Ming-Sing Si, Bruce A. Reitz and Dominic C. Borie. Transplantation Immunology Laboratory, Department of Cardiothoracic Surgery, Stanford University School ...
Investigational New Drugs 23: 21–29, 2005. C 2005 Springer Science + Business Media, Inc. Manufactured in The Netherlands.
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Inhibition of lymphocyte activation and function by the prenylation inhibitor L-778,123 Ming-Sing Si, Bruce A. Reitz and Dominic C. Borie Transplantation Immunology Laboratory, Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA 95305-5407, USA
Key words: prenylation inhibitor, lymphocyte activation, immunosuppression, interleukin-2
Summary Prenylated Ras GTPases transduce signals from the T cell receptor, CD28 costimulatory receptor and IL-2 receptor. Since signals from these receptors mediate T cell activation, proliferation and survival, we hypothesized that the prenylation inhibitor L-778,123 would impart immunomodulation. The effect of L-778,123 on T cell activation (CD71 or CD25 surface expression) was determined by flow cytometry. Peripheral blood mononuclear cell (PBMC) proliferation in the presence of L-778,123 and/or cyclosporine (CsA) was determined by [3 H]thymidine incorporation. The ability of L-778,123 to inhibit IL-2 receptor signaling was investigated by measuring IL-2 induced proliferation in CTLL-2 cells and IL-2 prevention of apoptosis in activated human PBMC. L-778,123 inhibited lectin induced expression of CD71 and CD25 with IC50 ’s of 6.48 ± 1.31 µM and 84.1 ± 50.0 µM, respectively. PBMC proliferation was inhibited by L-778,123 with an IC50 of 0.92 ± 0.23 µM, and addition of CsA did not increase the potency. L-778,123 did not inhibit IL-2 and IFN-γ production by T cells. L-778,123 abrogated IL-2 induced proliferation of CTLL-2 cells with an IC50 of 0.81 ± 0.44 µM. However, L-778,123 minimally reversed the prosurvival effect of IL-2 in activated lymphocytes. IL-2 ligand and receptor production during T cell activation are relatively unaffected by L-778,123. However, the activation and proliferative effects of IL-2 on T cells are potently blocked by L-778,123. These results reveal a selective blockade of the IL-2 cytokine axis distal to the IL-2 receptor by the L-778,123 and warrant evaluation of prenylation inhibitors in treating transplant rejection and autoimmune diseases.
Introduction Transduction of activation signals in T cells involves the redundant participation of the Ras superfamily of GTPases. TCR ligation, the first step, or so-called signal 1 of T cell activation, leads to the activation of the Ras/MAPK signal transduction pathway [1–3]. Following TCR engagement, Ras has also been shown to synergize with the serine-threonine phosphatase calcineurin to activate the nuclear factor of activated T cells (NFAT), a transcription factor critical for the transcription of interleukin (IL) genes [4]. More recently, it has been demonstrated that T cell receptor activation leads to phospholipase Cγ mediated translocation of the nucleotide exchange factor RasGRP1 to the Golgi apparatus where it activates Ras [5]. Costimulation through CD28 enhances TCR signals and is essential for robust T cell activation by providing the so-called signal 2. CD28 costimulatory receptor
ligation with its ligand B7 leads to the activation of signaling pathways including another member of the Ras family, the small GTPase Rac [6, 7]. Rac is involved in coupling both the TCR and CD28 signaling pathways and also regulates the actin cytoskeleton, which is involved in the formation of the immunological synapse, one of the early events of successful T cell activation by an antigen presenting cell [6–8]. Effective T cell activation and proliferation requires another signal, or signal 3, which is provided by the ligation of T cell growth factors to their corresponding receptors on the surface of activated T cells. Here also, members of the Ras superfamily are involved as downstream signaling from the IL-2 receptor requires the participation of Ras [9, 10]. Moreover, the β subunit of the IL-2 receptor has been recently shown to mediate NK cell survival and prevention of apoptosis through signal transduction pathways that involve Rac [11].
22 In order to function, the Ras superfamily of GTPases undergo prenylation [i.e., farnesylation or geranylgeranylation) which allows those molecules to insert into lipid membranes where they activate their target effector molecules [12]. Because prenylation is generally necessary for Ras GTPase function and uncontrolled activation of Ras GTPases are prevalent in many cancers, Ras inhibitors, such as farnesyltransferase inhibitors, have been primarily developed as anticancer agents (reviewed in [13,14]). One of those agents, the prenylation inhibitor L-778,123, was shown to have activity against both farnesyltransferase and geranylgeranyltransferase, although it has more activity against farnesyltransferase as compared to geranylgeranyltransferase [15–17]. In the wake of those studies and of others performed in our laboratory to characterize the potential of various immunosuppressive drugs [18, 19], we were interested in investigating the immunomodulatory properties of prenylation inhibitors. Here we describe the in vitro immunomodulatory properties of the prenylation inhibitor L-778,123. The expected abrogation of T cell activation by L-778,123, presumably because of its activity against Ras and Rac, was confirmed in this model. In addition, we were also able to demonstrate that L-778,123 selectively blocked the IL-2 cytokine axis distal to the IL-2 receptor with ensuing inhibition of IL-2 receptor mediated cell proliferation.
Materials and methods Materials and cells Buffy coats from normal human subjects were obtained from the Stanford University Medical Center Blood Bank. Peripheral blood mononuclear cells (PBMC’s) were isolated from buffy coats by Ficoll centrifugation. Cells were cultured in RPMI 1640 (Invitrogen Co., Carlsbad, CA), supplemented with 10% FBS (v/v), sodium pyruvate, nonessential amino acids, β-mercaptoethanol, glutamine, penicillin and streptomycin, at a concentration of 1 × 106 cells/ml. Murine IL-2 dependent CTLL-2 cells (American Type Culture Collection, Manassas, VA) were maintained in complete RPMI 1640 media with 10% (v/v) T-STIM (BD Biosciences Discovery Labware, Bedford, MA). All cells were maintained at 37◦ C and 5% CO2 in humidified air. Phytohemagglutinin D (PHA) was purchased from Calbiochem (La Jolla, CA). Concanavalin A (ConA) was purchased from Sigma (St. Louis, MO). Media supplements were from Sigma unless indicated otherwise. The prenylation inhibitor L-778,123 was a generous gift from Merck Research Laboratories. L-778,123 stock solution was made by dissolving the compound in DMSO (Sigma)
and aliquots were stored at –80◦ C. L-778,123 stock solutions were thawed and serially diluted in PBS prior to experiments. Final concentration of DMSO in experiments was less than 0.6% (v/v). Cyclosporine (CsA) was obtained from Sigma and a stock solution was made in methanol and serially diluted in PBS prior to experiments. Final concentration of methanol in experiments was less than 0.1% (v/v). All monoclonal antibodies (mAbs) and their appropriate isotype controls were purchased from BD Biosciences Pharmigen (San Diego, CA). Recombinant human IL-2 (rIL-2) was purchased from R&D Systems (Minneapolis, MN).
T cell activation surface markers To determine if L-778,123 inhibited T cell activation, the expression of activation-associated surface markers were measured after PHA stimulation. PBMC’s (1 × 106 cells/ml) in complete media were treated with incremental concentrations of L-778,123 for 30 minutes prior to stimulation with PHA (5 µg/ml) for 72 hours in growth conditions. This method and its variations across blood donor species has been described in detail previously by our laboratory [20–22]. Briefly, at completion of the stimulation period, PBMC’s were washed with PBS and subsequently stained with anti-CD3ε-PE, anti-CD71-FITC and antiCD25-CyChrome monoclonal antibodies. After staining, cells were washed in PBS and fixed with PBS-buffered 1% formalin (v/v) prior to analysis on a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA). Data were analyzed using CellQuestTM Pro (BD Biosciences Immunocytometry Systems, San Jose, CA).
PBMC proliferation assay Using protocols previously reported in detail [20–22], human PBMC’s were incubated with incremental concentrations of L-778,123 and/or CsA for 30 minutes in complete media, stimulated with PHA (5 µg/ml) and then dispensed into 96 well microtiter plates in quadruplicate fashion (105 cells/well). Cells were allowed to proliferate in growth conditions for 60 hours, pulsed with 1 µCi/well [3 H] thymidine (Amersham Biosciences, Piscataway, NJ) and incubated for 12 more hours before harvesting cells on glass filtermats with a multichannel cell harvester. Filtermats were placed in plastic pouches with scintillation fluid and read on a scintillation counter (Wallac LKB Microbeta plus 1450, Turku, Finland) and data recorded as counts per minute (cpm).
23 Cytokine production assay Using protocols previously reported in detail [20–22], intracellular cytokine staining was performed to evaluate the effects of L-778,123 on T cell cytokine production. PBMC’s were pretreated with incremental concentrations of L-778,123 prior to stimulation with ConA (7.5 µg/ml). One hour after stimulation, brefeldin A was added (10 µg/ml) and cells were placed in growth conditions for 7 hours. Cells were then washed with PBS, stained with anti-CD3ε-PerCP and then fixed, washed and permeabilized with the IntraPrep Permeabilization Reagents (Beckman Coulter, Inc., Brea, CA) according to the manufacturer’s directions. PBMC’s were then incubated with anti-IL-2-FITC and anti-IFN-γ -PE, washed in PBS and resuspended in 1% formalin prior to analysis on a FACSCalibur flow cytometer and software CellQuestTM Pro by first scatter-gating on the lymphocyte population and then gating on CD3 positive cells. The percentages of IL-2 positive and IFN-γ positive CD3+ T cells were then recorded. At least 7,500 CD3+ T cells were analyzed for each sample.
methanol and incubating with RNAse A and propidium iodide (PI). Cells were then washed and resuspended in 1% PI solution and analyzed on a FACSCalibur flow cytometer. Events were then collected in a scatter gate, and the FL-2 area and width signals recorded using the doublet discrimination module. A low threshold was used to exclude debris and an event gate was drawn around apoptotic, G0 /G1 phase, S phase and G2 + M phase singlet events. A histogram was then created from the FL-2 area and a tight cursor region was created to include events to the left of the G0 /G1 peak. Twenty thousand events were collected in the event gate on a low flow rate.
Data analysis Fifty percent inhibitory concentrations (IC50 ’s) were determined by mathematical modeling using the software WinNonlin (Pharsight, Mountain View, CA). Data between control and treatment groups were compared using the two-tailed Student’s t test.
Results IL-2 response assay T cell activation surface markers The proliferative response of CTLL-2 cells induced by IL-2 was used to determine the effects of L-778,123 on the IL-2 receptor signaling pathways. CTLL-2 cells were rested in complete media without IL-2 or T-STIM for 4 hours prior to experiments. CTLL-2 cells were pretreated with varying concentrations of L-778,123 for 30 minutes prior to the addition of rIL-2 (50 ng/ml). Proliferation was carried out for 24 hours and assessed by a 6 hour [3 H] thymidine pulse with subsequent measurement of incorporation as described above.
Apoptosis assay The effects of L-778,123 on the ability of IL-2 to prevent apoptosis in human PBMC’s were investigated using assays described in detail elsewhere [23–25]. Briefly, PBMC’s were stimulated with PHA (1 µg/ml) for three to six days in complete RPMI 1640 media, and media was changed and fresh PHA added every two days. Activated PBMC’s were then washed extensively in PBS and resuspended in RPMI 1640 media without serum. Recombinant human IL-2 (5 ng/ml) and incremental concentrations of L-778,123 were added and cells were incubated for another 7 days in growth conditions. PBMC’s were also incubated in the same conditions but without rIL-2 to serve as a positive apoptosis control sample. At the end of the incubation period, apoptosis in T cells was detected by fixing cells in 1% formalin, permeabilizing with
Human PBMC’s were treated with incremental concentrations of L-778,123 prior to a 72 hour stimulation with PHA. The levels of expression of the activation surface markers CD71 (transferrin receptor) and CD25 (IL-2 receptor) on CD3+ T cells were then measured by flow cytometry. Figure 1 summarizes the results of four different stimulations of PBMC’s isolated from four different donors. L-778,123 treatment resulted in a dose dependent decrease in the expression of CD71 and CD25. Expression of CD25 was less sensitive to L-778,123 treatment (IC50 = 84.1 ± 50.0 µM) as compared to that of CD71 (IC50 = 6.48 ± 1.31 µM). Proliferation assays PBMC’s were treated with incremental concentrations of L-778,123 and then stimulated for 72 hours with PHA. Proliferation was measured by [3 H] thymidine uptake. L-778,123 inhibited lectin induced PBMC proliferation with an IC50 of 0.92 ± 0.23 µM, although significant inhibition was seen only starting at 20 µM (Figure 2). Addition of CsA (200 ng/ml) resulted in significant additive inhibition with L-778,123 as reflected in the significant inhibition achieved at a lower drug concentration tested (2.0 µM). However, potency of this combination remained unchanged reflecting the lack of synergy between these two agents (IC50 of 1.1 ± 0.68 µM).
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Figure 1. L-778,123 modestly inhibits T cell activation. PBMC’s treated with incremental concentrations of L-778,123 were stimulated with PHA for 72 hours and CD3 T cell activation was measured by the expression of CD71 and CD25 using flow cytometry. L-778,123 inhibits the expression of CD71 and CD25 with IC50 ’s of 6.48 ± 1.31 µM and 84.1 ± 50.0 µM, respectively. Data shown are the average (±SD) of 4 independent experiments.
Figure 2. L-778,123 inhibits PBMC proliferation. Drug treated PBMC’s were stimulated with PHA for 72 hours and proliferation measured by [3 H] thymidine incorporation. L-778,123 inhibited proliferation with an IC50 of 0.92 ± 0.23 µM. Addition of cyclosporine (CsA) at 200 ng/ml was resulted in additive inhibition as demonstrated by a significant decrease in proliferation at 0.2 µM, however the IC50 for the drug combination was 1.1 ± 0.68 µM, suggesting lack of synergism. Data shown are the average (±SD) of 4 independent experiments.
Cytokine production assay
IL-2 response assay
PBMC’s pretreated with L-778,123 were stimulated with ConA. ConA, in lieu of PHA, was chosen as the stimulant in these experiments because this lectin induced the most IL-2 and IFN-γ production by T cells (data not shown). L-778,123 did not inhibit the production of IL2 or IFN-γ , even at 100 µM, the highest concentration tested (Figure 3a and b). Furthermore, longer drug preincubation periods (16 hours) did not result in any inhibition of cytokine synthesis (data not shown).
The CTLL-2 cell line requires ligand binding to the IL2 receptor for survival and proliferation. To determine if dual prenylation inhibition abrogated signals from the IL2 receptor, CTLL-2 cells were pretreated with L-778,123 and then stimulated with IL-2. Proliferation, as a marker for IL-2 response, was then measured after 24 hours. L778,123 inhibited IL-2 induced CTLL-2 proliferation in a concentration dependent manner with an IC50 of 0.81 ± 0.44 µM (Figure 4a). Significant inhibition of CTLL-2
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Figure 3. (a) L-778,123 does not affect T cell IL-2 or IFN-γ production. PBMC’s were stimulated with ConA for 6 hours and then the percentage of CD3+ T cells positive for intracellular IL-2 and IFN-γ were determined by flow cytometry. Representative dot plots of CD3 + T cells are shown for unstimulated, stimulated and 100 µM L-778,123. (b) L-778,123, even at the highest concentration, did not affect cytokine production. Data shown are the average (±SD) of 3 independent experiments. Preincubation for 16 hours with L-778,123 did not result in increased inhibition (data not shown).
proliferation was noted at all L-778,123 concentrations tested. This was in contrast to CsA, which showed no inhibition of CTLL-2 proliferation, even at high concentrations (Figure 4b). Apoptosis assay To determine if dual prenylation inhibition interfered with survival signals from the IL-2 receptors in lymphocytes, we evaluated the ability of L-778,123 to cause apoptosis in IL-2 treated, activated PBMC’s. Addition of rIL-2 to activated T cells resulted in an attenuated level of apoptosis as compared to T cells incubated in media without rIL-2 (Figure 5). Addition of incremental amounts of L778,123 to activated T cells did not significantly prevent the ability of rIL-2 to prevent apoptosis, although at high concentrations (100 µM) L-778,123 showed a trend towards increased apoptosis (Figure 5). Discussion Preliminary results obtained with prenylation inhibition in cancer and a variety of other diseases and conditions
have provided the proof of concept of modulating Ras GTPase function by prenylation inhibition as a new and potentially effective therapeutic approach [26–29]. Here we evaluated the immunomodulatory characteristics of a potent prenylation inhibitor, L-778,123. L778,123 has been shown to inhibit the farnesylation and geranylgeranylation of Ki4B-Ras, and to inhibit farnesyltransferase and geranylgeranyltransferase in vitro with IC50 ’s of 2 nM and 98 nM, respectively [15]. In a phase I evaluation of L-778,123, it was demonstrated that intravenous infusion of this compound in cancer patients resulted in the inhibition of the farnesylation of HDJ-2, a farnesylated heat shock protein that has been used as a pharmacodynamic marker of prenylation inhibitor activity, in PBMC’s isolated from these patients [17]. In the current studies, we verified that, as expected, L778,123 potently inhibited cell proliferation. Unexpectedly, however, the compound inhibited expression of T cell activation markers only at high concentrations and did not affect T cell IL-2 or IFN-γ production. Certainly, Ras inhibition by transfection of a dominant negative Ras, has led to the inhibition of IL-2 production [30]. To resolve this apparent contradiction of relatively intact T cell
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Figure 4. (a) L-778,123 potently inhibits proliferative signals from the IL-2 receptor. The IL-2 dependent murine T cell line CTLL-2 was rested and incubated with incremental concentrations of L-778,123 prior to stimulation with IL-2 (50 ng/ml) for 24 hours. Proliferation was measured by [3 H] thymidine incorporation. L-778,123 potently inhibited IL-2 induced CTLL-2 proliferation with an IC50 of 0.81 ± 0.44 µM. (b) On the other hand, cyclosporine, even at high concentrations, did not inhibit IL-2 induced CTLL-2 proliferation. Data shown are the average (±SD) of 4 independent experiments.
activation and immediate cytokine production yet potent inhibition of long-term (∼72 hours) proliferation, we hypothesized that treated T lymphocytes are altered in their response to proliferative and possibly survival signals mediated by IL-2. Previous investigation by others showing that Rac is involved in mediating Syk → Rac → Akt → bcl-2 survival signals from the β subunit of the IL-2 receptor in NK cells provided further, fundamental basis for this hypothesis [11].
The results of our second set of experiments involving the IL-2 dependent CTLL-2 T cell line partially support our above hypothesis. L-778,123 treatment of these T cells led to inhibition of IL-2 induced proliferation. Hence, L-778,123 is a unique immunomodulatory agent that has relatively few effects on T cell activation, yet selectively blocks signal 3 of T cell activation. This blockade of signal 3 deserves even further qualification because it occurs distal to the IL-2 receptor while
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Figure 5. L-778,123 does not affect the prosurvival effects of IL-2 on activated T cells. PBMC’s were stimulated for at least 3 days with PHA and then treated with L-778,123 and recombinant human IL-2 (rIL-2, 5 ng/ml) for 7 days. Apoptosis was measured by PI staining of methanol permeabilized and RNAse treated cell by using flow cytometry. Treatment with rIL-2 prevented apoptosis in activated CD3 T cells. L-778,123 did not result in a significant increase in apoptosis at all concentrations tested. Data shown are the average (±SD) of 2 independent experiments.
preserving IL-2 receptor (CD25) expression and IL-2 production. Prenylation inhibition by L-778,123 did not, however, block the survival signals from the IL-2 receptor in activated PBMC’s. One can speculate that L-778,123 may inhibit other Ras GTPases than that involved in antiapoptotic signals to explain the failure of this prenylation inhibitor to block the pro-survival signals from the IL-2 receptor, but further investigation will be needed to determine this. Another possibility is that the redundancy of the antiapoptotic mechanisms initiated by the IL-2 receptor (e.g., JAK3 independent and MAPK pathways) compensated for defects in the Syk → Rac → Akt pathway caused by L-778,123 [31]. Further investigation is needed to delineate the specific alterations in signal transduction from the IL-2 receptor caused by L-778,123. We and other investigators have shown previously that sirolimus (rapamycin), mycophenolic acid and the JAK3 inhibitor CP-690,550 are able to prevent acute and chronic allograft rejection in various animal models [19, 32–34]. Like L-778,123, CP-690,550, sirolimus and mycophenolic acid are able to inhibit the proliferative signals from the IL-2 receptor [19, 34–36]. Furthermore, L-778,123, sirolimus and mycophenolic acid do not affect the survival signals from the IL-2 receptor. A distinctive characteristic of L-778,123 is that it has little effect on CD25 expression, unlike what our laboratory found for mycophenolate mofetil [22], sirolimus [37] and more recently CP-690,550, which appears to inhibit the upreg-
ulation of CD25 on lectin stimulated T cells (Paniagua, R. et al., manuscript in preparation). It would be interesting to surmise that L-778,123 would have less effect on CD4+ CD25+ regulatory T cell generation than other immunosuppressive agents. CD4+ CD25+ regulatory T cells have been shown to promote tolerance after transplantation and prevent the development of allergy, graft versus host disease and hypersensitivity [38, 39]. Although the Ras superfamily of GTPases is probably the most studied group of prenylated signal transduction proteins, other important prenylated proteins may pose as important targets of L-778,123 in our experiments and thus may mediate these immunomodulatory effects via alternative mechanisms. Recently, the significance of farnesyltransferase inhibitors in the mitotic checkpoint have been suggested as a potential antiproliferative mechanism for these agents [40]. CENP-E is a farnesylated protein expressed during mitosis and regulates the progression of the cell cycle from G2 to M phase mitotic checkpoint. Treatment of cancer cell lines with the farnesyltransferase inhibitor SCH 66336 interfered with the ability of CENP-E to associate with microtubules [41]. The association of CENP-E with kinetochores was not studied here, and could certainly represent an additional mechanism by which L-778,123 prevented inhibition in lectin activated PBMC’s and IL-2 stimulated CTLL-2 cells. Finally, our experiments were not designed to determine the respective effects of farnesyltransferase and geranylgeranyltransferase inhibition by L-778,123 on
28 resulting lymphocyte activation and function. We can only speculate at this juncture that L-778,123’s inhibition of farnesyltransferase contributed more to the observed effects on lymphocytes because it has a 50-fold more potent activity against this enzyme as compared to geranylgeranyltransferase [15]. Further studies comparing the effects of very selective farnesyltransferase inhibitors and geranylgeranyltransferase inhibitors on lymphocyte activation and function are needed to address this question. It is conceivable that selective blockade of signal 3 of T cell activation may be beneficial in immune diseases that are mediated by detrimental activation of select populations of T cells such as in organ transplantation rejection which is due to graft reactive T cells or in autoimmune diseases which are due to self reactive T cells. Indeed, we have recently shown that the novel JAK3 inhibitor CP-690,550 which is a selective signal 3 blocker, effectively prevents allograft rejection in a preclinical model of kidney transplantation [19, 42, 43] which suggest that selective signal 3 inhibitors may lead the way to a new era in the field of small immunosuppressive drugs [44]. Furthermore, other JAK3 inhibitors, such as AG-490 and PNU156804 were shown to inhibit IL-2 induced T cell proliferation, albeit at concentrations 10 to 20 times higher than what we have measured in the current study for L778,123. Thus, we feel that the in vitro evidence presented here warrants careful investigation of L-778,123 and other similar compounds in animal models to determine if signal 3 blockade by these compounds is effective in treating and preventing autoimmune disease and transplant rejection. Acknowledgments The authors would like to acknowledge Merck Research Laboratories for generously supplying L-778,123 and Kathy Richards for assistance in the preparation of this manuscript. This work was supported by a NIH NRSA (1F32AI051094) and an American College of Surgeons Resident’s Research Scholarship awarded to MSI. The Transplantation Immunology Laboratory is supported by the Dr. Ralph and Marian C. Falk Medical Research Trust (Chicago, IL). References 1. Downward J, Graves JD, Warne PH, Rayter S, Cantrell DA: Stimulation of p21ras upon T-cell activation. Nature 346: 719–723, 1990 2. Franklin RA, Tordai A, Patel H, Gardner AM, Johnson GL, Gelfand EW: Ligation of the T cell receptor complex results in activation of the Ras/Raf-1/MEK/MAPK cascade in human T lymphocytes. J Clin Invest 93: 2134–2140, 1994
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