Cladribine (2-chloro-deoxyadenosine, CDA): an inhibitor of human B and T cell activation in vitro

Immunopharmacology, 26 (1993) 197-202

© 1993 Elsevier Science Publishers B.V. All rights reserved 0162-3109/93/$06.00

197

IMPHAR 00661

Research Papers

Cladribine (2-chloro-deoxyadenosine, CDA): an inhibitor of human B and T cell activation in vitro A n d r z e j G 6 r s k i a , P a w e t G r i e b b, G r a Z y n a K o r c z a k - K o w a l s k a a, P i o t r W i e r z b i c k i a, B a r b a r a St~piefi- S o p n i e w s k a a a n d T o m a s z M r o w i e c a aDepartrnent of Immunology, Transplantation Institute, Warsaw Medical School, Warsaw, Poland and b Foundation for the Promotion of Diagnostics and Therapy, Warsaw, Poland

(Received 5 February 1993; accepted 4 May 1993)

Abstract: We have previously shown that the novel immunosuppressive agent cladribine (CDA) inhibits human T and B cell lymphoproliferativeresponses and immunoglobulin synthesis in vitro, yet appears to be particulary efficacious as an inhibitor of B cell responses. We now report the effects of CDA on the human mixed lymphocyte reaction and on expression of T and B cell activation markers. CDA produced a significant inhibition of lymphocyte proliferation in human mixed lymphocyte reactions at a concentration of 10 nM. At concentrations of 10-100 nM the drug inhibited phytohaemagglutinin-inducedexpression of CD25 and HLA-D (by approximately 50%), but not phorbol myristate acetate-induced expression of CD69 on purified human T cells. At a concentration of 10 nM CDA totally abolished Staphylococcus aureus Cowan-induced expression of CD25 on purified B cells. These findings confirm that CDA is a potent immunosuppressive agent with some selectivitytowards B cells. The drug may have potentially wide applications in clinical immunosuppression. Key words: Cladribine; CDA; 2-Chloro-deoxyadenosine; T cell; B cell; Mixed lymphocyte reaction; Transplantation

Recent developments in immunosuppressive therapy have allowed a significant progress in clinical transplantation and treatment o f autoimmune disease. However, there is still a great need for new drugs acting primarily at the B cell level (Bach, 1991; K a u f m a n et al., 1992). Such drug could be o f value in the treatment o f chronic allograft rejection, which appears to be mediated primarily by humoral mechanisms (Paul and Fellstrom, 1992). Furthermore, a B cell drug is a necessary c o m p o n e n t o f immunosuppression in clinical xenotransplantation, where the current Correspondence to: A. G6rski, Transplantation Institute, War-

saw Medical School, 02006 Warsaw 22, Poland

protocol involves F K 506 with cyclophosphamide (Starzl et al., 1993). In addition, such an agent could also replace cyclophosphamide in other protocols used in therapy o f autoimmune diseases. A purine nucleoside analogue 2-chlorodeoxyadenosine (CDA, cladribine) has proven to be one of the most interesting c o m p o u n d s developed in recent years. The clinical properties o f the drug have been recently delineated (Beutler, 1992). Although not active against most solid tumors, C D A is clearly effective against a variety o f lymphoproliferative diseases (hairy cell leukaemia, B-cell chronic lymphocytic leukaemia, low and intermediate grade n o n - H o d g k i n ' s lym-

198 phoma, Waldenstr0m's macroglobulinaemia, and others). Given as a monotherapy in five to seven day courses, the drug produces a substantial rate of complete and/or partial remissions even in resistant and relapsed cases. In addition, encouraging preliminary results have been achieved in some autoimmune diseases (autoimmune haemolytic anaemia and multiple sclerosis) treated with the same dosing schedule as used in lymphoproliferative diseases. The side effects of CDA (chiefly thrombocytopenia and opportunistic infections) are rather mild compared to other chemotherapeutics. An unique feature of CDA is that it is an antimetabolite affecting both resting and proliferating lymphoid cells with seemingly similar efficacy. These properties are mediated by deoxycytidine kinase (dCK), the enzyme predominant in lymphoid tissue which activates the drug by phosphorylation, and by resistance of the drug to the deamination by adenosine deaminase (Carrera et al., 1990; Carson et al., 1980, 1983). Intracellular accumulation of CDA-triphosphate triggers programmed cell death (apoptosis) in susceptible cells through activation of an endonuclease and accumulation of DNA strand breaks (Hirota et al., 1989; Seto et al., 1985). Inhibition of DNA-polymerases 2 and/3, and of ribonucleotide reductases may also be involved (Parker et al., 1988). Although preliminary clinical observations cited above suggest that CD may also be efficacious in autoimmune diseases, very little is known about CDA as an immunosuppressive agent. It has been implicitly assumed to date that immunosuppression caused by CDA results from selective and transient elimination of lymphocytes and monocytes from circulating blood. Yet in our previous communication (G6rski et al., 1993) we have shown that CDA has potent immunosuppressive properties in vitro and invivo, some of which are certainly not secondary to lymphoid cell death. In nanomolar concentrations the drug strongly inhibited lymphoproliferarive responses of human purified T and B cells and Ig synthesis. Proliferation of T and B leu-

kaemic cell lines was also suppressed. When administered in vivo, CDA prolonged skin allograft survival in mice and caused suppression of specific (anti-sheep red blood cell, SRBC) antibody responses. Importantly, those in vivo effects were seen in both normal and presensitized host (animals which had rejected their primary skin transplant subjected to second grafting and mice re-challenged with the second dose of SRBC) (G6rski et al., 1993). Those data point to the potential role of CDA in transplantation. Therefore, we assayed the effects of the drug on alloantigen-elicited T cell proliferative responses in the Mixed Lymphocyte Reaction (MLR), an established in vitro model of allograft immunity (Milford, 1992). In addition, the results of our work cited above suggested that the B cell reactivity may be particularly susceptible to CDA. Thus, lower drug concentrations suppressed normal B cell proliferation, proliferation of a B cell line, terminal B cell proliferation to Ig secreting cells and Ig secretion than the concentrations needed to inhibit proliferation of normal and leukaemic T cells. To verify the hypothesis that indeed B cell activation may be more sensitive to the inhibitory action of CDA than T cell responses, we have compared the inhibitory effects of the drug on the expression of B cell and T cell activation markers.

Materials and Methods

Cell &olation Mononuclear cells (MNC) were isolated from heparinized blood of normal donors by FicollIsopaque gradient centrifugation. T and B cell preparations were separated from the same starting population of MNC. T cell-enriched fraction (>90~o C D 3 + cells, +5Yo m o n o c y t e s ) w a s obtained by MNC passage through nylon wool columns. To obtain B cell enriched fraction ( > 90Yo CD19 + cells) T cells were depleted by SRBC-rosetting and subsequent gradient centrifugation on Ficoll-Isopaque followed by monocyte depletion by plastic adherence. The purity of

199 T and B cell fractions was characterized by staining with monoclonal antibodies (anti-CD3 and anti-CD19, respectively, obtained from Becton-Dickinson), followed by anti-mouse Ig, (Fab')z-fragment (Dakopatts) and immunofluorescence examination, as described below. Mixed Lymphocyte Reaction (MLR) (MLR) cultures were established by coculturing 10s M N C with an equal number of irradiated (1500 r, U N P Cs 137, CELOR, Warsaw) allogeneic MNC in round-bottomed microtiter plates (Nunc) in medium RPMI 1640 with 10~o inactivated pooled human serum, 0.3 mg/ml L-glutamine and 25/~g/ml gentamicin. On day 5 of incubation the cultures were labelled with [3H]thymidine, harvested 16 h later and radioactivity assessed with the aid of a liquid scintillation counter (Betaszint, Germany). Activation antigens Purified lymphocyte fractions were cultured in medium RPMI with 107o fetal calf serum and other components listed above in the presence of appropriate activators. T cells were stimulated with phytohaemagglutinin (PHA, 1 gg/ml (Welcome) of phorbol myristate acetate, 10 ng/ml (PMA, Sigma) and B cells with Staphylococcus aureus Cowan (SAC, Calbiochem; an IgG crosslinking agent and a source of protein A), as described (Wa~sik et al., 1987). Following 72 h of culturing with PHA and SAC and 24 h with PMA (predetermined optimal timing) the number of cells positive for activation markers (HLA-D, CD25 and CD69 for T cells, CD25 for B cells) was determined by immunofluorescence (Pluta et al., 1990). The cells were washed and incubated with predetermined optimal concentrations of monoclonal antibodies against monomorphic determinant of human class II antigens (HLA-D, Becton-Dickinson), interleukin 2 receptor (CD25, Becton-Dickinson) and Activation Inducer Molecule (AIM, CD69, mAb TP 1/55, obtained from Dr F. Sanchez-Madrid, Universidad Autonoma, Madrid, Spain). Subsequently, the cells were stained with FITC-labelled anti-

mouse Ig, F(ab')z-fragment (Dakopatts). The specimens were examined using a fluorescent microscope equipped with epi-illumination (Jenalumar, Germany). 200 cells were counted on each triplicate slide prepared from a given cell sample and the results were averaged. Background staining (~o positive cells in control samples stained with normal mouse Ig and FITC-labelled anti-mouse Ig (not exceeding 1 7O) was subtracted to give the final readouts. The coefficient of variation between replicate counts did not exceed 15 ~oCladribine CDA (obtained from Foundation for the Development of Diagnostics and Therapy, Warsaw (99~o pure by HPLC) was diluted in isotonic saline, aliquoted and frozen at -20 °C. Final concentrations were prepared in a complete tissue culture medium and added at start of cultures. All concentrations of the drug assayed did not change significantly lymphocyte viability in culture up to 1000 nM. Statistics Statistical evaluation of the data was performed using the non-parametric Mann-WhitneyWilcoxon test and Statgraphics software.

Results

Fig. 1 shows the effects of CDA on T cell proliferation induced by alloantigens in the MLR. Significant inhibition of response was seen with 10 nM CDA. The same drug concentration caused consistent inhibition of PHA-stimulated T cell proliferation, while a 1 nM concentration was efficacious in approx. 30}o donors tested (not shown). As depicted in Fig. 2, CDA caused variable effects on mitogen-induced expression of T cell and B cell activation markers. PMA-induced T cell activation measured as expression of CD69 was resistant to the effects of the drug. In contrast, CD3-dependent T cell activation (triggered

200 mately 50 ~o) inhibition of their activation evoked by 1 nM of CDA (drug concentration which did not cause any apparent effects on T cells). Moreover, the expression of the B cell activation marker was totally inhibited at 10 nM, whereas the expression of T cell activation markers was inhibited to only about 50~o of control by this and higher concentration (100 nM) of the drug.

100 80 60 0 0

40 2O 0

i

,

0.1

1

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10 nM

Fig. 1. Effect of CDA on T cell proliferation induced by alloantigens in the mixed lymphocyte reaction. Mean values of 9 experiments _+SEM. The asterisk indicates significant difference (p < 0.005) versus control.

by PHA) was sensitive to CDA in concentrations of 10 nM and higher. In fact, 10 nM CDA caused approximately 50 ~o inhibition of T cell activation measured by the acquisition of activation antigens by cultured T cells (CD25, HLA-D). Further increase in CDA concentration (to 100 nM) caused only slight additive inhibitory effect. Furthermore, B cells were fond to be more susceptible to the immunosuppressive effects of CDA. This hightened susceptibility of B cells to the agent was manifested as significant (approxi-

I

100 ~

Discussion

The findings presented in this communication confirm and extend our earlier data indicating that cladribine is a potent immunosuppressive drug (Gdrski et al., 1993). Thus, CDA inhibits T and B cell activation which is manifest by markedly decreased ability of those cells to express class II and interleukin 2 receptor in the presence of the drug. Those effects are caused by CDA concentrations readily achievable in vivo in man (Liliemark and Juliusson, 1991). Interestingly, the drug does not inhibit CD3-independent activation of T cells involving protein kinase C-mediated pathway. Such a pathway operates during T cell activation by PMA leading to CD69 synthesis and expression (Cebrian et al., 1989). It is noteworthy that cladribine appears to be

I I

• CD 69

o

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0 0.1

1

10

100 nM

Fig. 2. The influence of CDA on the acquisition of activation antigens by T cells stimulated with PHA (HLA-D and CD25 and P M A (CD 69). (mean values of five experiments _+ SEM) as well as B cells stimulated with SAC (CD 25) (mean values of four experiments +_SEM). The asterisk indicates significant difference (p<0.005) versus control. Control values were: for TpH AHLA-D: 31 + 3%, TpHA-CD25:35 -- 8%, TpMA-CD69: 47_+ 3%, BsAc-CD25:33 + 2};.

201 very efficacious inhibitor of B cell activation, as inhibition of B cell triggering is achieved using CDA concentration 10-fold lower than those needed to impair T cell responses. This is in agreement with our previous work where we found that B cell proliferation and differentiation was much more susceptible to the inhibitory action of the agent. Our data demonstrating the B cell system as the primary target of immunosuppressive activity of CDA may have some relevance to particularly high effectiveness of the drug in the treatment of B-lymphoid malignancies (hairy cell leukaemia, B-cell chronic lymphocytic leukaemia, Waldenstr~3m's macroglobulinaemia). However, at the present time we have no data available concerning the relation between the immunosuppressive activity of CDA observed in our experimental systems and the mechanisms mediating apoptosis in lymphoid cells, which are responsible for the antileukaemic activity of the drug. This point deserves further study because one cannot exclude that the dosing schedule of the drug required to cause optimal immunosuppression would differ from that suited to obtain the maximal response in lymphoproliferative diseases. The fact that a novel and efficacious anti-B cell agent may be emerging is particularly important given recent developments in transplantation, especially xenografting, where novel anti-B cell agents are urgently needed (Starzl, 1993). It may well be that CDA could be used in that clinical setting replacing cyclophosphamide. Furthermore, CDA could be of value in the prevention of anti-idiotypic and anti-isotypic antibody responses neutralizing the beneficial effects of monoclonal antibody therapy. Naturally, the potential applications of CDA in transplantation are much broader and could involve using CDA as a adjunct drug with cyclosporine. This combination may be particularly efficacious as an immunosuppressive regimen containing primarily anti-T cells (cyclosporin) and anti-B cell (CDA) agents. The potential use of CDA in clinical transplantation is strengthened by our data indicating that

at moderate doses it inhibits mixed lymphocyte reaction, and in vitro model of graft sensitization and rejection. It could be anticipated that cladribine, an agent with relative low toxicity and high activity will become an important component of novel regiments of immunosuppressive therapy.

Acknowledgements This work was supported by grant Nr 4 1758 91 01p/03 from Committee for Scientific Investigation (KBN). We thank Dr F. Sanchez-Madrid, Universidad Autonoma, Madrid, Spain, for his generous gift of TP 1/55 and other monoclonal antibodies.

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