Interleukin-2 receptor β chain as a possible target for low doses of mafosfamide

The 7-day cytotoxic lymphocytes (CTL) induced in mixed lymphocyte culture express only the chain of the interleukin-2 receptor (IL-2R). In the present study this fact has been confirmed in a murine semi-allogeneic system. The ability of low doses of mafosfamide (Mf) to affect IL-2-induced CTL proliferation has been demonstrated. It was also shown that IL-2 activated resting suppressor cells. The pretreatment of the suppressor cells with either monoclonal antibodies (mAbs) against the p75 chain of IL-2R, or with Mf abolished the suppressive effect of these cells. No restoration of the proliferative response occurred when the anti-IL-2Rα mAb had been used. Flow cytometry analysis of 7-day CTL was carried out with mAbs against the α and β chains of IL-2R. CTL treatment with Mf inhibited anti-IL-2Rβ mAb binding. It may be assumed that the anti-proliferative effects of Mf which have been demonstrated in this paper, were a result of blocking the IL-2R β chain.


Introduction
Interleukin-2 (IL-2) is a T-cell derived lymphokine which participates in the manifestation of the normal immune response. IL-2 exerts its biological activities b binding to a specific receptor on a target cell. The IL-2 receptor (IL-2R) is composed of at least three cell-surface proteins, designated as the cz-, [3-, and ,-subunits. Different combinations of these subunits can define a number of states with differing affinities for IL-2 at the cell surface. - The interaction of IL-2 with its multi-subunit receptor can be a target for drugs with immunomodulating activities. For adequate immunocorrection it is very important to know what kind of IL-2R chain is affected by the immunosuppressive drug. For example, it has been shown that lymphocytes treated in vitro with cyclosporin A (CsA) show significant inhibition of the p55 chain (cz subunit) of IL-2R. 1-Previously, our group has shown that the spleen cells from mice of various genotypes have a different susceptibility to the antiproliferative action of alkylating agents. 14 The different sensitivity of lymphoid cells from different strains of mice to mafosfamide (a synthetic analogue of the alkylating metabolites of cyclophosphamide), and to the antiproliferative effects of cyclosporin A,.
has been also demonstrated. These interstrain variations have not been related to the inhibition (C) 1995 Rapid Communications of Oxford Ltd of interleukin 2 (IL-2) release but depend on differences in the expression of the IL-2 receptor (IL-2R). On the basis of these experiments we considered that the J3 chain (p75) of IL-2R was a possible target for mafosfamide (Mf) when administered in doses which were relatively low but sufficient for the inhibition of lymphocyte proliferation.
To test this hypothesis we needed an experimental system where the lymphoid cells expressed only the [3 subunit of IL-2R. Two experimental systems were used.
It is known that amongst cytotoxic lymphocytes (CTL) there exists a subset which expresses the [3 chain but not the cz chain of IL-2R.
Our unpublished data show that within CTL induced in a mixed lymphocyte culture, both cz and [3 chains of IL-2R occur, but beginning on the seventh day of cultivation the cells express the p75 chain only. This seventh day CTL culture was used as the first experimental system.
It is now evident that CD8 + cells reveal a significantly greater number of binding sims for anti-p75 than for anti-p55 monoclonal antibodies (mAbs). 7 Cell populations of thymocytes and splenocytes with phenotype CD3 +, CD4-, CD8 + express IL-2RJ3. s These data support the idea that resting suppressor cells exist among normal spleen cells and express the 3 chain of IL-2R.
This idea formed the basis of the second experimental system. Immunosuppressive agents: Mafosfamide (Asta Z7654, Asta-Werke, Germany) and cyclosporin A (Sandimiun, Sandoz, Switzerland) were used as immunosuppressants.
Cell cultures: The isolation of lymphoid cells from murine spleen and the inhibition of Con Ainduced lymphocyte proliferation by mafosfamide (Mf) and cyclosporin A (CsA) have been described in our previous publications. 4 '9 Briefly, mice were killed by cervical dislocation. Lymphoid cells were isolated from the spleens, washed, and resuspended in RPMI-1640 medium (Flow tab., UK) supplemented with 10% horse serum, 2 x 10 -3 M HEPES, 2 mM t-glutamine, 2.8 x 10 -6 M 2-mercaptoethanol, and 20 tg/ml gentamicin. Inhibition of Con-A stimulation by mafosfamide or cyclosporin A was evaluated at six different concentrations within the dose ranges 0.1 30 tg/ml and 0.03 10 lg/ml, respectively. The cells were incubated in fiat-bottomed 96-well plates (Nunc, Denmark) with different concentrations of the drugs for i h at 37C in humidified atmosphere containing 5% CO2. Then the plates were centrifuged, the supernatants were washed away by Transtar-96 (Costar, USA) and fresh culture medium with Con-A was added. The control wells incubated without drugs contained a culture medium with Con-A or culture medium only. The cells were incubated for 72h, pulsed with 40 kBq per well of [3H]-thymidine 4h before the end of cultivation, harvested with a cell harvester, and counted by using a liquid scintillation Suppressor cells were activated by recombinant IL-2 (rlL-2) treatment ( Fig. l(b)). Spleen cells were incubated in the presence of 10 IU of rlL-2 (Sigma, USA) for 24 h. For the evaluation of the suppressor activity, rlL-2-treated cells were co-cultivated with the fresh isolated syngeneic spleen cells in a ratio of 1:1. Co-cultivation of normal cells with the cells incubated without rlL-2 was used as a control. The cell mixture was stimulated with Con A and incubated for 72 h. As a result, the fresh isolated cells co-cultivated with the cells incubated without rlL-2 showed a normal proliferative response to Con A. In contrast, the mixture of normal spleen cells and cells preincubated with rIL-2 demonstrated a significantl decreased response. The suppressor cell sensitivity to Mf was evaluated by pretreating the cells with different doses of the drug for an hour. Then cells were washed and cultivated in the presence of rlL-2 as described above.

Results
The influence ofMf and CsA on CTL: Mf strongly suppressed the response of 7-day CTL to IL-2. In cOntrast, CsA did not suppress IL-2 stimulated CTL; nevertheless, we have shown a strong inhibition of Con A-stimulated spleen cell proliferation at significantly lower doses. The inhibition of IL-2-stimulated CTL with Mf was carried out in the same dose range as the spleen cell proliferative response induced with Con A (Fig. 2). Thus, the cell sensitivities for DBA/2 and C57BL//6 mice were significantly higher than those for BALB/c and CC57BR (p < 0.05). It is obvious that this model reveals the same interstrain differences as the experiments with the freshly isolated Con A-stimulated spleen cells.
Blocking of the suppressor cell activity by mAbs and Mf: The ability of IL-2 to stimulate sup- tion of the proliferative response was demonstrated with anti-p55 mAb. This level was also increased after the pretreatment of suppressor cells with low doses of Mf. The restoration of the proliferative response was dose-dependent and strain-specific. Our data demonstrated that exposure of C57BL/6 cells to Mf at 0.1 l.tg/ml resulted in the maximum increase of lymphocyte proliferation. In BALB/c mice, a dose which ensured a maximum level of restoration was ten times higher than in C57BL/6 mice (see Fig. 4).
The influence ofMf on mAb binding with and chains of IL-2R: The results obtained in this study are shown in Fig. 5 Fig. 5(a)). In  incubation with rIL-2 for 24 h was demonstrated. These residual cells were also sensitive to Mf treatment ( Fig. 5(b)).

Discussion
Our results show that 7-day CTL express the [ chain but not the a chain of IL-2R (see Fig. 5). Thus, in this study cells that expressed only constitutive IL-2R were investigated. It was shown that the exposure of CTL expressing the [ chain of IL-2R to relatively low doses of Mf resulted in a strong inhibition of the proliferative response to IL-2, whereas these cells were insensitive to the action of CsA. The different sensitivity of CTL to Mf and CsA may be explained by the lack of a specific target for CsA on 7-day CTL. The appearance of the target (the p55 chain of IL-2R) on the fresh isolated spleen cells stimulated with Con-A makes these cells sensitive to the antiproliferative effect of CsA. The spleen cell response to ConA was inhibited with the same doses of Mf as was the CTL response to IL-2. The experiments showed that both these cell populations revealed the same interstrain variations (see Fig. 2).
Our data indicate the widely accepted opinion that the mechanism of action of alkylating agents is the result of DNA-DNA linkage, 2 but does not explain completely the mechanisms of drug activity. It seems that the aforesaid mechanism is important for the suppression of malignant cell proliferation with high doses of alkylating drug, when there is no other alternative but irreversible damage to cell reproduction. In our case, the alkylating drug plays the role of immune response modifier but not the role of crucial cytostatic. The present data indicate that Mf can not only damage the proliferative response of cells but can also restore it by means of suppressor cell inhibition (see Figs. 3 and 4), it being shown that the suppressor cell activation was carried out by IL-2RI3. These data help to explain the well-known fact that the suppressor cells are a most sensitive subset to cyclophosphamide. [21][22][23] It should be noted that the increase in the proliferative response was more visible in C57BL/ 6 than in BALB/c mice (see Fig. 4). Previously, it was shown that the constitutive IL-2R were expressed in BALB/c mice clearly, whereas their expression in C57BL/6 mice was poor. 15 These data explain why Mf was able to affect cell suppressor activity in C57BL/6 to a higher degree than in BAtB/c mice.
In conclusion, our data suggest that the antiproliferative effect of Mf on lymphocytes activated with alloantigens or mitogens is a result of blocking the IL-2R 3 chain. Thus, an additional mode of action of alkylating drugs has been shown.