Differential influence of 2′-deoxyguanosine on the induction and expression of suppressor T lymphocytes in vivo

CELLULAR

IMMUNOLOGY

90,

531-538 (1985)

Differential Influence of 2’-Deoxyguanosine on the induction and Expression of Suppressor T Lymphocytes in Viva H. BRIL, TH. W. VAN DEN AKKER,

L. M. HUSSAARTS-ODIJK,

AND R. BENNER

Department of Cell Biology and Genetics, Erasmus University, 3000 DR Rotterdam, The Netherlands Received July 30, 1984; accepted September I 9, I 984 Subcutaneous (SC)immunization of mice with allogeneic spleen cells can induce delayedtype hypersensitivity (DTH) to histocompatibility antigens. Intravenous immunization with irradiated allogeneic spleen cells, on the other hand, induces suppressor T (Ts) lymphocytes. These Ts cells are capable of suppressing the host-versus-graft (HvG) DTH reactivity which normally arises after sc immunization. Moreover they can suppress the development of antihost DTH effector T cells during graft-versus-host(GvH) reactions. These models for HvG and GvH DTH reactivity were used to study the influence of 2’deoxyguanosine (dGuo) on the induction, further development, and expression of Ts cells in viva. It was found that administration of dGuo inhibits the proliferation-dependent induction and further develop ment of Ts cells, but not the suppression mediated by already activated Ts cells. o 198s Academic Press, Inc.

INTRODUCTION

The expression of immunodeficiency in patients with adenosine deaminase and purine nucleoside phosphorylase defects indicates a crucial role of the purine metabolism in the acquisition and expression of normal immune function (1, 2). A number of hypotheses have been put forward to relate the lymphocyte dysfunction to the biochemical abnormalities. The most current of these hypotheses ascribes a cardinal role to the accumulation of nucleotides [i.e., deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP)] in T lymphocytes, which results in an inhibition of the enzyme ribonucleotide reductase and subsequent inhibition of DNA synthesis (3). The group of Gelfand has described that the antigen-induced human suppressor T (Ts)-cell activity in vitro (4), as well as the in vivu-generated murine Ts-cell activity (5), can be abrogated by micromolar concentrations of 2’-deoxyguanosine (dGuo), but not by guanosine (Guo). Helper T cells that do not proliferate and precursor B lymphocytes, on the other hand, were found to have a more than lOOO-fold higher resistance to dGuo. Previously, we have reported (6) that dGuo, but not Guo, inhibited the generation of murine Ts-cell activity but not the generation and expression of proliferation-dependent delayed-type hypersensitivity (DTH) reactions. We also showed that dGuo acts via a direct effect on Ts cells and not indirectly, via contrasuppressor cells. In that study our attention was focused especially on the influence of dGuo on the induction of Ts cells and DTH-reactive T cells. The 531 0008-8749185$3.00 Cowright All xi&u

8 1985 by Academic Press, Inc. of reproduction in any fm reserved

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purpose of the present study was to investigate the influence of dGuo on the induction, further development, and expression of Ts cells, separately. This was done primarily under graft-versus-host (GvH) conditions becausethe different phases of generation of Ts-cell activity, i.e., induction, further development, and expression, can be studied separately in this model. MATERIALS AND METHODS Mice. (C57BL/Rij X CBA/Rij)Fl (H-26/“) and BALB/c (H-2d) female mice, 10 to 20 weeks old, were bred at the Laboratory Animals Centre of Erasmus University, Rotterdam. DBA/2 (H-8 female mice, 10 to 16 weeks old, were purchased from Bomholtgard, Ry, Denmark. BALB.B (H-26), BALBK (H-29, and (BALB/c X BALB.K)Fl (H-2@? female mice, 10 to 16 weeks old, were purchased from OLAC Ltd., Bicester, United Kingdom. Preparation of cell suspensions. The procedure for making cell suspensions has been described in detail in previous papers (8, 9). Host-versus-graft reaction (HvG). Responder mice were subcutaneously (SC)immunized with 1 X lo7 unirradiated nucleated allogeneic spleen cells, suspended in a volume of 0.1 ml BSS. These cells were equally distributed over both inguinal areas. In previous papers (8, 10) we have shown that immunization with H-2 and non-H-2 alloantigens according to this procedure induces maximal DTH responses. Graft-versus host reaction. Acute GvH reactions were elicited by intravenous (iv) injection of 1 X lo7 nucleated spleen cells into lethally irradiated allogeneic mice within 4 hr after irradiation. The cells to be injected were suspended in a volume of 0.5 ml BSS. Induction of suppression. Ts cells were induced by a single iv injection of 5 X lo7 irradiated (20 Gy) allogeneic spleen cells 4 days before using these mice as donors of spleen cells (11, 12). Control mice had been injected with the same number of irradiated syngeneic cells. Drug treatment. 2’-Deoxyguanosine, No. D-9 125, grade II, was purchased from Sigma Chemical Company, St. Louis, Missouri. Guanosine, No. C- 136, was purchased from Koch-Light Laboratories Ltd., Colnbrook, United Kingdom. Experimental mice received a daily intraperitoneal (ip) injection of 1 mg dGuo. Hydroxyurea (HU) was purchased from Calbiochem-Behring Corporation, La Jolla, California. Experimental mice received three ip injections of HU (1 g/kg body wt) in 0.5 ml of a balanced salt solution (BSS) 6 hr apart. SeeHodgson et al. (13) for details. Control mice received similar injections of BSS only. Assay for delayed-type hypersensitivity. The DTH assay for measuring HvG and GvH immune reactivity has been described in detail in previous papers (8, 10). HvG DTH responseswere elicited in previously immunized mice (see above) by sc injection of a challenge dose of 2 X lo7 unirradiated allogeneic spleen cells into the dorsum of the right hind foot. The DTH response to this challenge was measured as the difference in thickness of the hind feet 24, 48, and 72 hr later. In the figures, the 24-hr values are presented. The DTH responses at 48 and 72 hr were in harmony with those at 24 hr, but lower. The specific increase in foot thickness was calculated as the percentage increase in foot thickness of the immune mice minus the percentage increase in foot thickness of control mice which only received the challenge. The increase in foot thickness of these challenged control mice varied between 15 and 25%.

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For measuring the anti-host DTH reactivity under GvH conditions, a number of cells equivalent to the total cell yield obtained from spleen, inguinal, axillary and mesenteric lymph nodes from an irradiated and reconstituted recipient mouse were transferred iv into a normal secondary recipient syngeneic to the original spleen donor mouse 5 days after reconstitution. Thirty minutes before transfer, the secondary recipient mice were ip injected with 15 U heparin (Liquemine, HotfrnannLaRoche & Co. Ltd., Basel, Switzerland) to prevent embolism. The secondary recipient mice were challenged into the dorsum of the right hind foot with 2 X 10’ unirradiated spleen cells, syngeneic with the irradiated recipients. The subsequent DTH response was measured and calculated as described above for HvG DTH responses.Figure 1 shows the set-up of the assay for measuring the anti-host DTH response under GvH conditions. RESULTS Influence of dGuo on the Induction of Ts Cells Ts cells were induced by the protocol that previously has shown to be effective in suppression of HvG and GvH immune responses(11, 12). Thus, DBA/2 donor mice were suppressedby means of an iv injection of irradiated (C57BL X CBA)Fl spleen cells. Control DBA/2 mice received an iv injection of irradiated syngeneic spleen cells. Subsequently, both groups of mice received four daily injections of 1 mg dGuo, an appropriate dose to abrogate suppression (6). On the fifth day the spleen cells of these mice were used to elicit GvH reactions in lethally irradiated (C57BL X CBA)Fl recipients in order to evaluate the effect of dGuo on the Ts cells. Five days after irradiation and reconstitution, the anti-host DTH reactivity was determined as described under Materials and Methods. Figure 2 shows that dGuo treatment of spleen cell donors did not affect the development of anti-host DTH. The iv-induced suppression, on the other hand, was abrogated by this dose of dGuo. Three injections of suppressed DBA/2 mice with 1 mg dGuo 1 day before their use as donors of spleen cells to reconstitute (C57BL X CBA)FI recipients partially abrogated the iv-induced suppression in these donor mice (Fig, 3). Treatment of the

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534 I.V.

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FIG. 2. Influence of dGuo treatment of the allogeneic spleen cell donors upon the anti-host DTH reactivity in lethally irradiated (C57BL X CBA)Fl mice. (C57BL X CBA)Fl mice were inoculated with 1 X 10’ suppressed, 1 X 10’ nonsuppressed, or 1 X 10’ suppressedplus 1 X 10’ nonsuppressed DBA/2 spleencells, respectively. Anti-host DTH reactivity was determined 6 days after irradiation and reconstitution. The interval between induction of suppression in the donors and their use for reconstitution was 4 days. The dGuo treatment consisted of 1 mg per mouse per day. Anti-host DTH reactivity was determined on the sixth day after irradiation and reconstitution. Values represent the arithmetic mean of the DTH response -+ 1 SEM (n = 6).

suppressed donor mice with three injections of HU one day before reconstituting Fl recipients, which is known to kill all proliferating cells, completely abrogated the suppression, indicating that the Ts cells were proliferating at this time point (Fig. 3). The Injluence of dGuo on the Further Development of Suppressor T Cells under Both GvH and HvG Conditions The influence of dGuo on the further’development of already activated suppressor T cells was studied in mice subjected to GvH. Suppressed donors were used to

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FIG. 3. Influence of dGuo and HU treatment of suppressedand nonsuppressedspleen cell donors upon the anti-host DTH reactivity in lethally irradiated allogeneic mice. DBA/Z mice were iv preimmunized (“suppressed’) 4 days before their use as donors to reconstitute. lethally irradiated (C57BL X CBA)Fl mice. On the day Before their use as donors the suppressedas well as the nonsuppressed mice received three consecutive injections of 1 mg dGuo in BSS, HU (I gr/kg BW), or BSS only, 6 hr apart. For other experimental details, see legend to Fig. 2.

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reconstitute the irradiated allogeneic recipients; 4 hr after reconstitution the recipient mice received their first ip injection of dGuo, followed by a daily injection for 4 days. Other groups of irradiated allogeneic mice that were reconstituted with spleen cells from nonsuppressed donors were used as controls and received either dGuo or HU. The purpose of the HU treatment, which was performed on the second day after reconstitution, was to evaluate the proliferative activity of the T cells involved in DTH. As can be seen in Fig. 4, a marked anti-host DTH response was found after treatment with dGuo in the nonsuppressed group (second bar). After treatment with HU (third bar), marginal anti-host DTH reactivity was found, indicating that the DTH-reactive T cells were proliferating at the moment of treatment. Irradiated mice that had been reconstituted with spleen cells from suppressed mice and were treated with dGuo displayed a substantial anti-host DTH reactivity, in contrast to the BSS-treated control group (Fig. 4, fifth and fourth bars, respectively). The effect of dGuo on the proliferation and activity of Ts cells under HvG conditions was studied in DBA/2 mice that were iv injected with H-2 and non-H-2-incompatible (C57BL X CBA)Fl spleen cells. After 4 days, spleen and lymph node cells from these DBA/2 mice were transferred to syngeneic DBA/2 mice. Subsequently, these syngeneic DBA/2 mice were sc immunized with Fl spleen cells, daily ip injected with dGuo, and challenged with Fl spleen cells 6 days after the sc immunization. It was found (Fig. 5) that the dGuo treatment clearly inhibited the suppressor activity. Apparently dGuo selectively inhibits not only the induction of Ts cells but also their further development under GvH and HvG conditions. Influence of dGuo on the Expression of Suppressor T Cells In order to determine the sensitivity of Ts cells for dGuo, which had undergone activation and further development (see above), we made use of the phenomenon of bystander suppression that recently has been described by our group (14). Thus, after SCimmunization of “suppressed” mice with a combination of alloantigens comprising the antigen(s) used to induce the Ts cells as well as third party alloantigens, the DTH reaction against the third party alloantigens was effectively suppressed. This bystander suppression also occurs during the expression phase of DTH. Thus, after iv preimmunization with a particular alloantigen and induction

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FIG. 4. Influence of dGuo and HU treatment of mice subjected to GvH upon the anti-host DTH response. GvH was induced in lethally irradiated (C57BL X CBA)Fl mice by inoculation of 1 X 10’ spleen cells from either suppressed or nonsuppressed DBA/Z mice. Irradiated, reconstituted mice were treated with 1 mg dGuo in BSS or BSS only on Day 0, 1, 2, 3, and 4 after reconstitution. HU was given on Day 2 after reconstitution only. On Day 5 all mice were tested for anti-host DTH reactivity. For other experimental details, see legend to Fig. 2.

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FIG. 5. Influence of dGuo treatment on the further development of Ts-cell activity under HvG conditions. Suppressedand nonsuppressedspleen and lymph node cells were obtained from DBA/2 mice iv injected with 5 X 10’ irradiated (C57BL X CBA)Fl or syngeneic DBA/Z spleen cells 4 days earlier. The DBA/Z spleen and lymph node cells were iv transferred to syngeneic DBA/Z mice which were ip injected with 1 mg dGuo 4 hr before the transfer of the cells and daily during the following 6 days. Immediately after the transfer of the cells, the DBA/2 mice were sc immunized with 1 X IO’ (C57BL X CBA)Fl spleen cells. All mice were challenged with (C57BL X CBA)Fl spleen cells 6 days after sc immunization. All values represent the arithmetic mean of the DTH response + 1 SEM (n = 6).

of DTH effector T cells to a different alloantigen (e.g., during an acute GvH reaction), the DTH response can be completely suppressedby combining both sets of alloantigens in the inoculum used for challenge (A. T. J. Bianchi et al., in preparation). To determine the sensitivity of already activated Ts cells for dGuo, BALB.B mice were iv injected with irradiated BALB.K spleen cells in order to induce anti-BALB.K Ts cells. Subsequently, these BALB.B mice were used as donors of spleen cells to induce anti-BALC/c DTH-reactive T cells in irradiated BALB/c recipients. Five days later the anti-BALB/c immune reactivity was determined in dGuo-treated or BSS-treated BALB.B secondary recipients. This was done by challenging with spleen cells from (BALB/c X BALB.K)Fl mice. It was found that dGuo treatment of the secondary recipients did not influence the expression of Ts-cell activity (Fig. 6, first bar). DISCUSSION In this paper we show that administration of dGuo inhibits the proliferationdependent induction and further development of Ts cells, but not the expression phase of Ts-cell activity. As we showed previously (6), DTH-reactive T cells, the majority of which are probably identical to helper T cells (7), show a lOOO-foldhigher resistance to dGuo than do Ts cells. The same data were reported by Gelfand et al. for helper and Ts cells (4). It was concluded in that study that all T-cell proliferative events are susceptible to dGuo toxicity. These authors explained the differential effect of dGuo on helper and Ts cells by assuming that helper T cells, in contrast to Ts cells, do not need to proliferate in order to become functionally active. In our model, the induction and further differentiation of DTH-reactive T cells is dependent on proliferation (15, 16) and insensitive to daily doses of dGuo as high as 1 mg per mouse (6). On the other hand, the induction and further differentiation of Ts cells induced according to our protocol is also proliferation dependent (17). However, this process is sensitive to 1 mg dGuo. Furthermore, the expression phase of both Ts-cell activity and DTH-reactive T-cell activity is proliferation independent and not susceptible to dGuo. Thus we conclude from our studies that all Ts-cell proliferative events, but not the helper-T-cell proliferative events, are susceptible to

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FIG. 6. Effect of dGuo on activated Ts cells. BALB.B donor mice were suppressedby iv injection of irradiated BALB.K cells or received, as a control, BALB.B spleen cells. Four days after induction of suppression, 2 X 10’ spleen cells were transferred to irradiated BALB/c mice. Five days after reconstitution the spleen and lymph node ceils of the irradiated recipients were transferred to secondary BALB.B recipients, which had been treated for 3 days with 1 mg dGuo or BSS. These secondary recipients were challenged with (BALB/c X BALB.K)FI spleen cells. For other experimental details, see the legend to Fig. 2.

dGuo toxicity. Starting with the hypothesis mentioned in the Introduction, these data suggestthat DTH-reactive T cells and Ts cells have a different enzymatic make up with regard to the purine metabolism, resulting in inhibition of DNA synthesis by the accumulation of dGTP in Ts cells but not DTH-reactive T cells and helper T cells. Cloned mm-me helper and Ts cells seem to be the appropriate material for studying purine enzyme activities and accumulation of purine metabolites after administration of dGuo. With respect to differences between induction and expression phases of Ts-cell activities, similar results have been reported by Varey et al. (18). They found that the induction of the, probably, proliferationdependent Ts-cell activity in viva was sensitive to dGuo administration, whereas the in vitro, probably, proliferationindependent expression of Ts-cell activity was not. The validity of the hypothesis mentioned in the Introduction has been questioned by several recent studies. Martineau and Willemot (19) found in murine in vitro studies that mitogen-induced B-cell proliferation was at least as susceptible to inhibition by exogenous deoxyguanosine as T-cell proliferation. However, concomitant addition of deoxycytidine did not modify the inhibition by deoxyguanosine. Recently, Spaapen et al. (20) reported that human B-lymphocyte differentiation in vivo can be inhibited by dGuo, starting with degradation of dGuo by PNP, followed by guanosine salvage by HGPRT and possibly further phosphorylation of GMP into GDP and GTP. PNP-deficient B cells were shown to escapedGuo intoxication because of their lack of accumulation of GMP, GDP, and GTP. Spaapen et al. (21) also showed the involvement of two pathways contributing to dGuo-mediated inhibition of the proliferation of normal human lymphocytes, i.e., on the one hand, dGuo degradation by PNP, salvage of guanine by HGPRT, and possibly phosphorylation of GMP to GTP and, on the other hand, formation of dGTP by direct phosphorylation of dGuo by deoxycytidine kinase. Studies of Goday et al. (22) indicated that human B cells as well as T cells form deoxynucleotides from deoxyadenosine or deoxyguanosine. Since their T cells were derived from T-cell lines, which are mostly of malignant origin, and their B cells were derived from malignant B-cell lines, they suggest that the accumulation of high levels of deoxynucleotides in vitro are not dependent on the origin of the cells (T or B), but on whether they are malignant or not. In all lines they found a larger GTP than dGTP

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accumulation, supporting the above-mentioned pathway of dGuo degradation by PNP, salvage or guanine by HGPRT, and phosphorylation of GMP to GTP. It has recently been shown in rats (23) that macrophage-mediated suppression of the proliferative response of thymocytes to concanavalin A can be abolished by low concentrations of dGuo. The mode of action of dGuo on macrophages is unknown, but inhibition of ribonucleotide reductase by accumulation of dGTP is clearly not the case, because macrophages cannot accumulate dGTP due to their low dGuo kinase activity. In our systems, suppressor macrophages do not play a significant role. Anti-Thy-l plus complement treatment completely abolished the induction of suppressor cells, whereas the expression phase of suppressor activity was dependent on suppressor cells expressing Thy- 1 and Lyt-1 as well as Lyt-2 surface antigens (Bianchi et al., submitted). ACKNOWLEDGMENTS We gratefully acknowledge Mrs. M. Stout for typing the manuscript and the Dutch Kidney Foundation for their financial support.

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