The treatment of induced immune deficiency with interleukin-2

Immunology Letters, 10 (1985) 307-314

Elsevier Imlet 618

T H E T R E A T M E N T OF I N D U C E D I M M U N E D E F I C I E N C Y W I T H INTERLEUKIN-2 Paul J. CONLON, Theresa L. WASHKEWICZ, Diana Y. MOCHIZUKI, David L. URDAL, Steven GILLIS and Christopher S. H E N N E Y Immunex Corporation, 51 University Street, Seattle;, WA 98101, U.S.A.

(Received 6 February 1985) (Accepted 8February 1985)

1. Summary In vivo generation of alloreactive cytotoxic T lymphocytes (CTL) was found to be inhibited by treatment o f mice with either cyclophosphamide or the glucocorticoid, hydrocortisone acetate. The effects of these immunosuppressive agents could be overcome, however, by the in vivo administration of IL-2 from both murine and human sources. Both human IL-2 derived by recombinant DNA techniques as well as the natural protein from mouse and man all reverse the unresponsive state. A single injection of IL-2 was sufficient to reverse the effect of cyclophosphamide treatment, while additional injections with as little as 8/~g of protein ablated the steroidinduced suppression. Furthermore, the responder cells generated in vivo following IL-2 therapy were shown to be antigen specific in terms of their lytic capacity. Thus, IL-2 therapy appears to restore the in vivo responsiveness of immunosuppressed recipients to allogeneic tumor cell challenge. These data demonstrate the importance of IL-2 as an immunoregulatory molecule in vivo and suggest its future use as a potent therapeutic.

2. Introduction The role o f the T-cell-derived lymphokine, inKey words: immunosuppression - interleukin 2 - alloreactive - cytotoxicT lymphocytes

terleukin 2 (IL-2), in regulating the development and amplification of effector T-lymphocyte function has been well documented in both mouse and man [1-5]. In vitro studies have revealed an obligatory requirement for the presence of this molecule in effector T-cell differentiation and have suggested that IL-2 may play a pivotal physiological role as a regulatory protein [1-3]. This hypothesis has been supported by a number of in vivo studies [ 3 - 5 ] . Previously, this laboratory, using purified murine IL-2, has shown that both resident natural killer cell activity, as well as the generation of cytotoxic T-lymphocytes (CTL) to alloantigenic stimulation, could be significantly enhanced by the administration of IL-2 in vivo [5]. In an effort to further document the utility of in vivo IL-2 therapy, we attempted to use the lymphokine to restore immune responses in animals which had been immunosuppressed by pharmacological agents. To this end, both cytoxan and glucocorticoid hormones have been used to effect profound immunosuppression. The ability o f IL-2, from both recombinant DNA and natural sources, to reverse such immunosuppression has been studied. These investigations have demonstrated that both natural and recombinant-derived IL-2 are capable of restoring drug-induced immunodeficiency to response levels equal to, or exceeding, those o f normal animals. The implications of these findings for the potential therapeutic usefulness of II.,2 are discussed.

0165-2478 / 85 / $ 3.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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3. Methods and Materials

3.1. Mice and cell lines Female C57BL/6J (H-2 b) mice ( 6 - 8 wk of age) were obtained from The Jackson Laboratories, Bar Harbor, ME. They were housed in our animal facility and age-matched for each experiment. The DBA/2J (H-2 d) murine mastocytoma, P815, was passaged weekly in vivo by ascitic cell transfer into syngeneic recipients. LBRM 33-5A4 and Jurkat H33HJ-JA1 cells were grown in vitro as previously described [6]. 3.2. IL-2 production and purification Natural human IL-2, derived from the T-cell line Jurkat, was produced and purified to homogeneity as previously described [7]. Recombinant IL-2 (rlL-2) was produced and provided by a collaboration between Immunex Corporation and H o f f m a n n - L a Roche, Inc., Nutley, NJ. The material used was purified from cultures in which the IL-2 gene was expressed in an E. coli vector system. LBRM-33-5A4 tumor cells were used to produce murine IL-2. Its purification was carried out as previously described [6]. The production and purification of human IL-2 from Jurkat cells was carried out as previously detailed [7]. Briefly, following PHA stimulation for 24 h, cell-free supernatants were concentrated by differential ammonium sulfate precipitation. Ion-exchange chromatography, using CM Biogel A (LKB, Bromma, Sweden) further resolved contaminating proteins from IL-2. Two separations by reverse-phase high pressure liquid chromatography (HPLC) resulted in a homogenous source of IL-2, with a specific activity of 1 x 106 U/#g protein. All sources of IL-2 were suspended in phosphate-buffered saline pH 7.2 (PBS) containing human serum albumin (HSA) (1 mg/ml) as a carrier protein prior to in vivo administration. 3.3. In vivo generation of ailogeneic CTL Mice were immunized intraperitoneally (i.p.) with 2×107 P815. Ten days later, animals were killed, their spleens removed, and spleen cells used as effector cells in a lymphocyte-mediated cytolysis assay performed using 51C-labelled P815 cells. The percentage of specific lysis in a 4-h as308

say was then calculated as follows: percent specific release= experimental cpm-medium control cpm

× 100070

maximum cpm-medium control cpm The maximum cpm was determined by 4-h incubation o f target cells in solution containing 1 normal HCI, and minimum cpm was generated in cultures containing target cells without effector cells. Each experiment reported in this investigation was carried on at least three occasions; representative results are depicted. 3.4. Cytoxan (Cy) treatment At varying times following injection of allogeneic tumor cells, animals were given Cy (Sigma Chemical Co., St. Louis, MO) intravenously (i.v.) at doses of either 40 or 200 mg/kg body weight. 3.5. Hydrocortisone acetate (HC) treatment HC (Sigma Chemical Co., St. Louis, MO) was given i.p. in a PBS suspension (2.5 mg per animal) at varying times ( - 2 , 0, 2 or 7 days) prior to or following antigen injection. 3.6. IL-2 therapy At varying intervals animals were given either natural or recombinant IL-2 or an equivalent volume of a solution of PBS, containing HSA. IL-2 was administered i.p. to recipient animals. A unit of IL-2 was defined in standard IL-2 bioassays [8] as the reciprocal of the dilution that gave one-half maximal proliferative response (tritiated thymidine incorporation) of IL-2-dependent CTLL-2 ceils in a 24-h assay. The specific activity of the recombinant 1I_.-2 used was 106 U/#g protein.

4. Results

4.1. Studies with Cy 4.1.1. Timing of Cy administration. In order to induce a Cy-dependent unresponsive state, animals were injected with antigen (P815 cells)

on day 0, and at varying times thereafter, were given Cy i.v. at 200 mg/kg body weight (+2 h, day 3, day 6, and day 9). CTL responses in all mice were measUred on day 10. As can be seen in Fig. 1, administration of Cy, as soon as 2 h after P815 challenge, completely ablated the generation of splenic CTL when assayed 10 days later. Moreover, delay of drug administration for as long as 6 days still inhibited the generation of alloreactive T-lymphocytes. To ascertain whether low dosage of Cy affected the in vivo generation of alloreactive CTL, we attempted to reduce the dose of Cy required to ablate CTL development so as to be more consistent with dosages thought to inhibit helper Tcell function. By reducing the dose of Cy, we also hoped to address the possibility that the lack of responsiveness following high Cy dose administration might primarily be due to a reduction of tumor cell (antigen) stimulation. In experiments of this type, animals were given Cy at either 40 or 200 mg/kg body weight, 2 h or 6 days following i.p. injection of P815. While 200 mg/kg body weight of Cy was capable of inhibiting the development of cytotoxic activity when given at either time point, 40 mg/kg Cy administration was only effective if given on day 6. These results are consistent with the hypothesis that low-dose day-6 Cy treatment

reduces CTL development by effects on the immune response. By the same token, one concludes that suppression caused by high doses of Cy given around the time of antigen administration is largely at the level of diminished antigenic load. 4.1.2. Effects of IL-2 on immune responsiveness following Cy administration. Since it appeared that we could depress the in vivo generation of alloreactive CTL with either low- or high-dose Cy, we attempted to restore cytolytic responses by administration of IL-2. Animals were inoculated with allogeneic tumor on day 0, treated with Cy at either 40 or 200 mg/kg on day 6, and given rIL-2, 2 × 106 U i.p., on day 8. Two days later, animals were killed and spleen cells tested for lytic activity. As seen in Fig. 2, spleen cells from animals receiving either 40 or 200 mg/kg Cy on day 6, demonstrated a reduced lytic activity when measured 4 days after drug treatment. However, injection of rIL-2 within 48 h of Cy treatment had a dramatic effect on the in vivo generation of cytolytic activity. Animals given low-dose Cy followed by rIL-2 showed a significant increase in their lytic activity against radiolabelled P815 target cells (18-69°70 lysis). The restoration of the CTL response in animals receiving high-dose Cy 8C

80

6c (xl 60 I.4C

~ 40 "N

~ 2e

2c

P815 only

+2H

+3D

+6D

+9D

Cy Administration

Fig. 1. C57BL/6J females (three per group) were given 200 mg/kg Cy i.v. either 2 h, 3 days, 6 days, or 9 days following i.p. inoculation of 2x10 v live P815 tumor cells. On day 10, spleen cells were harvested and lytic activity directed against 5~C-labelled P815 determined in a 4-h assay. The data depict specific lysis at an E / T ratio o f 25:1.

P815 only

40 200 Cy (rng/kg)

40 200 Cy rIL-2 (rnglkg)

Fig. 2. C57BL/6J female mice were inoculated i.p. with live P815 tumor cells on day 0, followed 6 days later by i.v. treatment Cy at 40 or 200 mg/kg. Control animals received either no treatment or 2x106 U o f rIL-2 i.p. on day 8. On day 10, the splenic lytic activity was measured against P815. Data depict the lysis at an E / T ratio of 50:1.

309

was not as profound, in keeping with the suggestion that such doses have effects on antigen load, as well as on cells of the immune system. Nevertheless an immune response could be seen in such animals following IL-2 therapy. Thus, rlL-2 enhanced the in vivo generation of CTL activity in Cy-suppressed mice. Administration of placebo ( P B S - H S A ) to either low- or high-dose Cy-treated allo-tumor-challenged mice did not augment splenocyte CTL activity. 4.1.3. Therapy with natural or recombinant IL-2. As rlL-2 appeared to restore responsiveness in low-dose Cy-treated animals, the dose of rlL-2 needed to cause such effects was evaluated. Moreover, it was important to determine if natural (Jurkat tumor cell-derived) human IL-2 would also be effective in restoration of CTL activity in Cy-immunosuppressed mice. Subsequent experiments involved Cy treatment (40 mg/kg) 6 days following tumor injection, followed 48 h later by i.p. injection of varying concentrations of rlL-2 in P B S - H S A , natural (n)IL-2 in P B S - H S A , or P B S - H S A alone. The lytic activity of harvested splenocytes directed against radiolabelled P815 tumor targets was then measured on day 10 (2 days after lymphokine therapy). The resulting lytic activity of splenocytes against P815 is shown in Fig. 3. As seen previously, the cytolytic response to allogeneic tumor cells was depressed by Cy treatment on day 6 (depression from 70 to 35070 specific lysis), rIL-2 was seen to reconstitute the CTL response, in a dose-dependent fashion. Animals receiving 5 x l05 units of riL-2 on day 8 had a lytic response not remarkably different from the Cyonly treated group (43% instead of 35%). This response was also identical to that seen in animals receiving only carrier protein ( P B S - H S A , data not shown). However, if similarly suppressed animals were given either 2 or 10×106 units of rIL-2, a substantial degree of lysis was observed (93 and 98°/0, respectively). Jurkat cell-derived, or niL-2, at 2× l06 U per animal, was also capable of restoring the CTL response to levels seen in the untreated control group (62070 vs. 70070). This was somewhat less, 310

I00

8C

---q

~- 6C

I

~ 4C q)

a. 2c

P815 only

V Cy

rlL-2

nile2

Fig. 3. Animals were injected i.p. with 2×107 P815 cells followed 6 days later by i.v. treatment with either vehicle only, or 40 m g / k g Cy. Two days later, on day 8, groups received either vehicle only, natural h u m a n IL-2 (2×104 U per animal i.p.), or rlL-2 (5×105, 2x106, or 2x107 U per animal i.p.). On day 10, spleen cells were harvested, pooled, and tested for lytic activity against P815 tumor target cells. Data depict the lysis at an E / T ratio of 25:1.

however, than the activity seen with the recombinant-derived material. 4.1.4. Antigen-specificity of CTL response resulting from IL-2 therapy. Further experiments demonstrated that the splenic CTL population from IL-2-treated animals was able to lyse only for LPS-activated splenocytes syngeneic to the immunizing P815 allogeneic tumor cells. Thus, only H-2 d targets (77070 lysis), and not irrelevant H-2 k targets (0% lysis), were lysed by cell populations from IL-2-treated mice. It should also be noted that the capacity of IL-2 to augment alloreactive CTL generation in Cyimmunosuppressed mice was dependent on the presence of the immunizing antigen. Animals receiving as much as 5 × 106 units of IL-2 on day 6, in the absence of antigen stimulation, did not generate cells capable of killing the P815 target cells. Moreover, the effector cells from IL-2-treated animals were susceptible to anti-Thyl antibody and complement treatment. Thus, the IL-2-activated cell population appeared to be an antigen-specific T-cell. Contrary to previous reports [9, 10], we were unable, in this model system, to find any evidence for the in vivo existence of "lymphokine-activated" killer cells.

4.1.5. Duration o f rlL-2 effect in restoration o f immune responses in vivo. To determine the duration of the capacity of IL-2 to restore in vivo generation of alloreactive CTL, we conducted the following experiment. Animals were given allogeneic tumor cells i.p. on day 0, followed 6 days later by i.v. Cy treatment. Forty-eight hours later (day 8), rlL-2 (2 x 106 U per animal) was administered i.p. and the animals killed either 2 (day 10), 5 (day 13), 8 (day 16), or 12 (day 20) days later. Recombinant IL-2 given 48 h following Cy treatment restored the response of animals to allogeneic tumor cells. This augmented response lasted over 12 days following rlL-2 therapy; essentially for the entire time period tested. At every time point, spleen cells from rlL-2-treated mice exhibited augmented lytic activity indistinguishable from untreated, control groups which had not received Cy. Thus, in vivo administration of IL-2 to Cy-treated mice did not alter the kinetics of CTL generation in vivo. 4.2. Immunosuppression by H C 4.2.1. Timing of H C administration. Although glucocorticoid hormones are known to have a profound effect on the immune system [11, 12], little is known regarding their action on the generation of cytotoxic cells in vivo. Therefore, initial experiments were conducted to determine when, in relation to alloantigen administration, H C could be found to effect immunosuppression. Animals were given HC (2.5 mg suspension) i.p. 2 days prior to tumor cell injection (day - 2 ) , or either 2 or 7 days following antigen administration (day + 2 or day + 7). Additional groups of mice were g!ven injections of H C twice (on days - 2 and 0), or on three separate occasions (days - 2 , 0, and +2). Nine days following administration of allogeneic P815 tumor cells, the spleen cells from treated mice were harvested and cytolytic activity measured. Animals receiving no H C demonstrated potent lyric reactivity against P815 (86°70 lysis at an effector to target (E/T) ratio of 50:1). In contrast, C57BL/6 mice given H C 2 days prior to antigen (day - 2 ) showed a significant decrease in splenic cytolytic activity (3007o lysis at an E / T ratio o f 50: I). Interestingly, spleen cells

from animals treated with H C either +2 or +7 days following P815 injection had levels of cytotoxic activity equal to the untreated controls. HC, therefore, appeared to act early in suppressing the in vivo generation of alloreactive CTL. Animals receiving several injections of HC on days - 2 and 0, or days - 2 , 0, and + 2, were not different from those which had received a single injection o f steroid on day - 2 . Steroids have been shown to have a profound inhibitory effect on the in vitro generation of CTL responses. This effect has been overcome by the addition of IL-2 to the cultures. To ascertain whether IL-2 might be able to act in a similar fashion in vivo, we administered IL-2 to steroidtreated mice which had been challenged by antigen. As shown in Fig. 4, IL-2 therapy was capable of restoring the splenic CTL activity to the level of untreated control mice. HC-pretreated animals were given daily i.p. injections of either P B S - H S A solution or Jurkat cell-derived niL-2 (1 × 103, 1 × 105 or 5 × 105 U/day) starting 1 day after antigen administration (day 1) and continuing for 8 days (day 8). Spleen cells from P B S - H S A - t r e a t e d animals, as well as from 80

6O ...J

40

20

,50:1

25:1

17'.5:1

Effector to Target Ratio

Fig. 4. C57BL/6J females (three per group) were given either P815 tumor cells i.p. or pretreated twice with 2.5 mg HC (on days -1 and 0) prior to tumor cell injection. Beginning 1 day after antigen administration, and continuing for 8 days (days 1-8), animals were untreated (a) or were given PBSHAS ( o ) or varying amounts of niL-2, 1× 103 U/day ( • ). 1×105 U/day ( I ) or 5×105 U/day (e). Animals given P815 cells but no HC are shown by the symbol (a). Nine days after antigen administration, the animals were killed, their spleens pooled, counted, and the cytolytic activity against P815 measured in a 4-h s~Cr-release assay. 311

animals treated with 1 x 103 U of IL-2 i.p. daily over 8 days, displayed no significant increase in CTL compared to spleen cells from HC-treated mice. However, if similarly suppressed animals were given 1 x 105 U of IL-2, i.p. daily for 8 days, then spleen cells from such treated mice showed heightened CTL activity. An increase in the treatment dose of IL-2 to 5 × 105 U/day further increased the cytolytic activity o f spleen cells from treated mice to levels exhibited by splenocytes harvested from untreated control mice. A similar experiment was conducted to determine whether natural murine (LBRM-33-5A4 cell-derived) or human recombinant IL-2 were as effective as Jurkat-derived natural IL-2 in reversing the suppressive effects o f HC. Thus, steroidsuppressed, alloantigen-challenged, animals were given either P B S - H S A or 5 × l0 s U o f natural murine, or recombinant human IL-2 i.p. daily for 8 days, after which time splenic effector cells were harvested and tested for their capacity to kill the allo-immunizing tumor target P815. All types of IL-2 enhanced the in vivo generation o f allo-reactive CTL in steroid-treated mice to levels comparable to, or greater than, those observed in normal animals. Tile cytolytic activity observed in animals that were given rlL-2 daily following steroid pretreatment and antigen challenge, was antigen-specific. Furthermore, the lytic activity was eliminated by anti-Thy-1 antibody in the presence of complement. Further studies were then conducted to determine the frequency of IL-2 administration required to reverse steroid-induced suppression of alloreactive CTL. Groups of animals were treated with H C o n day - 2 and again on day 0, followed by i.p. injection o f 2)<107 P815. Beginning the following day, groups were given daily injections of r I ~ 2 (8 times, 106 U per injection, days 1-8), every other day (4 times, 2 × 106 U per injection, days 1, 3, 5, 7), every 4 days (2 times, 4× 106 U per injection, days 1 and 5) or once every 8 days (1 time, 8× 106 U per injection, day 1). All of the groups received a total of 8 x 106 U of rlL-2 with the only treatment variable being frequency of administration. On day 10, all mice were killed, splenocytes pooled, and tested for 312

alloantigen-directed cytolytic reactivity in vitro. The results of such an experiment are shown in Fig. 5. Clearly, animals receiving rlL-2 i.p. either daily, or every other day for the 8-day period, exhibited dramatic restoration of cytolytic activity, and were indistinguishable from the non-steroid-treatment control mice. However, when rlL-2 was given only once or twice over the 8-day period following antigen administration, no significant difference was evident in comparison to the control group which received steroid treatment alone. These results suggested that the biological effect of rlL-2 in vivo terminates, is inactivated, or is cleared between 48 and 96 h following administration. Although, at present, it is unknown whether IL-2 is removed or inactivated after administration, it remains clear from these experiments that the bio-availability of the molecule, i.e., the time after injection during which it can mediate its effect (some 2 4 - 4 8 h, based on the data detailed above), is significantly longer than its serum half-life [13]. No significant difference was observed in biological effect between natural murine, natural human or recombinant human IL-2, in terms of the capacity of these lymphokines to restore steroid-induced immunodeficiency. 10(3

A

8(;

LO Ld

4c ¢::

~- 2c r-] P815 only

HC

Treated

8

2

]

Number of Times

Fig. 5. HC-suppressed P815-injectedanimals were given rlL-2 i.p. daily (106U/day, days 1-8), every other day (2×106 U/day, days 1, 3, 5, 7), every 4 days (4x106 U/day, days 1 and 5), or once (Sx106 U, on day 1). On day 9, spleens were removedand their allo-directed CTL activity assessed in a 4-h 5~Cr-releaseassay. Data depict the lysis obtained at an E/T ratio of 6.25:1.

5. Discussion

The in vivo administration of IL-2 to both normal and immunoincompetent recipients has been shown to augment T-cell-mediated responses [3-5], as well as resident natural killer cell function [5]. As shown here, the ability of exogenous IL-2 to overcome both Cy- and glucocorticoid hormone-induced unresponsiveness is striking. In both cases, the reversal of immune responsiveness by IL-2 was dose dependent and resulted in an antigen-specific T-cell response. More importantly, the kinetics of the cytolytic response was similar to that seen with normal, untreated control mice. This is consistent with the contention that IL-2 therapy may act to overcome certain defects in the immune response that are deficient in T-helper cell function. The alkylating agent, Cy, has been shown to have widespread effects on murine lymphocytes when given prior to or after antigenic stimulation [14-18]. While B-lymphocytes are exquisitely sensitive to the actions of Cy [14, 15], T-cells that elicit delayed-type hypersensitive reactions [18, 19] and cytotoxicity to allogeneic cells [16, 17] are somewhat resistant to the drug. Indeed, as shown in vitro by Hurme [16], and in vivo (P.J.C., personal observation), injection of as much as 200 mg/kg Cy at the same time as antigen does not effect the generation of alloreactive cytotoxic T-lymphocytes. However, if administration of the drug is delayed until after antigenic challenge, then a significant effect on the generation of cytotoxic T-cells was observed. In delayed-type hypersensitivity T-cell responses [18], Cy given after antigen administration results in a decrease in cellular reactivity. In this instance, Cy-induced suppression is most profound if given at the time of maximum proliferation by draining lymph node cells [14]. In an analogous fashion, it was expected that cytotoxic cells would be most sensitive to Cy when they are rapidly dividing. This is consistent with the observation that low-dose Cy administration 6 days after antigen immunization completely inhibits CTL development when assayed on day 10, while the same dose of Cy at the time of tumor injection does not affect the CTL response.

Although Cy treatment following the injection of allogeneic tumor cells may have an effect on the tumor burden of the animal, we do not feel that this can explain the capacity of exogenous IL-2 administration to restore the generation of CTL in Cy-suppressed mice. The observation that exogenous IL-2 will overcome the Cyinduced immunoresponsive state, particularly when low doses of Cy are used, suggests that Cy affects the production of IL-2 by host cells, thereby limiting the replication or differentiation of host CTL or their precursors. This hypothesis is consistent with the observations of Merluzzi et al. [20], who demonstrated that human IL-2 given in vivo could augment the Cy-suppressed allo-response to L1210 tumor cells in lymph nodes draining the site of tumor cell injection. This group also demonstrated that in this Cyinduced unresponsive state, restored by IL-2 administration, only those lymph node ceils draining the site of tumor cell injection were capable of cytolysis. Contrary to the results contained in the present study, demonstrable cytolytic activity could not be found in the contralateral lymph nodes or spleen. We have shown that administration of rlL-2 is effective in restoring drug-induced suppression when given 48 h following Cy. As little as 2 #g protein given i.p. dramatically increases cytolytic responses to alloantigen. This restored response could remain for at least 12 days following lymphokine injection (which was the duration of the time period tested). Most importantly, IL-2 therapy for unresponsive recipients did not appear to alter either the specificity of the response, duration of reactivity, or the phenotype of the responding cell population. The effect of glucocorticoid hormones on human and murine cell-mediated immune responses has been well documented [11, 12, 21-23]. Previously, Gillis et al. [23, 24] reported that dexamethasone (10-6 M) completely inhibited murine T-cell proliferation in vitro. Similarly, the proliferation of human peripheral blood lymphocytes and rat splenocytes in response to lectin stimulation could be inhibited by the steroid. These authors further demonstrated that the dexamethasone-induced suppression of murine T313

cell proliferative responses c o u l d be o v e r c o m e by the a d d i t i o n o f IL-2. I n d e e d , recent e x p e r i m e n t s in o u r l a b o r a t o r y , a n d others, suggest t h a t dexa m e t h a s o n e has effects on IL-2 r e c e p t o r expression o n h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s s t i m u l a t e d with P H A (K. G r a b s t e i n a n d S. Dower, m a n u s c r i p t s u b m i t t e d ) . In vivo a d m i n i s t r a t i o n o f g l u c o c o r t i c o i d horm o n e has been s h o w n to result in h y p o r e s p o n siveness o f T-cells in b o t h h u m a n [25] a n d m u rine systems [11, 12]. B i l l i n g h a m et al. [11] d e m o n s t r a t e d t h a t steroid a d m i n i s t r a t i o n in mice resulted in r e t e n t i o n o f allogeneic skin grafts o n l y if the d r u g was given p r i o r to the graft. F u r t h e r m o r e , once allogeneic rejection was initiated, the a d m i n i s t r a t i o n o f steroid h a d no effect o n graft survival. These a u t h o r s h y p o t h e sized t h a t the steroid a c t e d o n the i n i t i a t i o n o f the cellular response a n d n o t o n the effector phase. This is in c o m p l e t e a g r e e m e n t with o u r results detailing t h a t s t e r o i d - i n d u c e d s u p p r e s s i o n o f the d e v e l o p m e n t o f alloreactive C T L occurs o n l y if the steroid is given p r i o r to antigen. F u r t h e r m o r e , we f o u n d t h a t the steroidi n d u c e d state c o u l d be o v e r c o m e by as l i t t l e as 1 × 105 to 1 × 106 units o f IL-2 a d m i n i s t e r e d daily. H u m a n n a t u r a l ( J u r k a t cell-derived) a n d r e c o m b i n a n t h u m a n IL-2, as well as m u r i n e n a t u r a l ( L B R M - 3 3 cell-derived) IL-2 were effective in restoring responsiveness. T h e frequency o f IL-2 a d m i n i s t r a t i o n in vivo which was required to reverse s t e r o i d - i n d u c e d imm u n e s u p p r e s s i o n suggests t h a t the r e s p o n d i n g cell p o p u l a t i o n requires the c o n t i n u e d presence of lymphokine. The restoration of immune responses in s t e r o i d - i n d u c e d i m m u n o s u p p r e s s e d mice, as d e s c r i b e d here, was d e p e n d e n t o n exo g e n o u s IL-2, as well as alloantigen. I n the absence o f either stimulus, no d e t e c t a b l e cytolytic activity was observed. F u r t h e r m o r e , it was h e a r t e n i n g to note t h a t a d m i n i s t r a t i o n o f as m u c h as 8 #g o f r l L - 2 p r o t e i n did n o t alter the a n t i g e n specificity o f the r e s p o n d i n g T-cells. F u r t h e r investigations with these m o d e l systems c o n c e r n i n g the role o f IL-2 in vivo m a y d o m u c h to increase o u r u n d e r s t a n d i n g o f regulat i o n o f the i m m u n e response. B o t h m o d e l s o f d r u g - i n d u c e d i m m u n o s u p p r e s s i o n d e s c r i b e d here 314

p r o v i d e systems w h i c h m a y better predict the efficacy o f IL-2 t h e r a p y in the current clinical trials o f IL-2.

References [1] Shaw, J., Monticono, U., Mills, G. and Paetkau, V. (1978) J. lmmunol. 120, 1974. [2] GiUis, S., Union, N. A., Baker, P. E. and Smith, K. A. (1979) J. Exp. Med. 149, 1460. [3] Cheever, M. A., Greenberg, P. D. and Fefer, A. (1981) J. Immunol. 126, 1318. [4] Wagner, H., Hardt, C., Heeg, K., Rollinghoff, M. and Pfizenmaier, K. (1980) Nature (London) 284, 278. [5] Hefeneider, S. H., Conlon, P. J., Henney, C. S. and Gillis, S. (1983) J. Immunol. 130, 222.

[6] Mochizuki, D. and Watson, J. D. (1984) in: Methods for Serum-free Culture of Neuronal and Lymphoid Cells. (D. W. Barnes, D. A. Sirbasku and G. H. Sato, Eds.) p. 97, Alan R. Liss, Inc., New York. [7] Urdal, D., Mochizuki, D., Conlon, P. J., March, C. J., Remerowski, M. L., Eisenman, J., Ramthun, C. and Gillis, S. (1984) J. Chromatogr. 296, 171. [8] Gillis, S., Ferm, M. M., Ou, W. and Smith, K. A. (1978) J. Immunol. 120, 2027. [91 Grimm, E. A., Mazumder, A., Zhang, H. Z. and Rosenberg, S. A. (1982) J. Exp. Med. 155, 1823. [10] Grimm, E. A., Gorelik, E., Rosenstein, M. M. and Rosenberg, S. A. (1982) in: Proceedings of the Third International Lymphokine Congress (S. Cohen and J. Oppenheim, Eds.), Academic Press Inc., New York. [11] Billingham, R. E., Krohn, P. L. and Medawar, P. B. (1951) Br. Med. J. 5, 4716. [12] Levine, M. A. and Claman, H. N. (1970) Science 167, 1515. [13] Donohue, J. H. and Rosenberg, S. A. (1983) J. Immunol. 130, 2203. [14] Turk, J. L. and Poulter, L. W. (1972) Clin. Exp. Im-

munol. 10, 285. [15] Stockman, G. D., Hein, L. R., South, M. A. and Trentin, J. J. (1973) J. Immunol. 110, 277. [16] Hurme, M. (1979) J. Exp. Med. 149, 290. [17] Taswell, C., MacDonald, H. R. and Ceroltini, J.-C. (1980) Thymus 1, 119. [18] Turk, J. L. and Parker, D. (1982) Immunol. Rev. 65, 99. [19] Sy, M.-S., Miller, S. D. and Claman, H. N. (1977) J. Immunol. 119, 240. [20] Merluzzi, V. J., Welte, K., Savage, D. M., Last-Barney, V. and Mertelsmann, R. (1983) J. Immunol. 131, 806. [21] Staug, L., Cohen, I. R. and Feldman, M. (1973) Cell. Immunol. 7, 310. 122] Tormey, D. C., Fudenberg, H. H. and Kamin, R. M. (1967) Nature (London) 210, 282. [23] Gillis, S., Crabtee, G. R. and Smith, K. A. (1979) J. lmmunol. 123, 1624. [24] Gillis, S., Crabtree, G. R. and Smith, K. A. (1979) J. Immunol. 123, 1632. [25] Cupps, T. R. and Fauci, A. S. (1982) Immunol. Rev. 65, 133.