Clin. exp. Immunol. (1990) 82, 108-113

Signal requirements for activation of leukaemic T cells from a chronic lymphocytic leukaemia (T-CLL) M. R. ZOCCHI*, A. POGGIT, S. HELTAI*, A. VILLA§, L. INVERARDI*, A. VICARI*, M. G. SABBADINIt & M. FERRARINI* *Istituto Scientifico San Raffaele, Milan, tDipartimento di Scienze e Tecnologie LRBiomediche, Cattedra di Immunologia Clinica, University of Milan, Milan, 1Istituto Scientifico Tumori, Genoa, and §Istituto di Tecnologie Biomediche Avanzate, CNR, Milan, Italy

(Acceptedfor publication 25 April 1990)

SUMMARY In order to define the signal requirements for leukaemic T cell activation, the proliferation and interleukin-2 (IL-2) production of peripheral lymphocytes from a patient with a HTLV-I-, CD4+, CD45RA+ CD45RO+ CD25- T-CLL were evaluated after the delivery of different stimuli. Unlike resting CD4+ normal T lymphocytes that can be activated only by a two-signal stimulation, T-CLL cells proliferated and released IL-2 in response to a pair ofanti-CD2 monoclonal antibodies (MoAbs) or concanavalin A (Con A) in the absence of both accessory cells (AC) and phorbol myristate acetate (PMA). The two stimuli were also able to induce CD25 expression within 12-20 h on the majority of T-CLL cells. A response to anti-CD3 and anti-CD28 MoAbs was detected only in the presence of PMA, similar to that observed in normal resting T lymphocytes matched for phenotype. Both Con Aand CD2-induced proliferation were strongly inhibited by the addition of anti-CD25 MoAb. Furthermore, T-CLL lymphocytes acquired anti-tumour lytic activity after culture in the presence of PMA and ionomycin. We conclude that HTLVI- CD25- T-CLL can be characterized not only by morphological and phenotypical studies but also on the basis of signal requirements for cell activation. Keywords leukaemia

lymphocytes

activation

INTRODUCTION Normal resting T lymphocytes can be activated via the antigenspecific CD3 T cell receptor (TcR) complex or via the CD2 molecule in the presence of accessory cells (AC) of the monocyte-macrophage lineage. Phorbol esters such as phorbol myristate acetate (PMA) can substitute for the AC requirement in such systems (Van Wauwe, De May & Goossener, 1980; Meuer et al., 1983, 1984a). A third pathway of T cell activation has been described involving the CD28 glycoprotein (Hara, Fu & Hansen, 1985). Several studies have demonstrated that at least two signals are required for the activation of resting T cells via the CD3/TcR complex, the first represented either by the antigen or by monoclonal antibodies (MoAbs) directed against the CD3/TCR complex and the second provided by AC (Meuer et al., 1983; Weiss et al., 1986). When T cells are activated via the alternative pathway, initiated through the CD2 molecule with combinations of anti-CD2 MoAbs, the requirement for AC varies depending on the specific pair of MoAbs utilized (Meuer Correspondence: Maria Raffaella Zocchi, MD, Laboratorio Immunoterapia Adottiva, Instituto Scientifico San Raffaele, Via Olgettina 60, 20132 Milan, Italy.

et al., 1983; Olive et al., 1986). MoAbs recognizing the CD28 molecule, originally reported to enhance lymphocyte proliferative responses to phytohaemoagglutinin (PHA) or allogeneic cells (Gmunder & Lesslauer, 1984; Ledbetter et al., 1983), can

activate T lymphocytes under appropriate conditions (Moretta et al., 1985). Conversely, activated T cells (such as cloned T lymphocytes in permanent culture) can be triggered by anti-CD3 MoAbs without the need of AC (Manger et al., 1985; Weiss et al., 1986). These signals lead to the upregulation of the interleukin-2 (IL-2) receptor (IL-2R) (CD25) which, upon interaction with IL-2, promotes proliferation (Robb, Munk & Smith, 1981; Meuer et al., 1984b). The IL-2/IL-2R network appears to play a role in the leukaemogenesis although differences exist, due to the type of leukaemia, namely in HTLV-I+ adult T cell leukaemias (ATL), cells derived from peripheral blood have a helper/inducer phenotype and express CD25 antigen but respond poorly to exogenous IL-2, whereas CD4+ T cells from HTLV-I-CD25+ T-CLL show a good proliferative response to IL-2 (Uchiyama et al., 1985; Tsudo et al., 1986). In contrast, leukaemic cells with a CD8+ phenotype neither express CD25 nor respond to IL-2, suggesting that the IL-2/IL-2R system is not involved in the

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Leukaemia lymphocytes activation actual proliferation of peripheral blood CD8+ leukaemic cells (Uchiyama et al., 1988; Umadome et al., 1988). In the present study we have examined the signal requirements for induction of proliferation, CD25 expression and IL-2 production in peripheral blood lymphocytes obtained from a patient with a HTLV-I-, CD4+CD25+ CD45RO+ CD45RA+, T-CLL. We show that the behaviour of these leukaemic cells significantly differs from that of resting T cells matched for phenotype, when the stimulation is delivered via CD2, resembling the response observed in cloned activated CD4+ cells. On the other hand, a behaviour indistinguishable from the one observed in normal resting CD4+ cells is recorded when the stimulus is delivered via CD3 or CD28 molecules. MATERIALS AND METHODS Patient The patient, a 62-year-old man, showed on admission hepatosplenomegaly and a few enlarged lymph nodes in the axillary areas, no anaemia and an increased lymphocyte count (204 x 109/l). May-Grunwald-Giemsa staining of peripheral blood smears showed homogeneous small lymphocyte population with a mature aspect and analysis of a bone marrow aspirate revealed a heavy, diffuse infiltration (not shown). A diagnosis of CLL, according to Rai, was made.

mentioned above. Alternatively, 10 yg/ml of either concanavalin A (Con A) or PHA (Sigma) or 250 ng/ml ionomycin (Serva, Heidelberg, FRG) were added to the culture. All the stimuli were tested in the presence or absence of PMA (Sigma) at submitogenic doses (0-5 ng/ml).

Proliferation assay Cells cultured with the various stimuli were pulsed with 1 pCi/well of 3H-methyl-thymidine (3H-TdR; Amersham International, Amersham, UK) for the last 18 h and then harvested. 3H-TdR uptake was quantified in a liquid scintillation counter (Packard Instruments, Downers Grove, IL). Proliferation was evaluated with time-points at 24, 48, and 72 h. All assays were performed in triplicates and results are expressed as mean ct/min x 10-3. Standard deviations were less than 2%. IL-2 measurement One-hundred microlitres of the supernatants were collected from cell cultures after 24 h of stimulation with either MoAbs or mitogens as described above. Murine CTLL-20 (IL-2-dependent cell line) were plated (4 x 103/well) in 96-well, U-bottomed microtitre plates with serial dilutions of test samples in triplicates. After 24 h, cells were pulsed with I pCi 3H-TdR for the last 8 h of culture, harvested and counted. Results were compared with those obtained from control samples with known IL-2

concentrations. Cell preparation Peripheral blood mononuclear cells (PBMC) were isolated according to Boyum (1968). AC were removed by two rounds of adherence to plastic. Normal CD4+ cells were obtained by removal of CD8+ cells from AC-depleted lymphocytes with panning techniques (Wysocki & Sato, 1978) using the anti-CD8 MoAb Leu2b (Becton Dickinson, Mountain View, CA). Cloned CD4+ T cells were obtained by limiting dilution as previously described (Moretta et al., 1983). Cloning efficiency was > 95%. Clones were grown and expanded in 20 U/ml recombinant IL-2 (rIL-2; Eurocetus, Amsterdam, The Netherlands) and they weakly expressed CD25 antigen.

Cytotoxicity assay T-CLL lymphocytes were tested in a cytolytic assay before and after culture with PMA (10-0-5 ng/ml) in combination with various amounts of ionomycin (1-0-25 pg/ml). The NKsensitive K562 cell line and the NK-resistant hepatoma cell line EPAI were used as targets. Lectin-dependent cellular cytotoxicity (LDCC) was performed versus the murine mastocytoma P815 cells in the presence of PHA (0-5 pg/ml). Lysis was tested in a 4-h 5"Cr release assay at the effector-to-target cells (E/T) ratio of 25/1. Results are expressed as percentage specific lysis as described (Pawelec et al., 1988).

Monoclonal antibodies For functional studies MoAbs were used at a final concentration of 0-5 pg/ml. The pair of mitogenic anti-CD2 MoAbs (CD2 1 and CD2-9) were kindly provided by Dr Daniel Olive (Marseille). Anti-CD3 (JT3A) and anti-CD28 (CK248) have been previously described (Moretta et al., 1987; Poggi et al., 1987). Anti-CD25 (anti-Tac) MoAb (kindly provided by Dr T. A. Waldman) and anti-CD7 MoAb (Leu9, Becton Dickinson) were used in blocking experiments at 1/100 up to 1/500 final dilution of ascites fluid or 100 to 1 pg/ml, respectively. Monoclonal antibodies used for cell staining were all purchased from Becton Dickinson, with the exception of UCHL1 (Technogenetics).

Characterization of T-CLL cells Molecular analysis showed the rearrangement of the T cell receptor fi gene (not shown). T-CLL cells were virtually all (99-8%) CD2+CD28+CD3+WT31+, CD4+CD45RA+ CD45RO+ HLA-DR-CD25- and CD71 -; the purity of T cells was further confirmed by flow cytometry using anti-CD14 (LeuM3) MoAb (not shown). Dual-parameter flow cytometric analysis of surface phenotype and DNA content showed that about 45% of T-CLL lymphocytes had entered the cell cycle (S/G2/M phases) while being CD25 negative (not shown).

Cell activation Purified CD4+ resting T lymphocytes, cloned CD4+ T cells or T-CLL lymphocytes were cultured in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS, Seromed, Berlin, FRG), penicillin (50U/ml), streptomycin (50 pg/ml) and 4 mM L-glutamine (Sigma) in a humidified 5% CO2 atmosphere at 37°C. Cells were cultured at 105/well in 0-2 ml ofculture medium in 96-well U-bottomed plates. Combination of anti-CD2, antiCD3 or anti-CD28 MoAbs were included in the medium as

Cell activation requirements. As shown in Table 1, T-CLL CD4+ lymphocytes gave a strong proliferative response to Con A and a pair of AC-dependent anti-CD2 MoAbs in the absence of PMA. The peak of 3H-TdR uptake was detected within 24 h and it did not increase after 36, 48 or 72 h. The proliferative response to PHA and anti-CD3 MoAb occurred only when PMA was present, whereas no proliferation was observed with IL-2. A slight 3H-TdR uptake was evident with ionomycin and anti-CD28 MoAb only in the

RESULTS

M.R. Zocchi et al.

110

Table 1. Signal requirements for T-CLL lymphocyte proliferation

Resting CD41 cells

Cloned CD41 cells

T-CLL CD4+ cells

Stimulus

-PMA

+ PMA

-PMA

+PMA

-PMA

+PMA

PHA (10 ug/ml) Con A (10 pg/ml) lonomycin (250 ng/ml) Anti-CD3* Anti-CD2* Anti-CD28* IL-2 (100 U/ml) None

2-1 2-8 04 1-4 1-8 1-9 04 0-3

7-2 24-2 20 7 22-1 49-7 37-6 106 10

66-0 48-0 2-1 82-1 87-0 10 33 0 2-8

141-8 70-0 67-0 170-9 153-3 10-2 58-0 3-4

7-7 31-0 0-2 2-6 43-6 1-7 1-8 0-5

28-2 40-0 10-7 35-2 45 8 9-7 2-6 2-0

Results of one experiment (representative of four independent experiments). Cells were cultured for 48 h and 3H-TdR was added during the last 18 h of culture. PMA was added at the sub-mitogenic dose of 0 5 ng/ml. Results are expressed as ct/min x 10-3 of 3H-TdR uptake. * Used at final concentration of 0 5 jug/ml. Table 2. Signal requirements for T-CLL lymphocyte IL-2 production

Resting CD4+ cells

Cloned CD4+ cells

T-CLL CD4+ cells

Stimulus

-PMA

+PMA

-PMA

+ PMA

-PMA

+ PMA

PHA (10 pg/ml) Con A (10 ,ug/ml) Ionomycin (250 ng/ml) Anti-CD3* Anti-CD2* Anti-CD28* None

0.1 0-2 0.1 0-2 0-3 0-7 03

16-9 46-9 29-4 37-4

36-5 10-2 0-2 41 1 25 5 0-4 09

108-6 18-1 11-4 115-7 109-8 10-7 0-6

09 4-1 0-8 0-8 9-9 09 0-4

23-8 12-6 3-8 102-3 104-9 13-6 0-5

45.7 27-5 09

Results of one experiment (representative of four independent experiments). Cells were cultured in the presence of the various stimuli as described in Materials and Methods. PMA was used at 0-5 ng/ml. Supernatants were collected after 24 h and tested for IL-2 activity on the IL2-dependent murine T cell line CTLL-20. Results are expressed as 3H-TdR uptake (ct/min x 10-3). (CTLL alone, 0 5 ct/min x 10-3; CTLL plus IL-2 100 U/ml, 55-3 ct/min x 10-3). * Used at final concentration of 5 0 yg/ml.

presence of PMA (Table 1). Highly purified (99-4%) resting CD4+ lymphocytes were able to proliferate regardless of the stimulus tested only in the presence of PMA, 3H-TdR uptake being detectable at days 2-3. Conversely, cloned CD4+ T cells gave a good proliferative response, starting from day 2, to all the stimuli tested without the need for PMA, with the exception of ionomycin and anti-CD28 MoAb that induced cell activation only if PMA was added (Table 1).

Induction of CD25 expression on T-CLL lymphocytes CD25 up-regulation was evaluated after 12 and 24 h from the beginning of the culture. When anti-CD2 MoAbs or Con A were used as stimuli, CD25 up-regulation was significant by 12 h, reached 100% by 24 h and did not require the presence of PMA. On the other hand, when anti-CD3 or anti-CD28 were used, cells needed PMA and the kinetics proved to be significantly slower (Fig. 1).

IL-2 production IL-2 production by T-CLL lymphocytes was observed after triggering cells via CD2, CD3, CD28 and with PHA or Con A in the presence of PMA. Lower but significant amounts of IL-2 were detected after stimulation with either Con A or anti-CD2 MoAbs also in the absence of PMA (Table 2). Resting CD4+ lymphocytes needed PMA to produce IL-2 regardless of the stimulus used (see Table 2), whereas cloned CD4+ T cells could produce it in the absence of PMA when triggering was provided via CD3, CD2 and with PHA or Con A. PMA had to be present when ionomycin or anti-CD28 MoAb were used.

Inhibition of T-CLL proliferation by anti-CD25 MoAb In order to verify that the IL-2/IL-2R interaction was responsible for the strong and early cell proliferation to anti-CD2 MoAbs and Con A, blocking experiments with anti-CD25 were carried out. T-CLL lymphocytes were cultured with either Con A or CD2 MoAbs in the absence or presence of anti-CD25 MoAb. Anti-CD2-induced proliferation decreased by 80% (Fig. 2) and 60% (not shown) by adding anti-CD25 MoAb at 1/100 or 1/500 final dilution, respectively. No inhibition was observed using anti-CD7 MoAb. Comparable results were obtained with regard to Con A-induced proliferation (Fig. 2).

Leukaemia lymphocytes activation

111 24 h

12 h b

a

d 0) .0

E

c

0 C,

0 C

Green fluorescence intensity

Fig. 1. Induction of IL2R (Tac, CD25) expression on T-CLL lymphocytes cultured for 12 h (left panels) or 24 h (right panels) with antiCD2 MoAbs (panels a, b), anti-CD3 MoAb (0-5 pg/ml) (c, d) or anti-CD3 plus PMA (0-5 ng/ml) (e, f). Results are expressed as green fluorescence intensity versus relative cell number. Dotted lines represent the negative control obtained with the fluoresceinated irrelevant MoAb.

Induction of cytotoxicity in T-CLL lymphocytes by PMA plus ionomycin To characterize further the functional properties of the T-CLL, cytotoxic activity was evaluated before and 24 h after culture in the presence of different stimuli. Neither resting nor activated T-CLL lymphocytes were directly cytotoxic to target cells. Leukaemic cells acquired the ability to lyse hepatoma tumour cells after culture with high doses of PMA or with the combination of PMA plus ionomycin (Table 3). Similar results were obtained using both K562 and P815 cell lines (not shown).

70 r 60 50 0 x C

40

E 30 C-

20

10 0

Medium

+ anti- CD25

DISCUSSION + anti-CD7

Fig. 2. Inhibition of Con A- and CD2-induced proliferation of T-CLL lymphocytes by anti-CD25 antibody. Leukaemic cells were cultured for 48 h with either 10 ug/ml Con A (open bars) or 0-5 pg/ml of anti-CD2 MoAbs (solid bars) in the absence or in the presence of anti-CD25 (1/100 dilution of ascites fluid) or anti-CD7 (100 pg/ml) MoAbs. Data represent 3H-TdR uptake of T-CLL lymphocytes (ct/min x 10-3).

We evaluated the signal requirements for activation ofperipheral blood lymphocytes from a patient with an HTLV-I - T-CLL. The progressive nature of the disease clearly indicates that leukaemic cells actively proliferate in vivo; conversely, in the absence of overt stimulation, no proliferation was observed in vitro as judged by thymidine incorporation. This can be explained,

112

M.R. Zocchi et al. Table 3. Induction of cytotoxicity in T-CLL lymphocytes by PMA plus Ionomycin

Ionomycin (pg/ml) PMA (ng/ml)

10 5 1 05 0

1

0-5

0 25

0

42 41 42 30 0

45 40 20 12 0

45 44

50 40 17 0 0

25 3

0

Cells were cultured for 48 h in the presence of PMA and Ionomycin, washed and used in a 4-h 5"Cr release cytolytic assay versus hepatoma tumor cells (E/T ratio 25/ 1). Results are expressed as percent specific lysis.

assuming that some growth factors or surface molecule ligands are missing in the in vitro system. The patient's T lymphocytes are characterized by a mature phenotype (e.g. CD3+CD4+ CD8-), express both the CD45RA and the CD45RO antigens (recognized by the 2H4 and the UCHL1 MoAbs, respectively) (Morimoto et al., 1985; Smith et al., 1986) and do not have the CD25 molecule on their surface. Despite this, half the lymphocytes show a DNA content that is typical of cell that have entered the cell cycle and proliferate. It is well-known that normal purified T lymphocytes need two signals in order to undergo cell activation and proliferation (Meuer et al., 1983; Weiss et al., 1986). Lectins, MoAbs directed against the CD3/ TCR complex or the CD28 molecule, some combinations of anti-CD2 MoAbs and the calcium ionophores have been shown to deliver one of the signals required, whereas compounds such as PMA can provide a second signal (van Wauwe et al., 1980; Meuer etal., 1983, 1984a; Hara etal., 1985; Moretta etal., 1985; Olive et al., 1986). Unlike resting cells, activated T lymphocytes can respond to a single signal, such as anti-CD3 MoAbs (Manger et al., 1985). Most of the CD4+ T-CLL studied so far are characterized by the expression of the CD25 molecule and by responsiveness to exogenously added IL-2 (Uchiyama et al., 1985; Tsudo et al., 1986; Uchiyama et al., 1988; Umadome et al., 1988); for this reason we thought it important to assess the behaviour of this CD25- leukaemia for its responsiveness to different stimuli. Interestingly, all leukaemic cells strongly expressed CD25 antigen within 20-24 h from the beginning of stimulation with a pair-wise combination of anti-CD2 MoAbs. Conversely, CD25 appearance on normal lymphocytes require at least 48 h of activation and occur only in the presence of AC or PMA (Manger et al., 1985; Olive et al., 1986; Yong et al., 1988). On the other hand, CD25 up-regulation on T-CLL after triggering via CD3 or CD28 was consistently slower and required the addition of PMA, as did normal T cells. It is noteworthy that T-CLL cells, but not normal lymphocytes, showed a very low threshold of activation via CD2. This fact was further supported by the observation that small T-CLL lymphocytes acquired morphological features of blast cells within 10 h after triggering with anti-CD2 MoAbs (not shown). These findings are in keeping with the proliferation data. In fact, T-CLL cells gave an early (24 h) and strong proliferative response via CD2 in the absence of AC resembling the

behaviour of activated cloned T cells rather than purified normal CD4+ lymphocytes that needed AC or PMA and showed a maximum of proliferation after 72 h. Analysis of IL-2 production in response to MoAbs and lectins, both with and without PMA, and studies of inhibition with anti-CD25 MoAb provided evidence that proliferation of T-CLL cells following activation with the various stimuli was sustained by an IL-2-mediated autocrine mechanism. Since interleukin-4 (IL-4) has also been reported to promote T cell growth in some T-CLL (Uchiyama et al., 1988; Umadome et al., 1988), we evaluated the possibility that in this case the proliferative response to anti-CD2 MoAbs was also partially due to IL-4. This does not seem to be the case, as IL-4 neither induced T-CLL growth nor increased the CD2-driven proliferation of leukaemic cells; moreover, the addition of anti-IL-4 MoAb to the culture medium did not enhance the inhibition due to anti-CD25 MoAb (not shown). Nevertheless, T-CLL lymphocytes needed activation prior to expressing CD25 antigen, producing IL-2 and proliferating. This signal could be delivered in vivo by one of the natural ligands of the CD2 molecule. At least two ligands for the Tl 1I I epitope of the CD2 surface antigen have been described: TlI TS and LFA-3 which are widely distributed on many cell types (Hinig et al., 1986; Selvoraj et al., 1987). LFA-3 plays an important role in cell-to-cell adhesion and is believed to be capable of triggering T cells via CD2 (Tiefenthaler et al., 1987). Thus it is conceivable that these T-CLL lymphocytes may be activated by the direct contact with tissues and other cells in the bloodstream. It is of note that these T-CLL lymphocytes co-expressed high levels of CD45RA and CD45RO antigens. Recently it has been suggested that cells that express high levels of CD45RA antigen might belong to the subset of naive cells and show low responsiveness to anti-CD2 MoAbs, whereas they strongly proliferate to mitogens. In contrast, memory cells are CD45RO+CDw29(4B4)+ and CD45RA- or CD45RAd'm and display a preferential activation via CD2 or CD3 (Sanders, Mangoba & Shaw, 1988). The T-CLL population that we examined resembles the functional behaviour of a memory cell despite the expression of an antigenic typical of naive cells. Nevertheless, these leukaemic lymphocytes, unlike CD45R+ cells, were brightly stained with anti-CD2 MoAbs, like cloned CD4+ lymphocytes (not shown). High levels of CD2 antigen expression have been reported to affect the functional capabilities of T cells (Sanders et al., 1988). Thus, the presence of the CD45R molecule associated with a preferential response to anti-CD2 MoAbs represents only an apparent contradiction. It should be noted that T-CLL cells acquired the CDw29 and lost the CD45RA antigen within 24 h after activation via CD2 (not shown). This finding suggests that these leukaemic cells might be 'frozen' in vivo at an intermediate stage of cell activation between naive and memory cells. Finally we demonstrate that these leukaemic cells can be induced to become cytolytic either with high doses of PMA or with the combination of PMA and ionomycin. A recent report showed that human CD4+ T cells can be divided into two groups on the basis of their potential to acquire anti-tumour activity when stimulated with phorbol esters and ionomycin (Pawelec et al., 1988). In that case the cytotoxic activity required cell activation because even lectin target approximation with PHA did not result in target cell lysis. Although we cannot rule out the possibility that other T-CLL show a different behaviour, our findings strongly suggest that leukaemic T cells can be ascribed

Leukaemia lymphocytes activation to a certain subset of normal T lymphocytes not only on the basis of surface phenotype but also on the basis of signal requirements for activation. For this reason, we believe that studies concerning the signalling systems in leukaemic cells could provide a powerful tool for a better understanding of the pathogenesis of leukaemias.

ACKNOWLEDGMENTS This work was partially supported by a fund from Associazione Italiana per la Ricerca sul Cancro.

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