerz & Bevan, 1985) was tested as target cell lysis of Fc receptor (FcR)-bearing P815 in the presence of anti-CD3 mAb (CLB-T3/4. 1, 1: 1000 dilution of ascites). Anti-CD3-mediated cytotoxicity permits detection of CTL activity, regardless of the antigen specificity of the CTL. LAK cell activity was tested on Daudi target cells, NK cell activity on K562 target cells. Alloreactive CTL activity was determined using allogeneic BLCL as target cells. Target cells were labelled for 1 hr at 37° with

Na251CrO4 (Radiochemical Centre, Amersham, Bucks, U.K.) followed by three washes with assay medium (IMDM supplemented with 10% FCS and antibiotics). Triplicate cultures of effector cells were incubated with 5 x 103 target cells in a final volume of 200 p1 assay medium in round-bottomed microtitre plates (Costar, Cambridge, MA). The tests were performed at effector: target (E: T) ratios as indicated in the text. After a 4-hr incubation at 37°, the supernatants were collected with the Titertek supernatant harvesting system (Flow Laboratories, Rockville, MD) and counted in a gamma counter. The percentage of cytotoxicity was calculated as: 100 experimental release -spontaneous release menx maximum release -spontaneous release

CTL induction by stimulation with immobilized anti-CD3 mAb

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Spontaneous release is c.p.m. release in the absence of effector cells and maximum release is determined by adding saponin to a final concentration of I %. The standard error of the mean percentage lysis did not exceed 3%.

Table 1. Immobilized anti-CD3 mAb initiate CTL differentiation in purified T cells

Proliferation assay T cells (50,000/well) were cultured in 96-well flat-bottomed microtitre plates in a total volume of 100 pl in IMDM supplemented with 15% human pooled serum and antibiotics. Cytokines were added as indicated using final concentrations as mentioned above. Where indicated irradiated (2000 rads) autologous monocytes were added (5000/well). T-cell proliferation was measured by [3H]thymidine ([3H]TdR) incorporation at Day 4; 0 2 pCi (7 4 kBq) [3H]TdR (2 Ci/mmol, Amersham, Amersham, Bucks, U.K.) was added during the last 4 hr of culture. The results are represented as the mean of triplicate cultures. SEM was less than 10%.

Stimulus

RESULTS

Immobilized anti-CD3 mAb initiate monocyte-independent CTL differentiation The ability of plastic-immobilized anti-CD3 mAb (CLB-T3/3) to induce the generation of CTL in highly purified (>99% CD3 +) T cells, which were stringently depleted for CD1 6+ and CD 1 b+ cells to circumvent involvement of non-specific (LAK and NK cell)-mediated cytotoxicity, was investigated. T cells were stimulated with an optimal concentration of coated antiCD3 mAb (5 pg/ml) during a 4-day culture period. As a control, T cells were cultured without stimuli. CTL activity was tested as target cell lysis of FcR-bearing P815 target cells in the presence of anti-CD3 mAb. Through bridging the effector CTL to the target cell FcR by anti-CD3 mAb, this system permits detection of CTL activity regardless of antigen specificity of the CTL (Leeuwenberg et al., 1985; Staerz & Bevan, 1985). As is shown in Table 1, high levels of cytolytic activity were generated in coated anti-CD3 mAb-stimulated cultures. Lytic activity against P815 target cells without addition of anti-CD3 mAb was not observed. No cytolytic activity was observed in unstimulated lymphocyte cultures. The absence of LAK and NK cell activity in the anti-CD3 mAb-stimulated T-cell fractions was confirmed by the absence of Daudi and K562 target cell lysis (Table 1).

Target cells*

Nonet

Anti-CD3

E:T ratio

P815

P815/anti-CD3

K562

Daudi

20:1 5:1 1:1

1t 0 1

6 2 1

0 0 0

3

20:1 5:1 1:1

2 1 0

48 23 12

0 0 0

6 2 1

1 1

* CTL activity was tested as target cell lysis of P815 in the presence of anti-CD3 mAb. Lysis of P815 without anti-CD3 mAb represents background lysis. LAK-cell activity was tested on Daudi target cells; NK-cell activity on K562 target cells. t The generation of CTL activity was tested in anti-CD3 mAbstimulated T cells and, as a control, in unstimulated purified T cells at the indicated E: T ratios. One out of four experiments is shown. t Percentage target cell lysis. Using undepleted PBMC, K562 and Daudi, target cell lysis ranged in a donor-dependent manner from 20% to 60% at E:T 20: 1. P815: spontaneous release (SR), 867 c.p.m.; maximum release (MR) 12155 c.p.m. K562: SR, 687 c.p.m.; MR, 12480 c.p.m. Daudi: SR, 353 c.p.m.; MR, 10268 c.p.m.

Table 2. Immobilized anti-CD3 mAb induce the generation of alloreactive CTL

B-LCL target cells* E:T ratio

Ml

100:1 50:1

2

19

39 19

55 29

15 8

43 23

49 32

14 8

PI

Immobilized anti-CD3 mAb induce alloreactive CTLp differentiation

100:1 50:1

7 6

6 3

6 3

37 19

11 4

30 12

11 5

64 35

G29

Next, it was investigated whether antigen specificity of immobilized anti-CD3 mAb-induced CTL could be demonstrated. Differentiation of influenza virus-specific CTL could not be detected, which might be due to relatively low influenza virusspecific CTLp frequencies (data not shown). In addition, the generation of EBV-specific CTL could not be demonstrated using syngeneic B-LCL as target cells (Table 2). However, after 4 days of stimulation with immobilized anti-CD3 mAb (5 pg/ml), high levels of cytolytic activity were generated against several allogeneic B-LCL targets (Table 2). A donor-dependent variance in the recognition of allogeneic B-LCL was consequently observed (Table 2). Next, allogeneic target cells sharing identical HLA-A and -B locus antigens with the effector CTL were analysed (Table 3). Effector cells of donor G29 (HLAA1,3; B7,8) and C6 (HLA-Al,2; B7,8) recognized HLA-A and

100:1 50:1

35 17

36 18

2 2

53 29

34 12

43 24

51 28

43 17

* Alloreactive CTL activity was measured as target cell lysis of allogeneic B-LCL. As a control, syngeneic target cell lysis was tested. t PBMC of donors Ml, PI and G29 were stimulated during 4 days with immobilized anti-CD3 mAb (5 jg/ml) and used as effector cells in an 51Cr-release assay. t Percentage cytotoxicity. Serologically defined HLA-A and -B typing: Ml (HLA-A2,-; B8, 39); PI (HLA-A2,24; B7,40); G29 (HLA-A1,3; B7,8); RI (HLA-Al 1,32; B35,44); V19 (HLA-A2,8; B27,35); S25 (HLA-A1,-; Bw57,-); Zi (HLAA2,-; B7,44); LI (HLA-A24,-; B44,w55). Ml: spontaneous release (SR), 129 c.p.m.; maximum release (MR), 1670 c.p.m. PI: SR, 140 c.p.m.; MR, 1536 c.p.m. G29: SR, 151 c.p.m.; MR, 1760 c.p.m. RI: SR, 155 c.p.m.; MR, 2190 c.p.m. V19: SR, 240 c.p.m.; MR, 2326 c.p.m. S25: SR, 156 c.p.m.; MR, 1607 c.p.m. ZI: SR, 148 c.p.m.; MR, 2095 c.p.m. LI: SR, 157 c.p.m.; MR, 1729 c.p.m.

Donort MI

P1 G29 RI V19 S25 Zi Ll

5t 35

R. De Jong et al.

360

Table 3. Induction of alloreactive CTL with specificity for HLA-A and -B locus antigens upon stimulation with immobilized anti-CD3 mAb

Effector cells* B-LCL targett

HLA-At

HLA-Bj

Donor G29

Donor C6

ua.40k 40 p -y 8_ 30


98%.CD8+ (a) and CD4+ (b) cells were stimulated with immobilized anti-CD3 mAb (04 pg/ml) during 4 days. In parallel cultures, immobilized anti-CD3 mAb-stimulated lymphocytes were costimulated withrIL-2 and rIL-4 as indicated (concentrations are described in the Materials and Methods). After the culture period, viable cells were counted and CTL activity was measured as target lysis of P815 in the presence of anti-CD3 mAb (hatched bars) and, as control, in the absence of anti-CD3 mAb was determined (closed bars). E: T ratios, 20:1, 5:1 and 1:1. P815: spontaneous release, 1105 c.p.m.; maximum release, 9524 c.p.m.

specific (LAK- and NK-cell mediated) cytolytic activity in the anti-CD3-mediated cytotoxicity assay was excluded by deplet-

ing for CDII b+ cells (Dianzani et al., 1989) and CD16+ cells (Kabelitz, 1989; Lanier et al., 1989), respectively. The absence of LAK- and NK-cell activity in the anti-CD3 mAb-stimulated Tcell fractions was functionally confirmed by lack of Daudi and K562 target cell lysis. Furthermore, preferential CTL induction in activated T cells was excluded by depletion of HLA classI1+

cells. The results indicate that addition of cytokines is not required for the generation of optimal levels of CTL activity in purified T cells when high-density anti-CD3 mAb stimulation is provided. In addition, CTL induction in optimal anti-CD3of mAb stimulated cultures was not enhanced byal.,addition monocytes. It has been reported (Van Lier et 1989) that immobilized anti-CD3 mAb (CLB-T3/3) do not induce detectable levels of IL-4 in purified lymphocytes and that proliferative responses to immobilized anti-CD3 mAb are mediated via an IL-2-dependent pathway. Here it is shown that addition of antiCD25 mAb (CLB-IL-2R/1) largely blocked the development of cytolytic activity, suggesting that CTL differentiation induced by high-density immobilized anti-CD3 mAb is also mediated via an IL-2-dependent pathway. By lowering the triggering events via the CD3/TcR complex, i.e. upon suboptimal anti-CD3 mAb stimulation, exogeneous cytokines appeared to be required for

IL-1,

CD4+

CTL induction by stimulation with immobilized anti-CD3 mAb ACKNOWLEDGMENTS We thank Drs C. Lucas, F. T. M. Rotteveel and L. de Waal for careful review of the manuscript. This work was supported by a grant from the Dutch Kidney Foundation (grant no. 87-710). F. Miedema is a senior fellow of the Royal Netherlands Academy of Arts and Sciences.

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