J Cancer Res Ctin Oncol (1991) 117:425-430

C~ii~:erResearch Clinical 9

017152169100074J

9 Springer-Verlag1991

High release of tumor necrosis factor interferon and interleukin-6 by adherent lymphokine-activated killer cells phenotypically derived from T cells * J. Koberda, L. Bergmann, P. S. Mitrou, and D. Hoelzer Department of Internal Medicine Division of Haematology, J. W. Goethe University, Frankfurt/M., Federal Republic of Germany Received 20 December 1990/Accepted 27 April 1991

Summary. Adherent lymphokine-activated killer cells (A-

Introduction

LAK) are highly potent cytotoxic cells, which are shown to be derived not only from natural killer ( N K ) / K cells but phenotypically also from T cells. The generation and phenotypical and functional characterisation of these Tcell-derived A - L A K are described. In contrast to non-adherent cells (NA-LAK) and unseparated L A K (UNLAK), these mostly CD3 +CD56 +CD8 + cells display a high degree o f expansion following initial interleukin-2 (rIL-2) activation and further culturing in autologous conditioned medium. A comparison of cytotoxic activities o f cultured cells reveals a significantly higher oncolytic ability of A - L A K cells against both K562 and Daudi cells than that of cultured controls of N A - L A K and UNLAK. In addition, A - L A K are characterised by a marked endogenous cytokine release of interferon 7, tumour necrosis factor e and IL-6 as well as by their shedding of p55 IL-2 receptor after exposure to IL-2. The results demonstrate A - L A K to be the lymphocyte subpopulation with the most cytotoxic activity and endogenous cytokine release after exposure to IL-2. The improvement of techniques for long-term cultures may be of interest for future therapeutic approaches.

Interleukin-2 (IL-2) alone or in combination with the transfer of cytotoxic cells activated in vitro (lymphokineactivated killer cells, LAK) may induce turnout rejection in turnout-bearing hosts or cancer patients (Rosenberg et al. 1987; Lafraniere et al. 1985; West et al. 1987; Bergmann et al. 1990 a). This new therapeutic approach with IL-2, however, is accompanied by remarkable toxicities and the clinical results are not yet satisfactory (Bergmann 1990). Developing strategies to improve the therapeutic results, the administration of purified populations o f L A K cells together with lower levels IL-2, may provide an alternative and presumably better approach with fewer side-effects (Schwarz et al. 1989). In this context a subpopulation of lymphocytes is of interest that was found to become adherent to plastic after IL-2 exposure. These so-called adherent L A K (A-LAK) cells are characterised by a high proliferation rate in the presence of IL-2. In addition, A - L A K were found to be highly enriched in large granular lymphocytes (LGL) with natural killer cell (NK) phenotype and they express higher cytotoxic activity than cultures of unseparated L A K cells (Molder etal. 1988; Vujanovic et al. 1988; Gunji et al. 1988). It has been confirmed that the similar generation o f A - L A K cells with elevated oncolytic potential and a high rate of expansion can be performed from lymphoid cells of tumour-bearing patients (Whiteside et al. 1988; Schwarz et al. 1989, Verfaillie et al. 1989). Furthermore, mononuclear cells (MNC), obtained from peripheral blood of cancer patients and expanded after adherence to plastic in the presence of recombinant (r)IL-2, were used in immunotherapy of these patients (Whiteside et al. 1990). Moderate toxicities and encouraging therapeutic effects in some patients were observed. Recently large-scale preparation methods of A - L A K cells for adoptive immunotherapy have been developed (Molder et al. 1989). In our studies we demonstrate another adherent L A K cell population with a mature T cells phenotype, which may also be more effective in tumour treatment than unfractionated IL-2-stimulated lymphocytes.

Key words: L A K cells - Cell adherence - interleukin-2 Cytotoxic T cells - Cytokines

* This work was supported by the grant 01GA8802 of the Bundesministerium f/Jr Forschung und Technologic (BMFT) Abbreviations used." A-LAK, adherent, lymphokine-activated killer cells; NK, natural killer; CTCM, complete tissue-culture medium; IL-2, interleukin-2; IFNT, interferow; MNC, peripheral blood mononuclear cells; TNFc~,tumour necrosis factor Offprint requests to: L. Bergmann, Division of Haematology, Department of Internal Medicine, University Clinics, Theodor-SternKai 7, W-6000 Frankfurt/M., FRG

426

Materials and methods Cell separation. Peripheral blood mononuclear cells (MNC) were isolated from heparinized venous blood of normal volunteers by Ficoll/Hypaque separation (Biochrom KG, Berlin, FRG). The celt concentration was adjusted to 4 x 106/ml in complete tissue-culture medium (CTCM), consisting of RPMI-1640 medium supplemented with 5 m M L-glutamine, 100 U/ml penicillin, 100 gg/ml streptomycin (Gibco, Scotland) and with 10% (v/v) heat-inactivated AB human serum, and cells were incubated in a horizontal position for I h at 37 ~ C in a humidified atmosphere of 5% COz in air. The cells were washed out with warm medium and adjusted to the concentration of 2 x 106/ml. In some experiments the nylon-wool (Fenwal Laboratories, USA) depletion of monocytes was performed (McFadden 1987). Briefly, peripheral blood MNC resuspended in CTCM were loaded onto prewarmed nylon-wool columns and incubated for 1 h at 37 ~ C in an atmosphere of 5% CO2 in air. The cells were washed out with 20 ml warm medium, concentrated by centrifugation and adjusted to the concentration of 2 x 106/ml.

Table 1. Expansion of adherent (A), non-adherent (NA), and unseparated (UN) lymphokine-activated killer (LAK) cells obtained from peripheral blood of normal volunteers observed in ten longterm cultures Time in culture (days)

Expansion ( fold; median, range) A-LAK

NA-LAK

UN-LAK

7 14 21 28 35 42

8.7 33.3 83.0 50.0 65.2 18.0

1.0 0.9 1.2 1.1 1.1 0.5

1.1 1.4 1.9 2.9 2.7 1.4

Cell phenotyping by flow cytometry and cell morphology. A-LAK cells were detached from plastic flasks by scraping with a disposable cell scraper (Costar, Cambrige, Mass., USA). Fresh or cultured cells were incubated with fluorescein isothiocyanate-or phycoerythrin-labelled monoclonal antibodies (Becton-Dickinson GmbH, Heidelberg, FRG) including CD2, CD3, CD4, CD8, CD16, CD20, CD25, CD56, HLA-DR, CD14 (LeuM3). After the appropriate preparation of cells, two-colour flow cytometry was performed using a fluorescence-activated cell analyser (FACScan; Becton-Dickinson GmbH, Heidelberg, FRG). In some particular cultures of A-LAK and N A - L A K cells, cytospin preparations were performed and the cells were stained with May-Grunwald-Giemsa stain and examined in the light microscope. Cytotoxicity assay. Cytotoxicity was measured in a 4-h s 1Cr-release assay as described elsewhere (Bol et al. 1986) using 96-well culture dishes at 2 x 104 cells/ml. All tests were done in triplicate at various effector/target ratios in a final volume of 200 lal. After incubation, 100 lal supernatant of each well was counted in a gamma counter to determine the experimental release. The spontaneous release never exceeded 20% of the total release. The percentage cytotoxicity was

(0.9-2.5) (0.7-1.3) (0.7-1.6) (0.7-1.5) (0.8-1.2) (0.4-0.7)

(0.7-1.4) (1.2-1.6) (1.4-2.9) (1.3 3.7) (1.1 3.0) (0.62.1)

calculated as follows: experimental release-spontaneus release total release-spontaneous release

Separation of adherent LAK cells. The monocyte-depleted M N C were incubated in CTCM containing 1000 U/ml (Cetus units) rIL-2 (EuroCetus, Frankfurt/M., FRG) in a plastic flask (Inter Med Nunc, FRG) positioned on its flat side for 24 h. Supernatant containing cells not adhering to plastic ( N A - L A K ) w a s subsequently decanted from the flask, centrifuged and resuspended in fresh CTCM plus 1000 U/ml riL-2 and cultured for several weeks. The cells adherent to plastic (A-LAK) were washed twice with prewarmed CTCM to remove residual non-adherent cells. The plasticadherent LAK (A-LAK) cells were supplemented with CTCM containing 50% autologous conditioned medium (i.e. the supernatant recovered by centrifugation of NA-LAK and unseparated LAK cells) and 1000 U/ml rIL-2 and also cultured for several weeks. Unseparated MNC were cultured as described above as controls. The cultures of A-LAK, N A - L A K and unseparated LAK (UNLAK) cells were monitored for cell proliferation by counting in a Neubauer camera. The cell concentrations were maintained at (1.52.0) x 106/ml by supplying fresh CTCM containing 1000 U/ml rIL-2 as needed. With the aim of enumerating the A-LAK cells at 24 h of activation, three fields of A-LAK cells were counted per flask using a 40 • objective, and the mean number of A-LAK cells per flask wa s calculated by dividing the mean number of cells per field by the area of one field in cm 2 and then multiplying this by the area of the flask in cm 2. The degree of expansion (-fold) of A-LAK cells was determined on the basis of cell counts performed two or three times per week compared to the number of adherent cells after 24 h of initial activation in rIL-2.

(3.5- 13.0) (16.4- 74.2) (28.6-160.0) (16.3-103.4) (34.0- 82.1) (12.1- 35.1)

x 100=lysis (%).

Cytotoxic (lytic) activity was determined from various E/T ratios, and I lytic unit (LU) was defined as the number ofeffector cells required to cause 20% or 50% specific 51Cr release from 2 x 104 target cells; the cytotoxicity in LU/107 effector cells was computed. The cytotoxic activity of cultured cells was tested against the NK-sensitive cell line K-562 and the NK-resistant line Daudi, which were always in the log phase of growth.

Cytokine levels. In culture supernatants the endogenous cytokine release of tumour necrosis factor c~(TNF c0, interleukin-6 (IL-6) and interfereon 7 (FNT) was determined using commercially evaluable enzyme-linked immunosorbent assays (ELISA) or radioimmunoassays (Medgenix, Dfisseldorf, FRG). Shed c~chains of the IL-2 receptor (IL-2R) were measured in the supernatants by an ELISA (T-cell science, Mass. USA).

Results

Growth o f L A K cells in long-term cultures After separation of peripheral blood MNC three types of culture - A-LAK, NA-LAK and UN-LAK - were established. T h e c o n d i t i o n s o f c u l t u r e w e r e the s a m e in all cases, e x c e p t f o r the A - L A K c u l t u r e s , w h i c h w e r e initially supplemented with autologous conditioned medium ( 5 0 % v/v). F o l l o w i n g s e v e r a l w e e k s o f o b s e r v a t i o n , the e x p a n s i o n s o f c u l t u r e d cells w e r e c a l c u l a t e d ( T a b l e 1). fold expansion 100

10

3

5

7

9

11 13

15

17 19 day

21 23 25 27 29

31 3 3 35

Fig. 1. Expansion of adherent (E3), non-adherent (~), and unseparated (I) LAK cells observed during one of the representative experiments. All cultures were incubated in the presence of 1000 U/ ml rIL-2

427 Table 2. Flow cytometry characterisation of cultured unseparated (UN), adherent (A) and non-adherent (NA) lymphokine activated killer cells Time in culture (days)

Cell type

10

Positive cells (%) CD2

CD3

CD4

CD8

UN A NA

94 99 99

86 69 94

44 9 50

47 43 47

24

UN A NA

95 97 96

63 41 56

14 1 20

8

UN A NA

95 98 98

82 72 88

12

UN A NA

97 98 95

90 43 88

CD20

CD14

CD16

6 4 6

6 0 3

5 46 4

75 74 69

3 1 3

5 1 0

43 3 46

43 68 37

12 1 8

48 4 42

41 54 45

7 1 6

A-LAK cells, which represented about 1% of the initial population of LAK cells after 24 h rIL-2 activation, clearly showed higher expansion than other cells (Fig. 1). Their expansion often reached 60- to 120-fold while the growth of NA-LAK and UN-LAK cells was not so marked. The most dynamic growth of A-LAK cells was observed after 3 weeks of culturing. However, in the 4th and 5th weeks of incubation the expansion of these cells did not progress in most cases and in the following weeks we have found a decrease in cell numbers despite continuous rIL-2 and CTCM supplementation. Also, a stimulation of A-LAK cells, using both submitogenic (1 ng/ml) and mitogenic (10 ng/ml) doses of CD3 monoclonal antibody prior to rIL-2 activation, as well as 3 weeks of culturing with IL-2 did not improve their expansion.

With the aim of determining the nature of the cultured cells, their surface characteristics were investigated by FACScan analysis using fluorescent monoclonal antibodies (Table 2). A remarkable proportion of A-LAK cells expressed both CD3 + and CD56 + antigens. Many cultures of these cells also displayed CD16 and a predominance of CD8 + cells. Characteristic for almost all A-LAK cells was their high expression of activation markers as class II major histacompatibility complex (MHC) antigens on CD3 (CD3+DR +) or on CD8 (CD8+DR +) cells and of Table 3. Flow-cytometry characterisation of A - L A K cells after depletion ofmonocytes on nylon wool and 10 days incubation with recombinant interleukin-2 (rIL-2)

1 2

CD16+CD56 C D 3 + D R

CD3-DR

CD25

28 76 22

4 38 4

20 66 36

8 18 4

4 9 4

30 55 43

72 87 67

30 54 42

49 40 31

29 54 19

1 i

ND ND ND

ND ND ND

56 100 63

ND ND ND

28 64 32

8 11 7

3 7 3

0 0 0

13 42 9

14 99 30

5 42 7

29 41 32

4 31 5

2 3

CD25 +. The rare expression of CD14-antigen-bearing cells confirmed the effectiveness of monocyte depletion. Morphological analysis of May-Grunwald-Giemsastained smears indicated that these cells had the characteristic morphology of large granular lymphocytes with azurophilic cytoplasmic granules. 5~ 2600F

mill. cells against K662

A

2000 1600 1000 600

Characterisation of cultured L A K cells

Expt.

CD56

0

.....

day 11

day 13 m

2~ 300r

A-LAK

CD2

CD3

CD4

CD8

CD16

CD56

ND 89

60 55

32 1

23 32

11 17

21 81

~NA-LAK

day 26

~UN-LAK

mill. cells against DAUDI

B

260 200' 160 100 60 0

.

day 12

.

.

.

.

.

day 13 IBm A-LAK

Positive cells (%)

day 14

~

day 26 NA-LAK

~

day 42

UN-LAK

Fig. 2. Generation of cytotoxic activity against K562 cell line (A) and Daudi tumour cell line (B). All cultures were assayed for cytotoxicity after different times of incubation. Lytic activities, denoted by day numbers, are unrelated to each other. Lytic units (LU) ofcytotoxic activity were determined from various E/T ratios and were shown as L U 2~ (A) or L U 5~ (B) calculated per 10 effector cells

428 Table 4. Comparison of cytotoxic activities of unseparated (UN), adherent (A) and non-adherent (NA) LAK cells following different time of incubation with rIL-2

Time in culture (days)

Cell type

10

UN-LAK A-LAK NA-LAK UN-LAK A-LAK NA-LAK UN-LAK A-LAK NA-LAK

11

13

14

K-562 lysis (%)

UN-LAK

26

12

42 Effector-target cell ratio b Effector-target ratio = 5:1

a

Table 5. Cytokine induction of unseparated (UN), adherent (A) and nonadherent (NA) LAK cells in independent cultures calculated for a cell concentration of 1 x 106 cells

Time in culture (days) 8

10

13

21 a TNFcq tumour necrosis factor c~; 1FNT, interferon ~; IL-6, interleukin-6; IL-2R, interleukin-2 receptor b ND, not done

35

10:1 a

1:1

0.25:1

10:1

1:1

0.25:1

44 54 31 78 75 78 ND 85 82

12 27 5 77 81 72 ND 80 44 41 50 29 55 75 35 66 72 71 87 74 89

3 7 1 44 50 32 ND 52 16 11 31 10 19 34 12 43 69 45 75 72 67

ND ND ND 14 18 12 ND 48 33

ND ND ND 2 3 2 ND 4 0 47 70 37 2 4 1 5 11 5 5 10 2

ND ND ND 1 1 3 ND 0 0 24 31 19 0 1 0 0 2 0 8 1 1

53 b

A-LAK

71 b

NA-LAK

56 b

UN-LAK A-LAK NA-LAK UN-LAK A-LAK NA-LAK UN-LAK A-LAK NA-LAK

81 85 80 69 66 69 82 83 83

Cell type

Cytokine levels/1 x TNFc~ (pg/ml)

UN-LAK A-LAK NA-LAK UN-LAK A-LAK NA-LAK UN-LAK A-LAK NA-LAK UN-LAK A-LAK NA-LAK UN-LAK A-LAK NA-LAK

I n some cases we p e r f o r m e d m o n o c y t e depletion using n y l o n wool before rIL-2 s t i m u l a t i o n , with the aim o f c o m p a r i n g the n a t u r e of A - L A K cells a n d their CD3 a n t i g e n expression (Table 3). A b o u t 5 0 % - 6 0 % of AL A K cells p r e p a r e d in this way displayed the CD3 + membrane marker.

Cytotoxic activity of cultured LAK cells I n vitro cytotoxic activity o f c u l t u r e d L A K cells against K-562 a n d D a u d i t u m o u r cell lines is presented in T a b l e 4. Cytotoxic activity o f A - L A K cells was comp a r e d to t h a t m e d i a t e d by s t a n d a r d u n s e p a r a t e d L A K cells a n d N A - L A K cells in a p p r o p r i a t e effector/target ra-

Daudi lysis (%)

11729 26674 2733 ND b 606 180 3731 17190 381 57 2206 237 543 5931 820

10 6

57 b

72 b 56 b

28 42 23 41 55 49 9 59 28

cells"

IFN7 (U/ml) 316 1266 189 5 31 6 217 1 500 63 ND 606 180 < 2 51 < 2

IL-6 (pg/ml)

IL-2R (U/ml)

1644 10477 263 3235 1020 913 ND 19970 20 46 1737 16 543 5931 820

1250 1714 563 ND ND ND 1 000 500 44 47 714 139 ND 1572 917

tios. It is o b v i o u s that A - L A K cells d e m o n s t r a t e d the highest levels o f cytotoxic activities against b o t h targets tested, this being especially visible a n d accented in the lower effector/target ratios. The cytotoxic activity o f N A L A K a n d U N - L A K cells did n o t differ greatly. F i g u r e 2 illustrates the calculated oncolytic activity in lytic u n i t s generated f r o m each k i n d of culture, a n d d e m o n s t r a t e s the clear superiority o f A - L A K .

Cytokine levels C y t o k i n e levels were d e t e r m i n e d in the various c u l t u r e s u p e r n a t a n t s . T o m a k e a c o m p a r i s o n o f cytokine levels

429 possible, the quantities of cytokines were corrected for the cell concentrations and calculated for i x 10 6 cells. ALAK were found to secrete excessively high endogenous TNFc~, IL-6 and IFN 7 after exposure to IL-2, whereas the cultures with UN-LAK and NA-LAK revealed much lower cytokine levels (Table 5). The secretion of TNFe, IL-6 and IFN7 in A-LAK cultures exceeded that of NALAK and UN-LAK by more than ten times. In addition, the shedding of e chains from the IL-2 receptor (IL-2R) was highly elevated in the cultures of A-LAK. The NALAK were found to have the lowest cytokine release and shedding of IL-2R e chains.

Discussion

Stimulation of monocyte-depleted peripheral blood MNC with rIL-2 developed the ability of a small subpopulation oflymphocytes to adhere to plastic. These so-called A-LAK were characterised by a high proliferative ability, which, however, could only be maintained for a few weeks of incubation despite continuous rIL-2 and CTCM supplementation. Additional CD3 monoclonal antibody stimulation with submitogenic doses at the end of the third week of culture did not improve the growth, and the cell numbers decreased after 3 weeks of incubation in the same manner as CD3-unstimulated cells (data not shown). Yang et al. (1990) have characterised OKT3-initiated lymphokine-activated effectors which, expanded with low concentration of IL-2 and TNFe, resulted in a higher cell recovery (50- to 3300-fold) compared to the number of cells in the initial culture. In our experiments, CD3 stimulation (10 ng/ml) 48 h prior to rIL-2 activation of monocyte-depleted MNC resulted in lymphocytes lacking their ability to adhere to plastic (data not shown). It may be speculated that CD3 monoclonal antibodies blocked the receptor responsible for plastic adherence of cultured cells, as extensively described by Melder et al. (1990). Flow cytometry characterisation of cultured cells revealed that the A-LAK cell population was highly enriched with CD3 +CD56 + and activation markers such as DR- and CD25-expressing lymphocytes. The pronounced expression of CD25 on A-LAK corresponds to an excessive shedding of IL-2R a-chains. High expression of activation markers was previously desdribed by Melder et al. (1988). The relatively high proportion ofCD3 + T cells within the A-LAK population in our experiments is in contrast to results reported by others (Melder et al. 1988), who found non-Tcells (CD3-CD56 +) or phenotypical NK cells, to be the predominant population of lymphocytes that became adherent to plastic after an initial rIL-2 activation. We supposed that the method of isolation of A-LAK cells (plastic adherence or nylon-wool separation) may be responsible for these differences. Therefore, we repeated the phenotypical analysis of A-LAK after depletions of monocytes on nylon wool. This was the method of preparation mainly used by other authors (Melder et al. 1988; Vujanovic et al. 1988). However, this

modification of A-LAK cell separation did not reveal any serious change of phenotype. On the other hand, the Tcell-derived A-LAK phenotype has also been described to be generated from blood of patients with brain tumours (Whiteside et al. 1988 b) but not from healthy volunteers. Administration of chemotherapy and steroids prior to LAK cell examination was suspected to be responsible for the preferential adherence to plastic and expansion of IL-2-activated cytotoxic T lymphocytes in these patients (Whiteside et al. 1988 b). A small subset of NK cells (~2.5%) in peripheral blood, which displays a mature T cell phenotype (CD3 + CD56+CD8-+), was described by Schmidt et al. 1986). A comparison of the morphology of CD3 CD56 + and CD3+CD56 + lymphocytes with the functional characteristics of NK cells revealed that both subtypes had the typical appearance of large granular lymphocytes and similar cytotoxic activity against K-562 target cells (Schmidt et al. 1986). Lotzova (1989) described these cells as T lymphocytes with NK-like or MHC-unrestricted cytotoxicity. Consistent with these findings, high proportions of CD3+CD56 + cells have been reported in LAK populations after in vitro culture of MNC (Grimm et al. 1982; Testa et al. 1990). Furthermore, phenotypical analysis of lymphocytes after IL-2 administration in cancer patients confirmed the contribution of CD3+CD56 + cells within the LAK population (Weidmann et al. 1989). LAK activity was found to be significantly diminished by antibodies defining T cell markers (Grimm et al. 1982). These data suggest that our T-derived A-LAK cells with the CD3+CD56 + phenotype may be generated from a subpopulation of NK-like cells of MNC that displays mature T cell determinants. The findings may suggest that the population of our A-LAK cells contains a mixture of subpopulations with various membrane determinants but accented T cell predominance. Therefore, it may be postulated that most cultured A-LAK cells have one of following phenotypes: CD2 +CD 3 +CD56 + CD 8 + or CD2+CD3-CD56+CD8 +. During IL-2 treatments, induction of high release of endogenous cytokines such as IL-6, IFN 7 and TNF has been described (Bergmann et al. 1990 b; Boccoli et al. 1990). However, it is not clear which subpopulations of IL-2-activated cells are the predominant source for cytokine release. Our results indicate that A-LAK cells seem to be the lymphocyte subset with the most effective secretion ofIL-6, IFN 7 and TNF ~ after exposure to IL2, whereas NA-LAK and UN-LAK secreted for fewer cytokines. In addition, A-LAK was the population with the highest levels of shed IL-2 receptors. This cytokine release may be responsible for some of the excessive antitumoral effects of A-LAK. In conclusion, the A-LAK cells may be generated out of lymphocytes phenotypically derived from T cells as well as out of NK cells, and are characterised by an excessive proliferation and cytotoxic efficacy after IL-2 exposure. The marked endogenous cytokine release underlines the significance of this subset for tumour surveillance or rejection. Further trials will have to focus on the improvement of techniques for long-term cultures for future therapeutic approaches.

430

Aknowledgements. The authors thank Mrs. S. Christ, Mrs. S. Fuck, Mrs. Ch. Happ, and Mrs. B. Wiirz for skilful technical assistance.

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