Inhibition of human LAK-cell activity by the anti-depressant trifluoperazine

ELSEVIER

Inhibition

Immunopharmacology

29 (1995)

1- 10

of human LAK-cell activity by the anti-depressant trifluoperazine

Lucia de La Rocque a,b, M&rcia M. Campos a, Beni Olej a, Frederic0 Castilho a, Isabella F. Mediano”, Vivian M. Rumjanekb3* “Basic Research Center, National Cancer Institute, Rio de Janeiro. Brazil b Laborutcirio Imunobiologia. Institute de Biofisica Carlos Chagas Filho, CCS. UFRJ, Cidade Universitciria,

de Imunologia de Turnores. Programa de Ilha do Fundrio. 21949 Rio de Janeiro, Brazil

(Received 21 July 1993; accepted 2 May 1994)

Abstract The anti-depressive drug trifluoperazine (TFP) was studied on in vitro immune responses. TFP proved to be an inhibitor of lymphokine-activated killer (LAK) cells in its generative step, as well as in its effector phase. Natural killer (NK) activity and interleukin-2 (IL-2) or mitogen-induced lymphocyte proliferation were just as sensitive to the drug effects, whereas the division of tumor cells was more resistant. The mechanism through which TFP suppresses these lymphocytic systems remains unclear. It does not, however, affect an early stage of cellular activation as the addition of the drug as late as 24 h after the start of the culture was still inhibitory for lymphocyte mitogenesis. Neither the expression of CD25, nor that of CDS6 was affected by TFP, and exogenous IL-2 was unable to overcome the suppression of proliferation. In relation to cell-mediated cytotoxicity, TFP partially interfered with the effectorjtarget binding. However, addition of lectin to the assay did not overcome the inhibition of lysis produced by the drug. Although further work remains to be done, the effect of TFP on immune responses must be taken into consideration when treating immunonosuppressed patients. Key lords: LAK; NK; Trifluoperazine;Tricyclic antidepressants; Lymphocyte proliferation; Immunosuppression

1. Introduction A lot of evidence has accumulated ing interdigitating points between

recently revealthe central ner-

vous system and the immune system. A complex feedback network of shared hormones, peptides and neurotransmitters enables these two-systems to

communicate extensively, resulting in mutual influence and modulation

* Corresponding author. Cam, caltnodulin; FCS. foetal calf serum; FITC. fluorescein isothiocyanate; [‘H]Tdr, thymidinc; IL-T, interleukin 2; Ig, immunoglobulin; LAK. lymphokine-activated killer; LFA 1. lymphocyte function associated antigen 1: MDD, major depressive disorder; MLR, mixed lymphocyte reaction; MoAb. mono-

Abbreviations:

clonal antibody; NK. natural killer; lymphocytes; PHA, phytohemagglutinin; pressant: TFP, trifluoperazine 0162-3 109/95/$9.50 0 1995 Elsevier SSDI 0162-3109(95)00037-9

PBL, peripheral TCA, tricyclic

Science

blood antide-

B.V. .411 rights reserved

(Miller

and Lakcner,

1989).

In view of the intense interelationship between these two systems, investigations were started on a possible interaction between tricyclic antidepressants (TCAS) and the immune system. Evidence suggestedthat major depressive disorder (MDD) may be associated with lowered cellular immune function (Scheifer et al., 1984), and, as TCAs are widely used to treat depression,it would certainly be

2

L. de La Rocque et al. / In~munopharnzacol~~g~

helpful to know the effect of these agents on the immune response when treating depressed patients. A number of these drugs are known to inhibit, in a cell-free system, the protein kinases, as well as to inactivate calmodulin and the phospholipases (Schleuning et al., 1989). TCAs have also been shown to downregulate several immune functions: chlorpromazine, promethazine, desipramine, amitriptyline and imipramine were able to inhibit either NK activity and/or mitogen-induced lymphocyte proliferation (Miller and Lackner, 1984; Rychlik et al., 1988; Martinez and Coleman, 1989). Furthermore, chlorpromazine inhibited the mRNA expression in human thymocytes (Schleuning et al., 1984) whereas other workers detected its capability to augment polyclonal IgG synthesis in lectin-stimulated peripheral blood mononuclear cells (Martinez and Coleman, 1989). Our work thus focused on another anti-depressant, trifluoperazine (TFP) which, similar to the ones above, is a known antagonist of calmodulin, a protein kinase inhibitor (Brumley and Wallace, 1989) a modulator of Ca2 + fluxes through an effect on the plasma membrane (Hoenig et al., 1988) and endoplasmic reticulum (de Meis, 1991; de Meis and Inesi, 1992), and a reversor of the mechanism responsible for multidrug resistance (Ramu and Ramu, 1992). The inhibition by TFP of several immune functions, among them lymphocyte proliferation induced by lectins and by alloantigens, as well as NK activity, has already been described (Bachvaroff et al., 1984). We tested the effect of this drug on NK activity and compared it to the one exherted on IL-2 or PHA-induced lymphocyte proliferation, as well as on tumor cells. As LAK cell activity seems to involve proliferation besides cytotoxicity, we studied the effect of TFP on this system to see how it would affect this dual activation process.

2. Materials 2. I. Preparation

and methods of peripheral

blood (ljmphocytes

Peripheral blood lymphocytes (PBL) were obtained by the separation of heparinized blood from healthy volunteers on Ficoll-Hypaque density gradient centrifugation. The PBL fraction was

29 11995) I-10

washed twice and resuspendedin RPM1 culture medium with 594 foetal calf serum (FCS). The cell number was adjusted at 1 x lo6 cells/ml for the proliferative assay and at 5 x lo6 for the cytotoxic assay. 2.2. Cell lines

The cell lines K562 (human erythroleukemia) and Jurkat (human T-cell leukemia) were maintained by weekly passagesin RPM1 with 5% FCS supplemented with 5 x 10e5 M P-mercaptoethanol and antibiotics. 2.3. Proliferu tive assay

Measurements of cell proliferation were made by determining the incorporation of [ 3H]thymidine ([3H]TdR) into cellular DNA. PBL at 1 x lo6 cells/ml were plated in 96-well microtiter plates to which phytohaemagglutinin (PHA) at 5 pg/ml or interleukin 2 (IL-2) at various concentrations were added and left to proliferate at 37°C in a 5% CO, atmosphere for 72 h. 6 h before the end of the assay 0.5 PCi of [3H]Tdr was added to each well; at the end of the assay, cells were harvested and radioactivity assessedin a liquid scintilator counter. 2.4. Generation

of LAK

cells

Peripheral blood lymphocytes at a concentration of 2 x lo6 cells/ml were incubated with 2.5% IL-2 of a solution containing 1000 U/ml, for 72 h at 37°C and 57; CO,. After this, cells were washed twice, resuspendedat 5 x lo6 cells/ml and used as effector cells in the cytotoxic assay. 2.5. C$totoxic

assay

Cytotoxicity was determined using the “Cr release assay (Rosenberg et al., 1974). Target cells (1 x lo6 cells/ml) were incubated for 90 min at 37°C in the presence of 100 VCi of ‘lCr in a total volume of 150 ~1 (50 ~1 of FCS plus 100 ~1 of radiolabelled solution). At the end of the incubation period, the cells were washed twice and resuspendedto 5 x lo4 cells/ml. Different proportions of effector: target cells (lOO:l, 50:1, 25:1, 12.5:1) were incubated in micro-

L. de La Rocque

et al. / It~llmunopharnlucolog~l

titer plates for 4 h at 37 ‘C and 5 % CO*. After the incubation period, 100 ~1 of supernatant were collected and the radioactivity assessed. The percentage of specific lysis was calculated from the following formula: (experimental

release - spontaneous

(total label - spontaneous

release)

release)

x 100

Some experiments were performed in the presence of 2.5 pg/ml or 5 pg/ml of PHA as a binding enhancer.

2.6. Treatment with tr@operazine TFP was and further experiments. tions to the the cytotoxic in the text.

29 (1995)

3

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2.9. Reagents RPM1 1640, PHA, TFP and Ficoll-Hypaque were from Sigma (St. Louis, MO, USA); IL-2 (Lymphocult-T-HP 10.000 Units) was from Biotest Diagnostics (Biotest, Germany); FCS was from Cultilab (Cultilab, Campinas SP); Naz”CrO, (250 pCi/mg Cr) was from Comissao National de Energia Nuclear (Sao Paulo, Brazil) and [ 3H]TdR (76 $i/ mmol) was from Amersham International (Amersham, UK). Anti-CD25 and anti-CD56-MoAb (Monoclonal Antibodies) and goat anti-mouse Ig FITC were obtained from Becton-Dickinson.

(TFP)

put into solution in FCS just before use diluted in RPM1 medium for use in the TFP was added at several concentracultures, to the proliferative assays, to and to the binding assays as described

2.7. Flow cytometric analysis for CD25 and CD56 expression Analysis for surface immunofluorescence were performed on FACScan flow cytometer (Becton Dickinson). Cells (1 x lo6 cell/ml) to be analysed for immunofluorescence were incubated with saturating amount of anti-human IL-2 receptor (CD25) or antihuman Leu-19 (CD56) for 30 min at 4°C washed twice with chilled PBS and ressuspended in PBS containing 1% formaldehyde and 0.5% sodium azide. Non-specific binding was subtracted using appropriate controls.

3. Results 3.1. Inhibition of cellular proliferation

by TFP

Inhibition, in a dose-dependent manner, of IL-2, as well as PHA-induced lymphocyte proliferation (Fig. I), was observed when TFP was added at the concentrations of 5 and 10 PM to the beginning of the assay. This effect was also observed when the drug was added 24 h after the start of the PHAstimulated culture (Table 1). We wondered whether tumor cells would show the same inhibitory pattern towards TFP. K562 and Jurkat cells were, however, more refractory to the

B 3

2

2.8. Binding assay

:

3 ; i

1

Freshly prepared PBL and PBL stimulated for 72 h with 2.5% IL 2, at 1 x lo6 cells/ml, were incubated with K562 at an effecter/target ratio of 20: 1, in flat-bottom microtiter plates. One experiment was carried out in the presence or absence of PHA at 2.5 pg/ml. 60 min after the start of the culture, effector:target conjugates were counted under phasecontrast microscopy and compared in the different situations studied.

J TFP

(IIM)

Fig. 1. Effect of TFP on IL-2- and PHA-induced proliferation. TFP was added at the concentrations of 5 PM and 10 PM to the beginning of the proliferative assay. (A) PBL stimulated with PHA; (B) PBL stimulated with IL-2 at the dilutions of 10% (0); 5”~ (m); 2.5”~ (A). The data represent the mean of thymidine incorporation of a representative experiment.

L. de La Rocque et al. / fmmunopharmacolog):

4 Table

29 (1995)

l-10

1

Inhibitory Experiment

effect of TFP on lymphocyte TFP

proliferation

Time

of addition

(h)

(IN 0

24

0 5 10 15

47381C 9523 853 379

52311 5 545 418 416

0 5 10 15

54373 37 680 3317 326

54655 39843 3880 437

0 5 10 15

40 390 4685 1090 1414

49967 21586 4 997 2049

4'

1

2

3

* Results are expressed as cpm of [3H]thymidine incorporation. PBL were stimulated with 5 pg:ml PHA for 72 h, and TFP added either at the beginning of the culture or 24 h later.

effect of the drug at all culture times tested, and did not display, thus, the same sensitivy pattern of IL-2 or PHA-stimulated lymphocytes (Fig. 2). 3.2. Failure of exogenous IL-2 to restore the proh$erative response When IL-2 was added together with several doses of TFP at the beginning of the PHA-induced proliferative assay, no reversal of the drug-mediated inhibition of this mitogenesis was observed (Table 2). 3.3. TFP efSect on the cytotoxic assqv When TFP was added at the beginning of the NK assay, it was seen to inhibit this cytotoxicity in a dose-response manner, this system having displayed a sensitivity to this drug approximate to that characteristic of PHA-induced lymphocyte proliferation (Fig. 3). The dose of 15 PM completely abolished this cytotoxic response and, therefore, is not shown. LAK cells are known to be more potent than NK effecters in their cytotoxic activity; we thus wondered whether the former would show a different

0' 8'

E c 0

642oTFP

uM

Fig. 2. Effect of TFP on the proliferation of cell lines. TFP was added at several concentrations: 2,5 PM, 5 PM, 10 FM and 15 pM at the beginning of the proliferative assay. Cell lines K562 (A) and Jurkat (B) were harvested at 24 (A) and 48 (e) h. The data represent the mean of thymidine incorporation, and were compiled from three separate experiments carried out in triplicate.

pattern of sensitivity to TFP when compared to the latter. The addition of this drug to the LAK cell assay, however, showed it to be just as sensitive to TFP as the NK cell assay (Table 3). As previously seen for NK activity, 15 PM completely suppressed this cytotoxicity.

Table

2

Effect of IL-2 addition stimulated lymphocyte

on the TFP-mediated proliferation TFP

Control PHA PHA + IL-2

(5%)

inhibition

of PHA-

bM)

0

5

10

15

727* 18627 18438

744 12969 10 153

789 3 906 6 674

569 903 844

* Results are expressed as cpm of [‘Hlthymidine incorporation. PBL were cultured for 72 h (Control) or stimulated for the same amount of time with 5 pg/ml PHA, and both TFP and IL-2 added at the beginning of the culture.

L. de La Rocque et al. / Imnzunopharmucolog~ 50

r

29 (1995)

l-10

5

4. Discussion

25:l

50 Effector

1

target

IOC

1

ratlo

Fig. 3. Effect of TFP on NK activity. TFP was added at several concentrations to the beginning of the 4-h “Cr release assay against K562 cells as targets. Data are expressed as ‘;; of cytotoxicity and taken from one representative experiment.

3.4. The efSect of TFP on LAK cell generation TFP was able to inhibit cellular proliferation, and as LAK cell generation seems to involve proliferation as well as differentiation, we investigated if this drug would be able to inhibit LAK cell generation. Results in Table 4 show that LAK cell generation displays a similar susceptibility pattern to the other systems studied. The exception was the concentration of 15 PM, which was not able to completely abolish this response.

3.5. Efect of TFP on surface molecules expression When TFP was added to IL-2- or PHA-stimulated cultures, no change was seen in the expression of either CD25 or CD56 when measured 24 h after the start of the cultures (Figs. 4 and 5):

3.6. TFP interference on eflector:target coqhgate forma tion TFP partially inhibited binding of both NK and LAK effecters to their targets at the concentrations of 5 PM and 10 PM (Table 5). The addition of PHA to this binding assay was able to restore almost completely conjugate formation, although the aspect of the assay with PHA in the presence of TFP did not display the usual clumping promoted by the lectin. Killing function, however, could not be reestablished by adding lectin to the assay, despite the fact that this substance is capable of augmenting NK activity (Table 6).

We found that TFP was able to inhibit LAK cell activity generation and the effector phase of this cytotoxicity. The same sensitivity to this drug was displayed by NK activity and IL-2- or mitogen-induced lymphocyte proliferation; the division of tumor cells, however, was more resistant to the suppressive effect of this inhibitor. The fact that almost all lymphocytic activation systems studied by us displayed the same susceptibility to TFP is in accordance with the findings of other authors carried out with different TCAs. The dose range in which desipramine, amitriptyline and imipramine suppressed both NK activity and mitogen-induced lymphocyte proliferation was approximately the same (Miller and Lackner, 1989). Chlorpromazine also abolished lectin-induced lymphocyte mitogenesis (Martinez and Coleman, 1989) in approximately the same concentrations that we and others (Bachvaroff et al., 1984), working with TFP, have found to be efficient to inhibit both mitogen-stimulated lymphocyte proliferation and NK activity, and that, in this paper, has also been shown to be capable of abolishing IL-2-induced lymphocyte proliferation, LAK cell generation and the effector step of this cytotoxicity. It is difficult to ascertain the mechanism(s) by which TFP could be affecting these lymphocytic systems of activation. Lymphocyte proliferation depends on the production of IL-2, as well as IL-2 receptor expression, which represents independent events that are used as landmarks of the activation process. The possibility that TFP could be acting by inhibiting IL-2 production was dismissed by the fact that addition of exogenous IL-2 did not reverse the inhibitory effect. IL-2 receptor expression was also not affected by TFP, as shown by CD25 expression analysis. Consequently, the modulatory effect observed is probably independent of the IL-2 pathway and this is in accordance with the finding that even when the addition of the drug was carried out 24 h after the start of the culture, inhibition of lymphocyte mitogenesis was just as effective as when it was added at the beginning of the assay. Inhibition was reported to be only detected when the drug was added up to 5 h after the start of the culture (Cheung et al., 1983);

6 Table TFP

L. de La Rocque et al. / Immunopharmuco1og.v

Control

cytotoxicity

5 PM 10 PM 15 PM

assay 1

Experiment

3

5O:l

25:l

too:1

50: 1

25:l

1OO:l

50: 1

25:l

28b 71 24 15 SF

25 13 21 9 SR

16 57 14 2 SR

6 61 47 18 SR

5 40 21 8 SR

SR 15 SR SR SR

12 50 58 39 5

3 38 45 9 SR

SR 20 19 4 SR

nevertheless, there were others who, in agreement with our results, stated that the effectiveness of TFP was independent of the time of addition of the drug after initiation of the cultures (Bachvaroff et al., 1984). Chlorpromazine, however, was effective in mouse lymphocytes only when added during the first 24 h of stimulation (Ferguson et al., 1975). The lymphocytic activation systems studied result in different effector responses through biochemical pathways that share some key events, susceptible to modulation by drugs. Some of these may modulate them diversely, like ouabain, which has a much more profound effect on lymphocyte proliferation than on NK cytotoxicity (Moraes et al., 1989). TFP, how-

TFP

Experiment

2

1OO:l”

” Effectorjtarget ratio. b I’; of cytotoxicity. ’ Spontaneous release. PBL at the concentration of 2 x 106:ml were incubated for 72 h without at the concentration of 5 x 10h/ml for use as effecters in the LAK activity using IL-2-activated cells as effecters

Table

l-10

(1!85J

3 added to the LAK

Experiment

IL-2 TFP TFP TFP

29

(Control) or with 2.5 “; IL-2 (IL-2), washed and resuspended assay. TFP was added at the beginning of this cytotoxic assay

ever, was, in our hands, able to affect in a similar way almost all lymphocytic activation processes studied, which points towards the involvement of this drug in a very basic activation step, shared by all these responses. TFP is known to affect many cellular functions. It is a calmodulin antagonist and, although the role of this protein is already well established in lymphocyte mitogenesis (Cheung et al., 1983), it is not so well defined for NK and LAK activities. Other Ca’ + binding protein inhibitors, such as cyclosporin, despite showing a very marked effect on lectininduced lymphocyte proliferation do not produce such an outstanding effect on cytotoxicity (di Padova, 1989).

4 added

to the generative Experiment

Control IL-2 TFP 5 PM TFP 10 PM TFP 15 PM

step of LAK

cytotoxicity

1

Experiment

2

Experiment

3

1OO:l”

50: 1

25: 1

1OO:l

so:1

25:l

1OO:l

5O:l

25:l

12b 50 37 13 SR’

3 38 17 5 SR

SR 20 17 10 SR

35 6 46 23 12

21 53 25 14 4

I 24 6 2 2

27 48 37 27 SR

11 44 20 14 SR

6 32 8 4 SR

&‘ See Table 3. PBL at the concentration of 2 x 10h/ml were incubated for 72 h without (Control), or with 2.5”% IL-2 (IL-2) in the presence or absence of TFP. After the incubation period, cells were washed and resuspended at the concentration of 5 x 106/ml for use as effecters in the LAK activity assay.

L. de La Rocque

et al. / It~lmunopharmacolog},

29 (19951 I-10 t

39.32

log

a

9.4%

e

b

13.4;.

f

fluorescence

Fig. 4. Effect of TFP on CD25 expression. Fluorescence profile of PBL bound with anti-CDZSMoAb. Cells were stimulated with either PHA-5 pg/ml (b to d) or IL-2 at 2,5 Y; (f to h) for 24 h and then labelled with anti-CD25-MoAb and stained with anti-Ig-FITC. (a) Control, (b) PHA-stimulated, (c) PHA-stimulated in the presence of 5 PM TFP, (d) PHA-stimulated in the presence of 10 FM TFP, (e) Control, (f) IL-2-stimulated, (g) IL-2-stimulated in the presence of 5 pM TFP, (h) IL-2-stimulated in the presence of 10 PM TFP.

On the other hand, the relative resistance to TFP on the tumor cells detected in this work could result from a greater calmodulin content of these cells compared to normal ones (Chafouleas et al., 1981; Takemoto and Jilka, 1983) or even from the secretion of calmodulin (Croker et al., 1988). In this case, the drug would bind to this secreted protein or to the augmented intracellular store, preventing its action

on other molecular targets. Calmodulin antagonism, however, seems unlikely to explain the inhibition,of the proliferative step in LAK cell generation, considering that Ca2+ mobilization is not involved in the biochemical events triggered by IL-2 binding to its receptors (Minami et al., 1993). TFP could also be acting on the lymphocytic activation systems studied through its inhibitory effect

L. de Lu Rocque

3 I

et ul. : Immunopharmucolog~v Table

a

29 (1995)

l-10

5

Effect of TFP on the binding

capacity

Presence of lectin in assayh

Effector

of effector

cells

O0 of target conjugates‘

cells forming

TFP

b 3%

PBL” PBL PBL cultured for 72 h PBL cultured for 72 h with IL-2

+ -

(PM)

0

5

10

56 100 65 100

17 ND 21 10

8 90 6 4

” Freshly prepared. b 2.5 pl PHA added to the beginning of the assay. ’ Target cells (K562) with one or more effector cells attached to them. Conjugate formation was assessed 60 min after effector:target cells (20: 1) were put together in hat-bottom microtiter plates.

d

log

fluorescence

Fig. 5. Effect of TFP on CD56 expression. Fluorescence profile of PBL bound with anti-CD56-MoAb. Cells were stimulated with either PHA-5 pg/ml for 24 h and then labelled with anti-CDShMoAb and stained with anti-Ig-FITC. (a) Control. (b) PHAstimulated, (c) PHA-stimulated in the presence of 5 PM TFP, (d) PHA-stimulated in the presence of 10 FM TFP.

on the protein kinases (Brumley and Wallace, 1989) since increasing evidence seems to point towards the involvement of these proteins in both NK and LAK cytotoxicities (Kvanta et al., 1990; Gibboney et al., 1992), whereas its role in lymphocytic proliferation is already well established (Friedrich and Gullberg, 1988). Furthermore, a recent report suggests that

IL-2 binding affects lymphocyte proliferation through the activation of a diacylglycerol kinase that leads to the accumulation of phosphatidic acid (Merida et al., 1993). More and more emphasis is now being placed on the role of cell-adhesion molecules (CAMS) in the deliverance of co-stimulatory signals for cellular activation (Oblakovski et al., 1991; Hogg and Landis, 1993). The main adhesion molecules involved in NK killing, CD2 and LFA-1 (Petranyi et al., 1986; Voltarelli et al., 1993), have not been investigated in

Table

6

Effect of Icctin, used as a binding agent, in the inhibition by TFP in NK cytotoxic assays Experiment PTFP Control PHA 2.5 pg/ml PHA 5 ,&rl

:;a 41

1 +5 PM TFP

SRb 12 9

Experiment -TFP 47 90 62

produced

2 +5gMTFP 14 30 33

a “; cytotoxicity. h Spontaneous release. PBL were used as effecters, at the effcctor:target ratio of 50: 1, in a 4-h 51Cr release assay against K562 target cells, with or without PHA as a binding agent, and with or without TFP, present throughout the assay.

the present work, but the expression of CD56, a surface molecule characteristic of NK and LAK cells (Robertson and Ritz, 1990) was unaffected by TFP. Nevertheless, the binding capacity of NK and LAK cells was impaired by TFP. The addition of lectin as a binding-enhancing agent, although re-establishing the binding capacity of NK cells, did not overcome the suppression exherted on the killing step of this cytotoxicity. These results suggest that TFP is capable of affecting not only the binding step of the cytotoxic process but also interferes with some biochemical events triggered by it. The fact that TFP was able to affect all these lymphocytic systems has certainly to be taken into consideration when treating depressed patients with immunosuppressive diseases such as cancer. LAK cell therapy is already being employed in tumors resistant to conventional chemotherapy (Rosenberg et al., 1985; Lotze et al., 1986), and it will probably be used when treating multi-drug resistant tumors, since no clear correlation was found between multidrug resistance and feasibility of lysis by LAK cells (Harker et al., 1990; Arienti et al., 1992). The fact that LAK cell therapy, both in its generative and effector steps, has been shown to be suscetible to TFP, has to be counterbalanced with the prospective beneficial effects antidepressants may cause to patients subjected to this kind of immunotherapy.

Acknowledgements We thank Conselho National de Desenvolvimento Cientifico e Tecnologico (CNPq) and Fundacao Ary Frauzino for financial support. We are specially indebted to Constantino Gonzalez Salgado for performing some of the preliminary experiments.

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29 /1995)

I-10

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5. Appendix TABLE

.4-l

Effect of TFP

on PHANo addition

Exp. 1 Exp. 2 Exp. 3

289 291 383

and IL-2

induced PHA

10305 8 745 18995

lymphocyte PHA + 5 I”M 325 848 5983

proliferation

TFP

PHA + 10 PM TFP

IL-2 (5”b)

IL-2 + 5 PM TFP

IL-2 + 10 FM TFP

328 239 496

1100 2003 853

635 789 569

323 243 418