The effect of immunosuppressive agents on lymphocyte subsets in rat peripheral blood

Int. J. lmrnunopharmoc,,

Vol. 9, No. 7, pp. 747 - 759, 1987.

(1192 (1561/87 $3.(RI4 .00 ..: 1987 International S~ciety for lmmunopharma,:ology,

Printed in Great Britain.

THE EFFECT OF I M M U N O S U P P R E S S I V E A G E N T S ON L Y M P H O C Y T E SUBSETS IN RAT P E R I P H E R A L BLOOD S. C. THOMPSON*, K. M. BOWEN~ a n d R. C. BURTON* *Discipline of Surgical Science, Faculty of Medicine, University of Newcastle, Newcastle, N.S.W. 2308; 'Department of Endocrinology, Royal Newcastle Hospital, Pacific Street, Newcastle, N.S.W. 2300, Australia (Received 16 April 1986 and in final form 17 November 1986).

Abstract - - A method for monitoring circulating lymphocyte subsets in the rat on an automated flow cytometer with monoclonal antibodies was used to ascertain in vivo effects of various doses of immunosuppressive agents. The agents tested were anti-lymphocyte serum (ALS), azathioprine (AZA), cyclosphosphamide (CTX), cyclosporin A (CsA) and methylprednisolone (MP). Each immunosuppressive agent varied in its capacity to induce changes in T cell subsets and B cell numbers. The rapidity of onset of action of the agents varied considerably; with ALS and MP maximal effects were seen within hours whilst the effects with CsA, cyclophosphamide (CTX) and azathioprine (AZA) took several days to develop. At.S had marked anti-T cell activity but did not selectively affect the T cell subsets. AZA and CTX both exerted their major effect upon the B cell (OX4") subpopulation. CsA administration was associated with the appearance of many circulating lymphocytes which expressed the pan-T marker (W3/13) but neither of the T cell subset markers (W3/25, OX8). With CsA there was no significant alteration in the W3/25:OX8 ratio, although a persistent decrease in the number of all T lymphocytes was observed after administration of this drug at a dose of 45 mg/kg had ceased. MP was the only drug which had a marked selective effect on a T cell subset. The numbers of circulating Class 11 major histocompatibility complex (MHC) reactive lymphocytes (W3/ 25" ) were significantly more depressed than the Class I MHC reactive subset (OX8"). This effect persisted fk~r up to 31 days after the single injection of a depot preparation of this drug, and was found to be associated with prolonged survival of precultured endocrine xenografts.

I m m u n o s u p p r e s s i v e agents have been used to modify the host response in a n u m b e r of clinical a n d experimental situations a n d have proved to be o f particular value when used to suppress the host allograft response after o r g a n t r a n s p l a n t a t i o n . U n f o r t u n a t e l y , these agents have adverse side effects when used to prevent a n d / o r reverse allograft rejection a n d when the graft recipient is in a precarious i m m u n e state, where u n d e r s u p p r e s s i o n can result in allograft loss a n d oversuppression in fatal infection. It is therefore i m p o r t a n t to optimize the dosage o f these drugs, whether used singularly or in c o m b i n a t i o n . The key role o f T (thymus derived) lymphocytes in the rejection o f t r a n s p l a n t e d tissue in m a m m a l s has been recognized for over a q u a r t e r of a century. O n e o f the two m a j o r T cell subsets, the Class II m a j o r h i s t o c o m p a t a b i l i t y complex ( M H C ) reactive (T

h e l p e r / i n d u c e r ) subset has been s h o w n to play a m a j o r role in allograft rejection and it follows that allograft survival m a y be p r o l o n g e d if the function of this s u b p o p u l a t i o n o f lymphocytes could be selectively impaired or suppressed. Therefore, two i m p o r t a n t aims o f current research in the area are to find more selective i m m u n o s u p p r e s s i v e drugs a n d to develop practical m e t h o d s of m o n i t o r i n g the biological effects o f i m m u n o s u p p r e s s i v e agents in allograft recipients. A successful simple m o n i t o r i n g technique would allow the dosages a n d frequency o f a d m i n i s t r a t i o n o f these drugs to be c o n t i n u o u s l y optimised for prevention o f allograft rejection. It is now possible to gain specific i n f o r m a t i o n regarding the selective effects o f i m m u n o s u p p r e s s i v e drugs o n i m m u n o c o m p e t e n t peripheral b l o o d T a n d B l y m p h o c y t e p o p u l a t i o n s by the use o f flow

Correspondence should be addressed to: Professor R.C. Burton, Discipline of Surgical Science, Faculty of Medicine, University of Newcastle, Shortland, N.S.W. 2308, Australia. 747

748

S.C. TttOMeSONet al.

cytometric monitoring with monoclonal antibodies. The potential of this approach is that it makes possible immunosuppressive therapy directed towards the destruction or inactivation of particular subsets of lymphocytes. This present study was designed to determine whether a technique we developed for monitoring of peripheral blood lymphocyte subpopulations in the rat (Thompson, Bowen & Burton, 1986) could be used to determine the effects of a number of immunosuppressive agents on circulating T and B lymphocytes, and might therefore be useful to allow the dosages and frequency of administration of these drugs to be continuously optimized for prevention of graft rejection. Further, we wished to determine whether any of these drugs showed selective effects on the Class I1 MHC reactive T lymphocyte subpopulation.

EXPERIMENTAL PROCEDURES

Male inbred Wistar rats aged between 8 and 20 weeks at the time of commencement of each experiment were obtained from the University of Newcastle animal house. Tail vein blood was collected into heparinised syringes while the animals were lightly anaesthetized with ether and the blood thus obtained was prepared for lymphocyte subset analysis using the following monoclonal antibodies (obtained from Commonwealth Serum Laboratories (CSL) Melbourne, Victoria): W3/13, (mature peripheral blood T cells); W3/25 (Class II MHC reactive T lymphocytes many of which have helper/ inducer function); OX8 (Class I MHC reactive T cells, many of which have cytotoxic/suppressor function), and OX4 (La positive cells such as B ceils, activated 7" lymphocytes and monocytes). The staining method has been described in detail previously (Thompson e t a l . , 1986), but briefly, 80/al of whole peripheral blood was reacted with I0 /A of monoclonal antibody and incubated for 15 - 30 min on ice. Red ceils were lysed in an ammonium chloride buffer (pH 7.35) for 7 - 10 min and then the cells were washed in phosphate buffered saline (PBS) before reaction for 15--30 rain at 4°C with a fluorescein conjugated (Fab'2) fragment goat antimouse IgG (Fc fragment) gamma-chain specific antibody (Cappel Laboratories, West Chester State, AP). After washing twice in PBS, ceils were resuspcnded in 1~.'0 paraformaldchyde in PBS and then positively stained cells were analysed using the Spectrum 11I automated flow cytometer (Ortho Diagnostic Systems, Raritan, JN), with the

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Fig. 1. Cytogram of rat peripheral blood leukocytes. The electronic gate shown encloses the lymphocyte cluster as defined by forward angle light scatter (Fw-Sc) vs right angle light scatter (Rt-Sc) on a Spectrum III flow cytometer analysis of rat peripheral blood following lysis of the erythrocytes lysed using a Spectrum ii1 flow cytometer. The monocyte cluster is above and the polymorph cluster to the far right of this gate. Red cell debris and platelets cluster near the origin and can be seen just above the electronic cut off for background noise. lymphocyte gate set as shown in Fig. 1. This electronic gate selectively encloses lymphocytes, and since activated T lymphocytes are rarely found in peripheral blood (Taniguchi, Miyawaki, Yachie, Ikuta 8,: Ohzeki, 1983), we have used OX4 as a marker for B lymphocytes. Baseline subset monitoring was performed on at least one occasion prior to the injection of the immunosuppressive agent to be studied into the animals of the test group. Both test and control groups of non-drug treated rats were subjected to the same bleeding protocol and monitored concurrently. Where drugs were given on a daily basis, bleeding was always performed prior to administration of the drug. D o s e response curves were constructed following investigation with the immunosuppressive drugs described below. The range of dosages selected for each drug was within and greater than those previously shown by other investigators to be appropriate for immunosuppression in the rat (Freircich, Gehan, Rail, Schmidt & Skipper, 1966; Kruckenberg, 1979). Rabbit anti-rat lymphocyte serum (AI.S) was obtained from Microbiological Associates (Bethesda, Maryvillc, l.ot No. 40642) and was

749

The Effect of Immunosuppressive Agents injected at doses of 1.0, 2.5, 5.0 and 7.5 ml/kg. Cyclophosphamide (CTX) (Bristol Laboratories, Crows Nest, N.S.W.) was administered over the dose range 5 - 300 mg/kg as a single i.p. injection on day zero. Azathioprine (AZA) (Wellcome, London, U.K.) was administered by intraperitoneal injection over the range 5-100 mg/kg. Initially, monitoring was carried out following a single injection, but subsequently monitoring was carried out following the daily administration of 2.5, 5 or 10 mg/kg AZA for a total of 10 doses. Methylprednisolone (MP) was given by intramuscular injection in two formulations, (Upjohn, Kalamazoo, Mi), methylprednisolone sodium succinate (Solu Medrol; SM) and a depot release preparation methylprednisolone acetate (Depo-Medrol; DM). SM was administered at a single dosage of 16 mg/kg IM, and the lymphocyte subset numbers analysed at 2, 5, 10, 24 and 48 h compared to rats which received a single dose of 16 mg/kg DM at time 0 and were monitored concurrently. An additional study was performed where animals were bled over a period whilst they received either ten daily injections of SM (16 mg/kg) or following the single administration of DM at a dose of 0, 2, 4, 8, 16 mg/kg on day zero. Cyclosporin A (CsA) was a gift from the Sandoz Company (Basel, Switzerland). It was dissolved in olive oil and administered by gastric lavage on a daily basis for a total of eight doses at a dose of 0, 15, 30 or 45 mg/ kg/day. Absolute lymphocyte counts (ALC) were determined using a Coulter S plus I1 counter and the number of a particular subset calculated by multiplying the ALC with the fraction of cells in the gated lymphocyte region stained with the appropriate monoclonal antibody. The absolute number of circulating lymphocytes is a direct measure of the recirculating pool, and thus significant alterations in particular circulating T lymphocyte subset numbers are likely to have immunological significance. In contrast, whilst the percentage of blood iymphocytes reactive with a particular MoAb may give valuable information about the differential effects of various doses of a drug, it does not give an accurate estimate of the recirculating pool. This is because the percentage could vary widely yet the re-circulating pool remain unaltered if the absolute number of circulating iymphocytes also simultaneously varied. Thus, the results reported in this paper are presented as absolute numbers of lymphocytes expressing a certain marker. Results are shown so that the data points on the graphs are the means for groups of animals (four to

seven rats per group). Standard deviations have been omitted for the sake of clarity. Statistical significance between groups was determined using the M a n n - W h i t n e y U-Test, and is shown as an asterisk (*) where statistical significance was P< 0.05 or less.

RESULTS

Anti-lymphocyte serum Wistar rats weighing 180- 200 g were bled at time zero and then five were injected with 1 ml ALS (5 ml/kg) intraperitoneally (i.p.) and 5 with normal saline (i.p.). T-cell subsets were monitored at 3, 6, 10 and 20 h post-injection. The absolute lymphocyte count fell markedly by 3 h with both the T cell (W3/ 13") (P< 0.05) and B cell (OX4') (P value not significant) populations being affected. The effect on T cells was much more profound. Furthermore, both the W3/25" and OX8" subpopulations of T cells were similarly affected, so that no statistical difference in the W3/25:OX8 ratio between test and control animals was seen at any time (data not shown). By 20 hours, B cell numbers had recovered, but the T cells had not (P< 0.05). The effect of ALS dosage on lymphocyte subset population was further investigated by monitoring the effects in four groups of animals (5 rats/group) which received a single injection of 0 (PBS control), 2.5, 5.0, or 7.5 mls/kg of ALS i.p. Essentially similar results were obtained at all three doses of ALS, with a profound T cell lymphopenia affecting both the W3/25' and OX8 ° T cell subpopulations within 6 hours and persisting for at least 9 days (Fig. 2) (P< 0.05). The effect on B cells was again only partial and transitory.

Azathioprine A pilot study where azathioprine was injected i.p. as a single dose of 0 (PBS control), 5, 10, 20 or 50 mg/kg demonstrated no effect on circulating lymphocyte subsets in the week following drug administration (data not shown). Since no effect was demonstrated even at a dosage of 50 mg/kg, (ten times greater than that usually given), AZA was administered on a daily basis for ten days at doses of 0 (PBS control), 2.5, 5.0 and 10 mg/kg/day to four groups of five rats. To put this in perspective, 2 - 3 mg/kg/day is the dose usually administered to rats in conjunction with corticosteroids to prevent allograft rejection (Thomas, Thomas, Mendez, Lower & Lee,

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1977). No significant differences between control and test animals were recorded until animals had received five doses of A Z A , when there was a significant decrease in the circulating number of OX4' lymphocytes (P< 0.05) in rats receiving 10 m g / k g A Z A / d a y (Fig. 3). This fall continued until the A Z A was ceased, when OX4' cell numbers gradually returned towards control values. There was no such effect on the T lymphocytes (Fig. 4), where total T lymphocyte and subset numbers were not significantly different to those of the controls, except for the 5 mg dose on day 5. The W 3 / 2 5 ' : OX8 ÷ ratio was not significantly different from the control group at any dose with the exception of the 10 m g / k g group which had a statistically significantly higher ratio (mean _+ S.D., 10 m g / k g

2.50 _ 0.28; controls, 2.17 -+_ 0.17, P< 0.04) on day 14.

Cyclophosphamide This drug was tested over the therapeutic and toxic dose ranges for the rat. Two out of two rats injected with a dosage of 300 m g / k g died at days 7 and 9 post injection, and one of two animals receiving 200 rag/ kg cyclophosphamide died 13 days post injection. T w o rats who received 100 m g / k g survived, but manifested conjunctivitis, loose stools and hair and weight loss. Therefore, a d o s e - r e s p o n s e study was performed using groups of five rats which received a single dose of 0 (PBS control), 5, 10, 20 and 50 rag/ kg CTX i.p. on day 0 (Fig. 5). This shows a dose-

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related effect maximally affecting lymphocyte subsets by day 4 post-injection, following which there was a gradual return towards control values. CTX produced a profound pan-lymphopenia so all lymphocyte subset numbers were depressed. On day 4, rats receiving 50 mg/kg had lymphocyte numbers which were 28070 for W3/13, 3207o for W3/25, 37070 for OX8 and 2.5070 for OX4 of the control rat lymphocyte numbers. By day 24 for rats receiving 50 mg/kg these had returned to 75070 for W3/13, 71070 for W3/25, 75070 for OX8 and 63070 for OX4 of the control rat lymphoctye numbers. At low doses (5, 10 mg/kg) of CTX, there was an initial increase in the W 3 / 2 5 : O X 8 ratio, due to a relatively larger depression of the O X 8 ' population, although this was not statistically significant. At higher doses (20, 50 mg/kg) the Class I1 MHC reactive T cell population was affected proportionately as much as

the Class I MHC reactive population so that the ratio was the same as the control.

Methylprednisolone Two different formulations of MP were compared. Initially, three groups of rats (4 rats/ group) which received PBS (control), 16 mg/kg methylprednisolone sodium succinate (Solu-Medrol; SM) or 16 mg/kg methylprednisolone acetate suspension (Depo-Medrol; DM) as a single I.M. injection. Animals were monitored six times during the first 24 h and then periodically until 41 days after injection. There was no statistical difference between cell populations of the control and two test groups at time 0. However, in the first 10 h after injection both methylprednisolone injected groups developed lymphopenia, selectively affecting the W3/25 + T cell population most, with relative sparing the OX8'

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cells so that there was a marked alteration of the normal W 3 / 2 5 : O X 8 ratio (Fig. 6). By 24 h the cell counts of SM treated rats had returned to control values. In contrast, beyond 24 h animals receiving DM continued to have significantly decreased lymphocyte counts, especially of OX4" and W 3 / 2 5 ' cells compared to both the control and SM treated groups. The number o f cells expressing OX8 was also lower, but was not depressed to an equivalent extent. Consequently, the W 3 / 2 5 ' : O X 8 ~ ratio remained profoundly depressed in rats receiving DM and remained that way for greater than four weeks after injection (Fig. 6) (P< 0.05 on day 31). D o s e - r e s p o n s e curves depicting the mean of seven rats per group monitored every few days following the administration of a single dose of 0 (PBS control), 2,4,8 and 16 m g / k g of Depo-medrol

are shown (Fig. 7). The lymphocyte subset profiles of a group o f rats monitored concurrently which received 10 days of daily SM (16 mg/kg) injection showed no statistical difference from the control group of animals (data not shown). With DM there is an obvious dose-related affect upon lymphocyte numbers, and the effect was most marked upon the W 3 / 2 5 ' and OX4" lymphocytes. When compared with the control group, no statistically significant difference was observed at a dose o f 2 m g / k g DM. At doses of 8 and 16 m g / k g DM, significantly different values (P<0.01) were observed for most lymphocyte subpopulations even up to day 23 post injection. By day 23, animals receiving 16 m g / k g DM showed a reduction in lymphocyte numbers of 40% for W3/13; 63% for W3/25; 20% for OX8 and 75% for OX4 compared to the control group. Thus,

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Cyclosporin A A single oral dose of 15, 30 or 45 mg/kg CsA produced no significant alteration of lymphocyte subsets. Therefore, groups of 6 animals/group which received daily oral CsA of 15, 30 or 45 mg/kg for 8 days were monitored. Compared to the control group, there was a significant elevation of all lymphocyte subsets by day 4 (Fig. 8). However, as shown, this rise had begun to return towards control values by Day 7, prior to the cessation of drug administration. Within 12 weeks of commencement of CsA administration lymphocyte numbers for all subsets were depressed. By day 19, rats which

received 45 mg/kg/day CsA for 8 days had lymphocyte numbers reduced to 60070 for W3/13, 50°70 for W3/25, 67070 for OX8 and 75070 for OX4, compared to control rats, all significantly lower (P< 0.05). An interesting observation was that the proportion of cells expressing the W3/13 marker increased markedly during CsA administration whilst those expressing either W3/25 or OX8, markers decreased significantly from control values. When analysed in terms of lymphocyte numbers we noted a significant increase in total T cell numbers over the controls. Further, the W3/13' (pan T) cell numbers were much more elevated than the subsets so that on day 4 of the experiment shown (Fig. 8) the means for lymphocyte numbers for the 45 mg/kg group were W3/13, test 7483 ± 1193, control 4096 ___791; W3/ 25, test 3756 ± 872, control 2419 +_ 371; and OX8,

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test 1425 +_. 305, control 1009 +__ 194. So, while the addition of W3/25 and OX8 numbers was close to the total T cell (W3/13) numbers for the control group (3428 vs 4096), for the test animals there was significant disparity (5181 vs 7483) (Student's t-test P < 0.0l). DISCUSSION Many pharmacological agents in current use can be assayed in plasma to determine whether a certain dosage is appropriate for a therapeutic effect whilst avoiding toxicity. In the area o f immunosuppression there is a need for an assay which enables in vivo monitoring of the powerful drugs in current usage. This study was designed to assess, in an animal model, the appropriateness of sequential flow cytometric monitoring of peripheral blood lymphocyte populations as an in vivo measure of the effect of the various immunosuppressive agents. This study showed a rapid lymphopenic effect within 6 h following 2.5, 5.0 or 7.5 m l / k g of a commercially available ALS preparation, with a gradual return to control values over some days. This

substantiates the observation that a single injection of ALS causes a profound and sustained T cell lymphopenia maximal after four hours, with a gradual return to normal values over several days (Rolland & Nairn, 1972). This study in addition, demonstrated that neither Class 1 nor Class II M H C reactive T lymphocyte populations were selectively affected over the dose range tested. The lymphopenic effect of ALS after 24 h has been reported to be dose dependant (Sacks, Filippone & Hume, 1964), but similar kinetics were demonstrated with the three dosages used here. In an additional experiment ALS was injected at an even lower dose (1 m l / k g i.p.). This showed a similar kinetics and no selective effect within the T lymphocyte populations (data not shown). Therefore, there was no evidence in this study that the ALS preparation tested was other than a pan-T cell immunosuppressive agent. This is how it is used in human renal transplantation where it has been shown that renal allograft rejection is u n c o m m o n when circulating T lymphocytes are profoundly depressed. (Cosimi, Colvin, Burton, Rubin, Goldstein, King, Hansen, Delmonico & Russell, 1981).

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Doses of 2 - 3 m g / k g / d a y of A Z A are used in most protocols of immunosuppressive therapy for human transplant recipients. Although, a single dose of up to 50 m g / k g of A Z A does not exert a differential effect on the numbers or proportion o f circulating lymphocyte subsets in this rat model, more prolonged exposure selectively affects the proportion and number of lymphocytes bearing O X 4 " , the Ia marker found on B cells and activated T lymphocytes. Since A Z A acts by competitive enzyme inhibition to block synthesis of inosinic acid and ultimately inhibits D N A synthesis, (Nydegger, 1985) it is not surprising that some days elapse before an effect is seen on the peripheral blood lymphocyte population. Azathoprine has been reported to have a preferential effect on T-cell mediated reactions but the present study showed a cumulative depressive effect on circulating l a ' cells with increasing dose and time o f administration. Its mode o f action

means A Z A exerts its most damaging effects on cells during mitosis and therefore is more powerful when administered at the time when antigen-sensitive cells are dividing. Since Ia" expression, a hallmark of lymphocyte activation, is synonomous with the synthesis of D N A leading to mitosis, A Z A will prevent the population of activated lymphocytes bearing receptors for the epitopes of the graft antigens from proliferating, and thus the normal rejection response will not proceed. Over the 0 - 50 m g / k g range the effect of C T X on lymphocyte subpopoulations was dose related. A transient depression in lymphocytes bearing the O X 8 ' marker (Class I M H C reactive) which resulted in an elevation of the Class II M H C reactive/Class I M H C reactive T lymphocyte ratio, was observed in these rats at 5 and 10 m g / k g C T X , although it did not achieve statistical significance. This confirmed reports from human studies o f the differential effects

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Fig. 8. Dose - response curves showing the effect on circulating lymphocyte subsets of an 8 day orally administered course of Cyclosporin A (CsA). The means of six animals per group are shown.

of low dose CTX on Class I MHC reactive T cells and hence on this ratio (Bast, Reinherz, Mauer, Lavin & Schlossman, 1983). There was a marked depression of circulating lymphocytes bearing the l a ' marker in CTX treated rats. Thus, CTX probably exerts some, at least, of its immunosuppressive effects through the mechanism described above for AZA. Because of their slow rate of recovery, these cytotoxic agents have a more pronounced effect on B than on T ceils. Since the l a' marker is found on B cells, macrophages and dendritic cells and possibly on other cells which have a key role in antigen presentation, the effect of CTX on la ~ cells may explain the role of donor pre-treatment with CTX in conjunction with organ culture in achieving allograft survival across a major histocompatability barrier (Lafferty & Prowse, 1984). Thus, administration of CTX days before removal of the donor endocrine organ, would reduce the l a ' v e cells bearing foreign class II antigen the graft and the susceptibility of these ceils to culture in a high oxygen environment may reduce the "passenger leucocytes" to below the critical threshold necessary to initiate graft rejection. These monitoring experiments using AZA and CTX were carried out on animals which had not received any deliberate primary antigenic challenge

and were therefore not in proliferative phase of an immune response, and thus the effect demonstrated might be even more magnified following a specific immune challenge. Administration of a single dose of glucocorticoid affects both the distribution and function of lymphocytes and Haynes & Fauci (1978) have established that the transient lymphopenia following glucocorticoid administration selectively affects different subclasses of lymphocytes. However, these and other studies to determine the mechanisms by which corticosteroids exert their immunological effects were performed before the advent of monoclonal antibodies and flow cytometry made possible the analysis of lymphoid subpopulations on the basis of their cell surface receptors (Claman. 1972; Fauci, 1975). This study has taken advantage of these recent developments to show a differential effect of glucocorticoids on lymphocyte subsets in peripheral blood. This is transitory following SM administration but sustained after DM administration. A relative sparing of Class 1 MHC reactive (OX8') suppressor/cytotoxic cells, and a marked depression of Class I! MHC reactive (W3/ 25') and l a' (OX4') lymphocytes in peripheral blood was demonstrated. Depo-medrol thus was shown to have a powerful prolonged

The Effect of Immunosuppressive Agents immunosuppressive effect at doses of 8 and 16 mg/kg. The effect of depressing circulating Ia'(B) lymphocyte numbers and hence impairing the antibody response against foreign graft antigens would also be beneficial in the prevention of graft rejection. The immunoregulatory potential of a drug which spares Class I MHC reactive (OX8") cells whilst depressing the Class II MHC reactive (W3/ 25') cells which provide help for the generation of donor-specific T cells that mediate rejection was felt worthy of further investigation. A series of transplantation experiments using DM in recipients of endocrine xenografts were performed using BALB/c murine donor tissue cultured in RPMI for 14 days in a 95°7o oxygen environment prior to transplantation under the renal capsule of male inbred Wistar rat recipients. All xenografted control animals rejected within 10 days. A single injection of 16 mg/kg DM on a day 0 retarded rejection so that 56070 (n = 9) of grafts were partially intact on day 14. Six animals which received ten daily injections of 16 mg/kg SM from the time of grafting had completely rejected their grafts on day 14 (P<0.05 Fisher's Exact Test). Administration of DM 16 mg/kg on day 0 and 16 mg/kg on day 5 resulted in 8007o of thyroid grafts intact when removed on day 21. Similar graft survival has been found when cultured fetal pancreas is used as the donor tissue in the same xenograft strain combination (Thompson et al., 1987). This profound and prolonged immunosuppressive effect of a single intramuscular injection of 8 or 16 mg/kg DepoMedrol on circulating Class II MHC reactive T cells and the associated prolongation of graft survival has not been previously reported. This may well have important implications for the use of steroids in human transplantation since the mechanism of action of glucocorticoids and its dampening effect on the immune system is similar in rat and man (Baxter & Harris, 1975). The administration of methylprednisolone in soluble and depot forms has been examined in man where prolonged serum drug levels and depression of serum cortisol following intramuscular or interarticular injection of DM have been demonstrated (Stubbs, 1975; Armstrong, English, Gibson, Chakraborty & Marks, 1981). These effects are entirely consistent with the prolonged effects of DM in rats reported in this study but unfortunately these clinical studies did not address the immunological consequences of sustained steroid levels, and further investigation of this is obviously needed. The dose-response data for CsA reported here was obtained by monitoring lymphocyte subsets

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following daily administration of CsA for 8 days. A 7-day course of CsA given orally at doses of 5 or 10 mg/kg has been shown to result in prolonged and sometimes indefinite survival of renal allografts in the rat even across strong histocompatability barriers (Homan, Fabre, Williams, Millar & Morris, 1980). Our results show that the significant differences in all lymphocyte subset populations following CsA administration took some days to develop, and persisted long after the administration of CsA had ceased. CsA reportedly blocks receptors for DR molecules on responding T cells, thus inhibiting the expression of IL-2 receptors on these cells and preventing clonal expansion (Palacios & Moiler, 1981). This mechanism may account for the delay in producing effects on lymphocyte subsets and slow return towards control values upon cessation of drug administration, in contrast to agents suich as SM and ALS which have effects via lympholysis and rapid redistribution to the extravascular compartment. In this study, la* lymphocytes were not selectively more affected than T lymphocyte populations. It has been proposed based largely upon in vitro studies, that CsA immunosuppression is mediated through alloantigen specific suppressor T lymphocytes which maintain specific immunologic unresponsiveness following discontinuation of CsA treatment. Hess, Tutschka & Santos (1983) proposed that CsA defines two T cell subsets; those that are sensitive to CsA (including helper and cytotoxic cells) and those that are insensitive to CsA (including suppressor cells). Unfortunately, the lack of monoclonal markers which distinguish between those lymphocytes which exert a suppressive rather than a cytotoxic effect does not enable our in vivo study to resolve this issue. Following this short course of CsA administration there was no significant difference in the ratios of Class I1 MHC reactive to Class I MHC reactive lymphocytes. Sweny & Tidman (1982) published results which showed no change in this ratio in the peripheral blood of renal allograft recipients who had received CsA for less than 2 months although a marked reversal (0.33) occurred in the group using CsA as the sole form of immunosuppression for more than 2 months. An unexpected finding was the initial rise in the proportion of lymphocytes expressing W3/13 the pan-T marker. Further, there were many lymphocytes which did not express either the T helper/ inducer or T cytotoxic/suppressor subset markers. Once CsA was ceased the number of these T cells dropped and the discrepancy between the number of cells bearing the pan-T but neither functional subset

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S. C. THOMPSON el al.

m a r k e r diminished. G o d d e n , Herbert, Stewart & Roser 0 9 8 5 ) reported the a p p e a r a n c e o f a novel large lymphocyte in the peripheral b l o o d of rats which were receiving CsA, but only if there was an allograft in place. The cell they reported bore b o t h W 3 / 2 5 a n d O X 8 lymphocyte subset markers, a p p e a r e d within seven days o f c o m m e n c i n g CsA a n d disappeared over 2 - 4 weeks after cessation o f treatment. It is difficult to reconcile this cell which the a u t h o r s claim to be derived from the periphery (since t h y m e c t o m y m a d e n o difference) a n d which expressed b o t h W / 2 5 a n d OX8 (normally reciprocal markers on peripheral T lymphocytes), with n o r m a l o n t o g e n y whereby the only ceils which regularly carry b o t h m a r k e r s in the rat are thymocytes. O u r data examining peripheral blood of u n g r a f t e d animals during CsA a d m i n i s t r a t i o n in fact show the opposite p h e n o m e n o n - - c e l l s labelling with W 3 / 1 3 but neither W 3 / 2 5 n o r O X 8 a p p e a r in peripheral blood. The sensitizing role o f the graft m a y be i m p o r t a n t in reconciling these discrepant findings. The i m m u n o k i n e t i c studies on i m m u n o s u p p r e s s i v e drugs reported here provide a f r a m e w o r k for a more rational a p p r o a c h to i m m u n o s u p p r e s s i v e therapy. If an a n i m a l is p r o f o u n d l y lymphopenic, it is u n a b l e to m o u n t an i m m u n o l o g i c a l rejection response to foreign graft antigen, but such non-selective i m m u n o s u p p r e s s i o n puts the recipient at risk o f s u c c u m b i n g to infection. Since a thymic-derived cellular suppression system has a central role in the

m o d u l a t i o n and regulation o f cellular and h u m o r a l i m m u n e response, lymphocyte subset m o n i t o r i n g has been used to examine the susceptibility o f different i m m u n o r e g u l a t o r y cell p o p u l a t i o n to various agents c o m m o n l y used in t r a n s p l a n t a t i o n . The interval between (graft) antigen presentation a n d drug administration(s) influences graft survival. Hence, this in vivo m e t h o d o f examining changes in lymphocyte subsets, which is a sensitive indicator o f the recirculating lymphocyte s u b p o p u l a t i o n s m a y enable i m m u n o s u p p r e s s i v e regimes to be optimized and monitored following t r a n s p l a n t a t i o n . In particular, the finding o f prolonged a n d p r o f o u n d depression o f the Class II M H C reactive lymphocytes when m e t h y l p r e d n i s o l o n e was given as a depot f o r m u l a t i o n , in c o n j u n c t i o n with its effect in prolonging endocrine graft survival in rats receiving thyroid or fetal pancreas allografts or xenografts, shows the potential of this a p p r o a c h in the assessment o f i m m o n o s u p p r e s s i v e therapy. Acknowledgements -- This work has been supported by grants from the National Health and Medical Research Council of Australia, Novo Laboratories and the Wellcome Research Foundation. Cyclosporin A was the generous gift of the Sandoz company, Basel, Switzerland. The co-operation of Arno Enno, Ross Richards and the Department of Haematology and the technical assistance of Michelle Corner and Kate Lenord are gratefully acknowledged. Thanks also to Ms. Margaret Finlayson for typing of the manuscript.

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