Published online before print September 15, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pfizer Global Research and Development, Department of Antibacterials, Immunology and Cancer, Groton, Connecticut
1 Correspondence: MS8220-2326, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340. E-mail: maryrose_j_conklyn{at}groton.pfizer.com
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
chain, an integral component of cytokine receptors of the interleukin (IL)-2 family, including IL-4, -7, -9, -15, and -21. CP-690550 is a JAK3 inhibitor with immunosuppressive properties under development for transplantation. We evaluated alterations in circulating lymphocyte subsets in cynomolgus monkey blood following chronic (3-week), oral CP-690550 administration. Natural killer (NK) and CD8+ T cell numbers were reduced in a dose- and time-dependent manner; the latter was a primary effect on memory subsets. CD4+ T and B cell numbers were unaffected or slightly increased, respectively. NK cell numbers were reduced 
80% (vs. 35% in vehicle-treated animals) and returned to baseline levels within 3 weeks following treatment cessation. CD8+ T cells declined by a maximum 43% (vs. 25% for vehicle-treated animals) but rebounded significantly (300%) within 2 weeks after the last dose. Although CP-690550 did not result in reduction of CD4+ T cell number, these cells also increased (225%) within 2 weeks of treatment cessation. IL-15 is important for maintaining homeostasis of these cell types, and CP-690550 inhibited IL-15-induced CD69 expression in NK cells [inhibitory concentration 50% (IC50)=48.0±8.4 nM] and CD8+ T cells (IC50=16.2±1.5 nM).
Key Words: memory T cells • IL-15 • CD69 • transplantation
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
chain (c), an integral component of receptors of the interleukin (IL)-2 cytokine family. Members of this family include IL-4, IL-7, IL-9, and IL-15, all of which use the JAK-signal transducer and activator of transcription pathway for intracellular signaling [3
]. These cytokines are critical for lymphocyte differentiation, survival, and function. The X-linked form of severe combined immunodeficiency (SCID) has mutations in c that resulted in the absence of T and natural killer (NK) cells [4
, 5
]. Similarly, a patient with defective expression of the IL-2/IL-15 receptor ß (IL-2Rß/IL-15Rß) subunit had a NK cell-deficient form of SCID [6
]. In mice, the genetic ablation of JAK3 or c leads to dramatic defects in T, B, and NK cell development [7
8
9
].
CP-690550 is a recently described selective, small molecule inhibitor of JAK3. It is a potent inhibitor of immune responses in such in vitro assays as IL-2-mediated human T cell blast proliferation [inhibitory concentration 50% (IC50)=11 nM] and mixed lymphocyte reaction (IC50=87 nM) [10
]. In vivo studies demonstrated that CP-690550 reduced a delayed-type hypersensitivity response and extended cardiac allograft survival in murine models [11
]. Analysis of circulating lymphocyte subsets in mice receiving chronic CP-690550 dosing revealed reductions in T, B, and NK cell numbers, which were consistent with the phenotype of c and JAK3 knockout (KO) mice [7
8
9
]. As IL-7 is an important regulatory cytokine for T and B cell development in this species, some of the lymphoid effects could be attributed to blockade of IL-7R signaling by CP-690550 [12
, 13
]. In addition, the loss of NK cells is consistent with the known role of IL-15 for their homeostasis [14
15
16
].
CP-690550 also demonstrated robust extension of life-supporting kidney allograft survival in cynomolgus monkeys [10 ]. Given species differences that exist for cytokines in lymphocyte development, we explored the effects of this novel JAK3 inhibitor on lymphocyte subsets in the peripheral blood of nonhuman primates following chronic oral administration. The selective reduction in NK and CD8+ T cells observed following JAK3 inhibition is consistent with the ability of the compound to block IL-15R signaling and function. These data may provide not only a biomarker for CP-690550 activity but may also improve our understanding of the mechanism(s) by which JAK3 inhibition prolongs allograft survival in this species.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(SP34) phycoerythrin (PE), CD4 (L200) peridinin chlorophyll protein (PerCP), CD8
(RPA-TA) fluorescein isothioycanate (FITC) or allophycocyanin (APC), CD8 (SK1) PerCP, CD16 (3G8) FITC, CD20 (2H7) FITC, CD28 (CD28.2) FITC, CD69 (FN50) PerCP, CD95 (DX2) APC, integrin ß7 (FIB504) PE; and for human studies, CD3 (SK7) PerCP, CD16 (3G8) PE, CD56 (B159) PE, CD8 (RPA-TA) APC. CD69 (CH/4) FITC from Caltag (Burlingame, CA) was used in the whole blood, IL-15-mediated CD69 induction assay. CD8ß (2ST8.5H7) PE was purchased from Immunotech (Marseille, France).
Animals
Mauritius-origin adult cynomolgus monkeys (Macaca fascicularis) were used in these experiments. Animals weighed between 3.5 and 8.8 kg, were in good clinical health, and acclimated to the venipuncture and oral dosing procedures prior to initiation of the studies. All procedures were in strict accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Numbers of male and female animals were evenly distributed between the treatment groups. CP-690550 was prepared in 0.5% methylcellulose in distilled water and administered twice daily (12 h apart) by oral gavage.
Lymphocyte phenotyping
Monkey whole blood was collected into EDTA microtainer tubes. Total leukocyte counts of cynomolgus whole blood were determined using an Abbott Cell-Dyn 3500 (Abbott Laboratories, Abbott Park, IL). The following antibody panels were used: CD16 FITC x CD3 PE x CD4 PerCP x CD8 APC or CD20 FITC x CD3 PE. Manufacturer-recommended isotype controls were used. Numbers of specific lymphocyte subsets were calculated by multiplying the total white blood cell counts by the percent total of each specific lymphocyte subset. Baseline values were determined by averaging two independent measurements obtained at least 2 weeks prior to treatment commencement. Effects of compound administration on lymphocyte subsets were expressed as a percent of the baseline determination.
IL-15-mediated induction of CD69 expression
Heparinized human or cynomolgus monkey blood was incubated with various concentrations of human recombinant IL-15 (R&D Systems, Minneapolis, MN) for 4 h at 37°C. The antibody panel used for human blood studies was CD69 FITC x CD16 PE and CD56 PE x CD8 APC. For cynomolgus blood studies, the panel used was CD8 FITC x CD3 PE x CD4 PerCP x CD69 APC. In these studies, cynomolgus NK cells were defined as CD8+ CD3– cells. To compare CD69 up-regulation on naïve versus memory CD8+ T cells, the following antibody panel was used: CD28 FITC x CD8ß PE x CD69 PerCP x CD95 APC. To determine effects of CP-690550, whole blood was first incubated at 37°C for 5 min with varying concentrations of compound then incubated with IL-15 (10 ng/ml) for 4 h at 37°C.
Fluorescein-activated cell sorter (FACS) analysis
Fluorescent immunostaining was quantitated using a Becton Dickinson FACSCalibur equipped with a 488 argon laser and a 635-nm red diode laser. Data acquisition and analyses were performed using CellQuestPro software (BD Biosciences). For phenotypic analyses, 10,000 lymphocytes gated by light-scatter profile were collected. For the IL-15-mediated CD69 up-regulation studies, data acquisition was terminated after 2500 gated NK cells were acquired.
Statistics
Statistical analysis of changes in lymphocyte subsets was performed by one-way ANOVA. Significance was defined as P < 0.05.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
3 weeks later. B cell numbers (Fig. 1C)
in monkeys receiving CP-690550 at 30 mg/kg/d were increased moderately relative to vehicle-treated animals throughout the entire observation periods. CD4+ T cells numbers (Fig. 1D)
were not significantly different between treatment groups for the first 21 days of treatment. However, after cessation of CP-690550 dosing, the CD4+ T cell number increased 225% over baseline, which was coincidental with the increase of the CD8+ cells. |
View this table: [in a new window] |
Table 1. Hematology Parameters in Cynomolgus Monkey following 21 Days of 30 mg/kg/d Administration of CP-690550
|
 View larger version (33K): [in a new window] |
Figure 1. Alterations in circulating lymphocyte subsets following chronic CP-690550 exposure. Cynomolgus monkeys received 0.5% methylcellulose vehicle (n=6) or CP-690550 (n=7) at 30 mg/kg/d for 21 days. Animals continued to be monitored for 7 weeks following cessation of dosing (indicated by  ). Circulating NK cells (A), CD8+ T cells (B), CD20+ B cells (C), and CD4+ T cells (D) were identified by the antibody panels as described in Materials and Methods. Data are expressed for each animal as a percentage of the average of two cell number determinations made prior to compound administration (% Baseline).
|
 View larger version (19K): [in a new window] |
Figure 2. Dose-dependent reductions in circulating NK cell numbers following chronic CP-690550 exposure. Cynomolgus monkeys received 0.5% methylcellulose vehicle (n=12) or CP-690550 at 1 (n=7), 10 (n=7), or 30 (n=7) mg/kg/d. Circulating NK cells were identified with the antibody panel CD16 x CD3 x CD8 as described in Materials and Methods. Data are expressed for each animal as a percentage of the average of two NK cell numbers made prior to compound administration (% Baseline).
|
 View larger version (22K): [in a new window] |
Figure 3. Flow cytometric analysis of cynomolgus CD4+ and CD8+ memory and naïve T cells. Memory and naïve circulating lymphocyte subsets were determined and defined by an antibody panel as described previously [17
]. The fluorescent profiles of gated CD4+ (A) and gated CD8+ (B) naïve (CD28med/highCD95neg/low, green dots) and putative central memory (CD28med/high CD95+, red dots) and putative effector memory T cells (CD28neg/lowCD95+, blue dots) are presented.
|
 View larger version (14K): [in a new window] |
Figure 4. Selective reduction in circulating effector memory CD8+ T cell numbers following 2 weeks of CP-690550 dosing. Cynomolgus monkeys received 0.5% methylcellulose vehicle (n=6) or CP-690550 (n=6) at 10 mg/kg/d for 14 days. Circulating CD4+ (A) and CD8+ (B) T cell subsets (memory and naïve) were quantitated using the antibody panel described in Figure 3
.
|
 View larger version (19K): [in a new window] |
Figure 5. IL-15 induced CD69 expression on monkey and human peripheral blood NK and CD8+ T cells. Heparinized whole blood from normal human donors (n=6) or untreated cynomolgus monkeys (n=6) was incubated with IL-15 for 4 h at 37°C. CD69 expression on NK cells (A) and CD8+ T cells (B) was then determined. Submaximal induction of CD69 expression was achieved at an IL-15 concentration of 10 ng/ml for NK and CD8+ T cells.
|
|
View this table: [in a new window] |
Table 2. IL-15-Induced CD69 Expression on Human and Monkey Peripheral Leukocytes
|
 View larger version (15K): [in a new window] |
Figure 6. IL-15 selectively induced CD69 expression on peripheral blood cynomolgus memory CD8+ T cells. Heparinized whole blood from untreated cynomolgus monkeys (n=4) was incubated with IL-15 at 10 ng/ml for 4 h at 37°C. CD8ß was used to identify CD8+ T cells. Memory and naïve cells were identified by CD95/CD28 expression as determined in Figure 3
. Expression of CD69+ cells in different CD8+ T cell subsets was examined (A). (B) The same data are expressed as fold increase in CD69+ cells relative to the buffer-treated control sample for each of the CD8+ T cell subsets.
|
 View larger version (15K): [in a new window] |
Figure 7. CP-690550 inhibited IL-15-induced CD69 expression in cynomolgus and human peripheral blood NK cells and CD8+ T cells. Heparinized whole blood from normal human donors (n=4) or untreated cynomolgus monkeys (n=4) was incubated with CP-690550 at 37°C for 5 min followed by 4 h incubation with 10 ng/ml IL-15 at 37°C. CD69 expression on NK cells (A) and CD8+ T cells (B) was determined and expressed as percent control samples that had not been treated with CP-690550. The IC50 for CP-690550 was 62.6 ± 10.0 for human NK cells and 48.0 ± 8.4 nM for cynomolgus NK cells. The IC50 for CP-690550 in T cells was 24.6 ± 2.3 nM for human T cells and 16.2 ± 1.5 nM for cynomolgus T cells.
|
 View larger version (20K): [in a new window] |
Figure 8. IL-15-induced CD69 expression in cynomolgus central and memory CD8+ T cells was equally sensitive to inhibition by CP-690550. Heparinized whole blood from untreated cynomolgus monkeys (n=4) was incubated with CP-690550 at 37°C for 5 min followed by a 4-h incubation with 10 ng/ml IL-15 at 37°C. CD69 expression was determined and expressed as percent control samples that had not been treated with CP-690550.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Despite the myriad of cytokines that affect lymphocyte homeostasis, the selective reduction in NK and memory CD8+ T cells observed following CP-690550 administration to cynomolgus monkeys is consistent with emerging data about the importance of IL-15 for the homeostasis of these cells. Mice genetically deficient in IL-15 or IL-15R subunits exhibited selective defects in NK and CD8+ T cells, the latter being primarily of the memory phenotype [15 , 16 ]. Conversely, transgenic mice that overexpressed IL-15 had expanded populations of the same cells [22 ]. Of the lymphocyte subsets affected by this cytokine, NK cell development appears most dependent, a response that may be related to the degree of CD122 expression on these cells [23 , 24 ]. In our experiments, the decline in peripheral NK cell numbers appeared within 1 week of initiating JAK3 inhibition and suggests effects on cell processes other than development alone. IL-15 has been shown to be important for the survival of mature NK cells, an effect that appears mediated via Bcl-2 [14 , 25 ]. This cytokine also prevented apoptosis of human NK cells in a dose-dependent manner and was capable of sustaining survival of these cells for up to 8 days in the absence of serum [26 ]. To verify that CP-690550 does affect IL-15 signaling, we demonstrated its ability to inhibit in vitro CD69 up-regulation in cell populations affected by the compound in vivo. In fact, CP-690550 plasma levels achieved following doses of 10 and 30 mg/kg/d were 19.9 ng/ml and 73 ng/ml, respectively, which is nearly equal to or exceeded the IC90 of the compound for inhibition of IL-15-induced CD69 up-regulation on NK cells (240±49.6 nM; n=4). NK cell expansion and differentiation are promoted by IL-21, another cytokine that uses JAK3 signaling and synergizes with IL-15 [27 ]. It is possible that CP-690550 produced some of its effects via inhibition of this cytokine’s signaling as well.
Chronic CP-690550 administration also significantly reduced the number of circulating memory CD8+ T cells. These data cannot simply be explained by the fact that memory lymphocytes have a more rapid background rate of proliferation than naïve cells [17
, 28
], as CD4+ memory T cell numbers were unaffected during the treatment period. At least two c cytokines have been shown to be responsible for CD8+ memory cell development: IL-7 and IL-15, the signaling of which would be affected by JAK3 inhibition. In the absence of IL-15, CD8+ memory T cells gradually decline in number as a result of reduced cell proliferation; it has been proposed that nearly 80% of memory phenotype CD8+ T cells proliferate within a 5-week period [28
]. Murine CD8+ memory cells can use IL-7 or IL-15 to undergo homeostatic proliferation, which fails to occur in the absence of both [29
]. Continued survival of memory CD8+ T cells also appears to rely on these cytokines. IL-15 modulates the expression of the antiapoptotic protein Bcl-2 in these cells [30
]. Depletion of IL-7 also reduces memory cell survival in vivo, although less consistently or potently as IL-15 [31
]. In fact, increased expression of the IL-7R chain identifies CD8+ T cells, which will differentiate into memory cells [32
]. It is interesting that a marked increase was observed in CD8+ T cell numbers following discontinuation of CP-690550 treatment. However, naïve versus memory T cells were not analyzed at this stage; therefore, it is unknown whether the increase represented a general proliferation of T cells or of specific subpopulations. It is possible that the rebound effect occurred as a result of increased IL-7 levels, which have been reported upon T cell depletion [33
] concurrent with reversal of blockade of IL-7R signaling. The cytokine has been shown to increase proliferation of peripheral T cells in nonhuman primates, thereby increasing peripheral homeostatic proliferation [34
]. Although CP-690550-treated animals did not demonstrate a decline in peripheral CD4+ T cell number during the 21 days of compound administration, an increase in these cells (>200% of baseline) occurred within 2 weeks after termination of compound administration. This effect was not observed in the vehicle control group and paralleled the CD8+ T cell increase, consistent with the hypothesis that there was an overproduction of a cytokine such as IL-7, which promotes CD4+ and CD8+ T cell proliferation [35
].
CP-690550 monotherapy significantly improved renal allograft survival in nonhuman primates when administered at doses comparable with those described here [10
]. Given the broad range of cytokines affected as a consequence of JAK3 inhibition, the precise molecular mechanism responsible for efficacy may be difficult to ascertain. However, it is clear that CP-690550 affected NK and CD8+ T cell numbers, both of which may play a role in allograft rejection. It has been suggested that long-term allograft survival in humans is impaired as a result of the persistence of memory T cells, which may be resistant to currently used immunosuppressive therapies [36
]. NK cells have been implicated in rejection of bone marrow allografts [37
] as well as solid organ transplants [38
]. Administration of CP-690550 to cynomolgus monkeys has also been shown to affect cell function as measured by its ability to attenuate IL-2-induced interferon- production, CD25 expression, and proliferation in lymphocytes [10
].
In conclusion, these studies have demonstrated a reduction in selected circulating lymphocyte subsets following JAK3 inhibition by the small molecule inhibitor CP-690550. These findings are consistent with the SCID phenotype observed in humans with disrupted JAK3 signaling. Interruption of the IL-15R as well as IL-7R signaling and hence their concomitant functions may play an important role in the immunosuppressive effects observed following CP-690550 administration. The depletion of NK cells and a subset of CD8+ T cells, in combination with other effects on lymphocyte functions described previously [10 ], contributes to the robust efficacy observed in nonhuman renal transplant models and supports the promise of CP-690550 as a valuable therapy for the treatment of human allograft rejection.
Received May 10, 2004; revised August 2, 2004; accepted August 17, 2004.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
 |
|
 |
|
  F. Erdmann, M. Weiwad, S. Kilka, M. Karanik, M. Patzel, R. Baumgrass, J. Liebscher, and G. Fischer The Novel Calcineurin Inhibitor CN585 Has Potent Immunosuppressive Properties in Stimulated Human T Cells J. Biol. Chem., January 15, 2010; 285(3): 1888 - 1898. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
