Effects of JAK3 Inhibition with CP-690,550 on Immune Cell... : Transplantation (original) (raw)
Advances in immunosuppressive therapies have contributed significantly to the success of organ transplantation. However, because of the ubiquitous distribution of their molecular targets, current immunosuppressive drugs have numerous side effects that lead to significant morbidity in organ transplant recipients (1–5). This has motivated the investigation and development of alternate targets of immunosuppression. We recently demonstrated that specifically inhibiting Janus kinase (JAK) 3, a key lymphocyte cytoplasmic tyrosine kinase, resulted in effective and well-tolerated immunosuppression in rodent and nonhuman primate (NHP) models of organ allotransplantation (6–11). Hence, work from our group and others (reviewed in (12–14)) clearly identifies JAK3 inhibition as a promising new modality of immunosuppression.
The JAKs are large cytoplasmic tyrosine kinases which are the most proximal element in a signal transduction cascade that transmits signals from the Type I cytokine receptors to the nuclei within minutes of ligand binding (15). Whereas some JAKs (JAK1, JAK2, Tyk2) are used by a variety of cytokine receptors, JAK3 is used only by Type I cytokine receptors which contain the common gamma chain (γc) (16). Interestingly, JAK3 has a restricted and regulated expression and is expressed at high levels in natural killer (NK) cells and thymocytes and is inducible in T-cells, B-cells and myeloid cells but is not expressed in resting T-cells (15). Targeting JAK3 would therefore, theoretically, offer immune suppression where it is needed, i.e., on cells actively participating in rejection, without resulting in any effects outside of these cell populations.
Following our in vitro and in vivo demonstration of the potency of the specific JAK3 inhibitor CP-690,550 in rodents and NHPs, we were particularly interested in the evaluation of its effects on the distribution and function of NHPs immune cell populations. Our laboratory has previously developed pharmacodynamic (PD) assays incorporating flow cytometric analyses that focus on end products of specific intracellular biochemical cascades in lymphocytes, including cell-surface markers of lymphocyte activation, cytokine production by T-cells and lymphocyte proliferation. The relevancy of these assays in the estimation of the resulting effects of candidate immunosuppressive drugs has been established in small (17–19) and large (20–22) animal models and was recently verified in human blood (20, 23–25) and in stable human recipients of kidney allografts (26).
In the current study, we investigated the effects of JAK3 inhibition on NHP immune cells using these PD assays in three different settings: (a) in vitro with CP-690,550 added to whole blood from naïve animals (b) in vivo with blood drawn from NHPs dosed orally with CP-690,550 and (c) in vivo from blood drawn from NHP recipients of kidney allografts treated with CP-690,550. In addition to these assays, effects of JAK3 inhibition on lymphocyte subsets—including pilot data on CD8+ memory T-cell populations—were studied. Our results show that JAK3 inhibition reproducibly results in a particular signature characterized by significantly reduced T-cells and NK cell numbers, alterations of lymphocyte activation and proliferation, and by significant attenuation of the capacity of T-cells to produce IFN-γ.
METHODS
Animals
Wild-caught, male cynomolgus monkeys (Macaca fascicularis) originating from Mauritius (4–6 kg) were purchased from Biomedical Resource Foundation (Houston, TX). The animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, prepared by the National Research Council and published by the National Academy of Sciences (National Academy Press, Washington DC, 1996) and Stanford University Animal Care Guidelines. This study was approved by the Institutional Animal Care and Use Committee at Stanford University.
Treatment of Blood In Vitro with CP-690,550
A stock solution (2.0 mM) was prepared by first dissolving CP-690,550 (MW: 313, Pfizer, Inc., Groton, CT) in dimethylsulfoxide (DMSO) (Sigma Chemical Co., St. Louis, MO) and then diluting in methanol (Fisher Scientific, Pittsburgh, PA). All working drug solutions were subsequently made by serial dilutions in culture medium. CP-690,550 was added to whole blood to produce final concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 μM. The concentration of DMSO in blood was ≤ 0.05% by volume, and did not affect lymphocyte function (data not shown). CP-690,550 was allowed to equilibrate in the blood for 30 min at 37°C in a humidified 5% CO2-air incubator to ensure homogeneous distribution prior to stimulation.
Dosing of Nontransplanted Animals with CP-690,550
Three naïve, nontransplanted animals dosed with CP-690,550 (treatment group) had blood drawn for PD assays. CP-690,550 was prepared in 0.5% a carboxymethylcellulose vehicle to obtain a final concentration of 15 mg/ml. Treated animals were sedated and dosed with CP-690,550 (15 mg/kg) twice daily (BID) by oral gavage for a total of 8 days at which time dosing was discontinued. Blood was collected and analyzed on days –6 and –4 prior to the first CP-690,550 dose to establish baseline (day 0) values. Blood was subsequently collected on days 1, 4 and 8 at 0, 3 and 12 hr following the morning CP-690,550 dose. Cell subsets were analyzed in the same animals on days 9, 15 and 21. Blood was drawn from two other naïve nontransplanted animals (vehicle control group) dosed with a 0.5% carboxymethylcellulose vehicle and analyzed at time points similar to the treatment group.
Treatment of Transplanted Animals with CP-690,550
Nine recipients of ABO blood group-matched and MLR-mismatched life-supporting kidney allografts were treated BID with CP-690,550 (average dosing; 17.4±9.6 (SD) mg/kg/day). Details on the clinical course of these animals are reported elsewhere (6). Blood was collected and analyzed on days –14 and –7 pretransplant to establish baseline (day 0) values. Subsequently, blood was collected and analyzed on a weekly basis. For the purpose of the current study, and to permit group analysis of animals with different survival times and outcomes, three individual posttransplantation time-points were considered: (a) two weeks after transplantation and initiation of drug therapy (Day 14), (b) two weeks prior to terminal rejection or completion of study (by design, 90 days posttransplantation) (Preterm), and (c) at terminal rejection or completion of study (Endpoint).
Reagents
Culture medium (CM) was prepared using RPMI-1640 (Life Technologies, Rockville, MD) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10 mM l-glutamine (all obtained from Sigma). Concanavalin A (Con A, Vector Laboratories, Inc., Burlingame, CA), phorbol 12-myristate 13-acetate (PMA, 100 μg/ml), brefeldin A (BFA, 5 mg/ml), propidium iodide (PI), ribonuclease A (RNAse) (all reagents from Sigma) and ionomycin calcium (Iono, ICN Biomedical, Inc., Costa Mesa, CA) were prepared as described elsewhere (20, 22, 27). Interleukin-2 (IL-2, Roche, Basel, Switzerland) was dissolved in CM and stored at –20°C. Concentrated phosphate-buffered saline (PBS) was from Coulter Corporation (Miami, FL), formaldehyde solution, from Fluka Chemie AG (Buchs, Switzerland) and methanol, from Fisher Scientific. Lysis and permeabilization buffers were prepared as described (22). The monoclonal antibodies (mAb) anti-CD3, anti-CD25, anti-CD71, anti-CD4, anti-CD8α, anti-CD8β, anti-CD16, anti-CD20, anti-CD28, anti-CD95, anti-interferon (IFN)-γ, and their respective isotype controls were purchased from Pharmingen (San Diego, CA). Anti-PCNA mAb (clone PC-10) was purchased from Dako Corporation (Carpinteria, CA) and the PCNA staining mixture was prepared as described previously (28).
Pharmacodynamic Assays
Assessment of IL-2-Mediated IFN-γ Production by T-cells
To determine if CP-690,550 inhibited Th1-specific cytokine production, the expression of intracellular T-cell IFN-γ production was measured after mitogenic whole blood stimulation as described previously (22, 28). In brief, blood was drawn from either treated or non-treated animals and stimulated in vitro with a combination of PMA (14.4 ng/ml), ionomycin (0.70 μg/ml) and IL-2 (9.3 ng/ml) in the presence of BFA. Samples were analyzed using an Epics XL-MCL flow cytometer using System II software (Coulter Corp., Miami, FL). Unstimulated blood cultures were used as negative controls. Specificity controls included replacement of primary mAbs with isotype mouse immunoglobulins. A minimum of 5000 events was analyzed per sample. IFN-γ data was expressed as the percentage of lymphocytes that were stained double positive for CD3 and IFN-γ (20, 22).
Assessment of T-cell Surface Activation Antigens and Proliferation
T-cell activation was assessed on treated and untreated, Con A-stimulated and unstimulated blood by the determination of cell surface antigens CD25 and CD71 as described in detail previously (22). T-cell proliferation was assessed by measuring the expression of proliferating cell nuclear antigen (PCNA) as described previously (18, 19). Proliferating cells were identified and enumerated in two-parameter DNA/PCNA distributions as PCNA+ cells with S/G2M-phase DNA content. Five thousand events were analyzed per sample. For simplicity, we refer to these data as % S/G2M cells.
Lymphocyte Phenotyping
To determine the effects of CP-690,550 on lymphocyte subsets, the following cell surface markers in unstimulated whole blood were analyzed using flow cytometry: CD3+/CD4+ (helper T lymphocytes), CD3+/CD8+ (cytolytic T lymphocytes), CD3−/CD16+ (NK cells), and CD3−/CD20+ (B lymphocytes). To assess the potential effects of CP-690,550 on the distribution of respective CD8+ memory cell populations, blood was drawn from a subset of four animals transplanted and dosed with the JAK3 inhibitor. Four-color flow cytometric analysis of CD8+ memory cell populations was performed on a FACSCalibur® flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA) and data was analyzed with the software CellQuestTM Pro (BD Biosciences Immunocytometry Systems). Analysis was performed by first scatter-gating on the lymphocyte population and then gating on CD3+CD8+ double-positive cells. CD95/CD28 dot plots were created to assess central memory (CD95+/CD28bright+), effector memory (CD95+/CD28−) and naïve (CD95−/CD28+) CD8+ T-cells as previously characterized in rhesus and cynomolgus monkeys (29, 30).
Statistical Analysis
All data were expressed as mean ± standard error of the mean (SEM). The software SPSS for Windows version 10.0 (SPSS Inc., Chicago, IL) was used to perform statistical analyses. Groups were compared using the two-tailed Student’s t test. Cell subsets changes were compared using a paired t test. A _P_-value of less than 0.05 was considered statistically significant.
RESULTS
In Vitro and In Vivo Exposure to CP-690,550 Significantly Reduces IL-2 Enhanced IFN-γ Production by T-cells
We determined the effects of CP-690,550 on T-cell IFN-γ production by first exposing peripheral blood to incremental concentrations of the compound for 30 min prior to stimulation and performing subsequent intracellular cytokine measurement. CP-690,550 inhibited IFN-γ production by CD3+ T-cells in a dose dependent fashion with an IC50 of 1.1 μM. At the greatest concentration tested (3.2 μM), CP-690,550 inhibited IFN-γ production by 55% (Fig. 1A).
Inhibition of T cell IFN-γ production upon whole blood exposure to CP-690,550. (A) Inhibition of in vitro IFN-γ production by CP-690,550. Incremental amounts of CP-690,550 were added to whole blood from naïve, nontransplanted cynomolgus monkeys (cynos). After a 30-minute incubation period, blood was stimulated by a combination of PMA, ionomycin and rh-IL-2 (see text for details). Intracellular IFN-γ production was assessed by flow cytometry and results for each drug concentration were normalized using results measured in stimulated and untreated blood. A dose-response effect is observed with an IC50 of 1.1 μM. (B) Inhibition of IFN-γ production by CP-690,550 in vivo. Three naïve untransplanted cynos were dosed with CP-690,550 at 15 mg/kg twice daily. Two similar control animals were dosed with the solvent for CP-690,550. Blood was obtained from all animals on the days indicated and stimulated as above. When compared to vehicle-control animals, significant inhibition of IFN-γ production in response to ex vivo stimulation was observed at all time points considered in animals that received CP-690,550. (C) Inhibition of IFN-γ production in cynos recipients of life-supporting kidney transplants immunosuppressed with CP-690,550. Nine immunosuppressed cynos had blood drawn at the indicated time points. At all considered time points, a statistically significant reduction of IFN-γ production as compared to baseline pretransplantation levels was observed. (D) Longitudinal monitoring of IFN-γ production in transplanted animals immunosuppressed with CP-690,550. *P<0.05, **P<0.001.
To verify that this inhibitory effect would still be seen following oral dosing, enteral absorption and metabolical processing of the compound, naïve animals were dosed orally twice a day with CP-690,550 which resulted in average blood trough and peak levels of 0.32±0.03(SD) and 4.1±2.1 μM, respectively. As early as one day after introduction of dosing there was a significant (_P_=0.002) reduction in IFN-γ producing T-cells as compared to animals that received only the vehicle for the compound (Fig. 1B). This inhibitory effect was up to 60% at the last time-point following completion of the 8-day treatment course.
To determine if this inhibitory effect would be maintained in the presence of an additional allogeneic stimulus, IFN-γ production by T-cells from transplanted animals was studied at various time-points during their postoperative course. Transplanted animals immunosuppressed with CP-690,550 with average blood trough and peak levels of 0.34±0.52 and 1.9±1.3 μM, respectively demonstrated a marked reduction of IFN-γ production by T-cells at all considered time points in comparison with pretreatment values (Fig. 1C and Figure 2). This significant reduction in IFN-γ production, when compared to pretreatment values, was as high as 63.2% on day 14 (_P_=0.001) and was, in general, maintained throughout the posttransplant course (Fig. 1D).
Inhibition of T cell IFN-γ production in peripheral blood of cynomolgus monkey kidney transplant recipients immunosuppressed with CP-690,550. Whole blood drawn from untransplanted and untreated animals or immunosuppressed transplant recipients was drawn at indicated time-points and stimulated ex vivo with a combination of PMA, ionomycin and rhIL-2. Dot plots from representative animals show that whereas the capacity of T cells to produce IFN-γ in untreated animals is unaffected (A), an important and sustained reduction of IFN-γ production is seen in transplanted and treated animals (B) (adapted from (8)).
In Vitro and In Vivo Exposure to CP-690,550 Significantly Affects T-cell Activation and Proliferation
To determine if CP-690,550 inhibited T-cell activation and proliferation, we first incubated whole blood from cynomolgus monkeys with incremental concentrations of CP-690,550 for thirty minutes prior to stimulation with Con A. Activation and proliferation were measured at 48 and 72 hr, respectively, by flow cytometric methods. CP-690,550 inhibited the surface expression of the activation markers CD25 and CD71 in a concentration-dependent manner (Fig. 3A and B). CD25 surface expression was more potently inhibited than that of CD71 (IC50 of 0.18 μM and 1.6 μM, respectively). Lymphocyte proliferation as measured by PCNA expression was also inhibited in a concentration-dependent fashion with an IC50 of 0.87 μM (Fig. 3C).
Effect of CP-690,550 on lymphocyte activation and proliferation in whole blood assays. Incremental amounts of CP-690,550 were added to whole blood from naïve, non transplanted cynos prior to stimulation with concanavalin A (Con A) (see text for details). The up-regulation of the surface expression of the lymphocyte activation markers CD25 and CD71 and the intracellular concentration of proliferating cell nuclear antigen (PCNA) were measured with flow cytometric methods. In naïve blood spiked with incremental concentrations of the drug (top row), CP-690,550 inhibits surface expression of CD25 (A) and CD71 (B) and expression of PCNA (C) in a concentration-dependent manner. Naïve untransplanted animals (middle row) were dosed with either CP-690,550 (open circle) or its vehicle (closed circles). Over a course of nine days, treated animals demonstrated a significant reduction of surface activation markers expression (D and E) and lymphocyte proliferation (panel F) when compared to untreated animals. Transplanted animals were dosed with CP-690,550 and had blood drawn at the indicated time points. A significant reduction of the expression of surface activation markers was observed two weeks after transplantation and introduction of treatment but waned off at the subsequently tested time-points (G and H). To the contrary, significant inhibition of lymphocyte proliferation as reflected by the inhibition of the expression of the PCNA was observed at all time points tested (I). *P<0.05, **P<0.001.
These in vitro inhibitory effects of CP-690,550 on lymphocyte activation and proliferation were also observed in naïve animals administered CP-690,550 for an 8-day course and in transplanted animals treated with CP-690,550 during their posttransplant course. Naïve animals exposed to the compound displayed a significant reduction of both lymphocyte activation (Fig. 3D & E) and proliferation (Fig. 3F) markers (all, P<0.01) when compared to vehicle control animals. Expression of both CD25 and CD71 T-cell surface antigens was also significantly blocked (59.1% and 51.1%, respectively, P<0.05) early in the postoperative course of transplanted animals but not at the later time points investigated (Fig. 3G & H). Lymphocyte proliferation as measured by PCNA expression was also significantly reduced (74% as compared to day 0, P<0.001) at day 14 and this effect was maintained at the subsequent time points (Fig. 3I).
Exposure to CP-690,550 in Transplanted Animals Results in Significant Reduction of NK Cells and T-cell Numbers
Because of the particular T−B+NK− phenotype observed in SCID patients with JAK3 mutation(s), we investigated the effect(s) of a short course exposure to CP-690,550 on T-, B-, and NK-cell populations. Naïve primates exposed for a week to 15 mg/kg CP-690,550 BID did not display significant changes of lymphocytes, granulocytes and monocyte populations (data not shown). Those animals however displayed a 63±13% averaged reduction of NK cell percentages by day 9 (Fig. 4A). At that time, NK cell percentages were lower than those observed in vehicle-treated animals although not significantly. The contraction of the NK cell population in naïve animals dosed with CP-690,550 was reversible upon cessation of treatment. Two weeks after dosing was discontinued, NK cell percentages were near baseline levels (Fig. 4A). No significant changes were noted in CD4+ or CD8+ T-cell and B-cell percentages upon short-term exposure to CP-690,550 (data not shown).
Effects of CP-690,550 on immune cell populations and subsets. (A) Short-term exposure to CP-690,550 in naïve animals results in reduction of NK cell percentages that is reversible after interruption of treatment. (B) Trends in lymphocyte, monocyte and granulocyte cell numbers in cynos recipients of renal transplants immunosuppressed with CP-690,550. (C) Peripheral blood mononuclear cell numbers at indicated time points after transplantation. Monocyte numbers are unaffected. Granulocyte numbers are significantly increased at the two-week time point after transplantation but return to normal at subsequent time points. Lymphocyte numbers two-weeks prior to study completion (Preterm) and at study completion (Endpoint) are significantly lower than pretransplantation numbers. (D) Immune cell subsets characterized in transplanted animals at indicated time-points. NK cell numbers are significantly reduced as compared to baseline at all time points tested. At late time point tested, significant reduction of both CD4+ and CD8+ T cells. Absolute CD20+ B-cell numbers are unaffected by treatment. Trends in NK cells (E) and CD4+ and CD8+ T cell (F) numbers in animal graft recipients immunosuppressed with CP-690,550. *P<0.05, **P<0.001.
Similar studies were performed in transplanted animals. Analysis of complete blood cell counts revealed a significant decline over time of lymphocyte numbers (P<0.01) whereas monocyte numbers were not affected (Fig. 4B and C). By virtue of both percentages and absolute numbers, NK cells were significantly reduced (P<0.01) at all time-points tested as compared to pretreatment values (Fig. 4D). Reduction in NK cell percentages was observed throughout the postoperative course during which animals were exposed to CP-690,550 without interruption (Fig. 4E). At study endpoint, NK cells percentages were reduced to nearly 10% of the pretransplantation values. By virtue of percentages of lymphocytes, both CD4+ and CD8+ T-cell populations did not show significant changes during the postoperative follow up in transplanted animals whereas significantly increased CD20+ B-cell percentages where observed two weeks prior to completion and at completion of study (data not shown). When absolute numbers where considered, however, T-cell subset populations were significantly affected (Fig. 4D & F). Whereas changes were not significant two weeks following introduction of the drug and organ transplantation, absolute numbers for both CD4+ and CD8+ cells were significantly reduced (P<0.05) at the later time-points tested (Fig. 4D). At neither time point tested were CD20+ absolute numbers significantly different from baseline, pretransplantation numbers (Fig. 4D).
JAK3 Inhibition in Transplanted Animals Does Not Result in Significant Changes in CD8+ Effector Memory T-cell Populations
Four NHP recipients of kidney allografts had blood collected and analyzed for the phenotypic characterization of central memory (CD95+/CD28bright+), effector memory (CD95+/CD28−) and naïve (CD95-/CD28+) CD8+ T-cells (Fig. 5A). Three of those animals experienced terminal rejection at days 29, 38 and 73 and one animal completed the 90-day study period without allograft rejection. Exposure to the drug, as reflected by median area under the time-concentration curve (AUC0-12), varied from 134 to 425 ng.h/ml (Table 1). No significant change in the distribution of CD8+ naïve and effector memory cell populations was demonstrated within this set of animals (Table and Figure 5). To the contrary, the percentage of CD95+/CD28bright+ central memory CD8+ cells at necropsy (19.7±4.1%) was significantly higher than that measured prior to transplantation (8.5±2.8%, _P_=0.01).
Longitudinal analysis of CD8+ memory cell populations in transplanted animals immunosuppressed with CP-690,550. (A) Four-color flow cytometric analysis of cynomolgus monkeys CD8+ memory T-cells. Central memory (CD95+/CD28bright+), effector memory (CD95+/CD28−) and naïve (CD95−/CD28+) CD8+ memory T-cells populations are characterized using panels previously reported in rhesus and cynomolgus monkeys (29, 30). (B) Data collected from 4 transplanted animals comparing baseline, pretransplantation data, to data collected at necropsy does not show significant changes of naïve and effector memory cell populations whereas central memory cell populations are significantly increased at necropsy (*; P<0.05). (C) Longitudinal assessment at various indicated time points after transplantation of respective CD8+ memory T-cells populations showing a progressive increase in frequency of central memory T cell populations.
Clinical characteristics and CD8+ memory T-cell subsets distribution in cynomolgus monkey recipients of life-supporting kidney allografts immunosuppressed with CP-690,550
DISCUSSION
To characterize the immunomodulatory effects of the JAK3 inhibitor CP-690,550, naïve untransplanted animals and recipients of kidney transplants treated with the drug were monitored with pharmacodynamic assays that we used in previous studies to assess the immunosuppressive effects of candidate immunosuppressive drugs (18–23, 25–28). In vitro and in vivo treatment with CP-690,550 resulted in inhibition of lymphocyte activation and function. In assays carried out in whole blood, effects on immune cells were characterized by a significant reduction of: (a) T-cell production of IFN-γ in response to IL-2/PMA/ionomycin stimulation, (b) lymphocyte proliferative response to lectin stimulation, and (c) expression of the activation markers CD25 and CD71 in response to lectin stimulation.
We first demonstrated that in vitro whole blood exposure to CP-690,550 resulted in significant, dose-dependent reduction of IFN-γ production by T-cells. Production of IFN-g is classically regulated by IL-12, a cytokine which does not require JAK3, but rather JAK2/STAT4 for signal transduction (31, 32). Hence, the precise mechanism(s) for this effect remains to be determined although previous studies demonstrated that blockade of the IL-2R signaling resulted in inhibition of IFN-γ production through both IL12-dependent—i.e., significant reduction of IL-12 production—and IL-12-independent mechanisms, the latter of which directly related to blockade of IL-2R signaling (33). In our whole blood cultures, impaired IFN- g production may also have resulted from altered signaling from cytokines recently demonstrated as critical (e.g., IL-15, IL-21) for the production of this TH1 cytokine in T-cells and NK cells (34) and which effects are expected to be blunted by JAK3 inhibition. In support of this is the recent demonstration that both NK cells and T-cells exposed to CP-690,550 failed to up-regulate the activation marker CD69 in response to IL-15 stimulation (30). At levels of drug exposure consistent with those tested in vitro, reduction of IFN-γ production was subsequently verified in animals chronically dosed with CP-690,550 in which the capacity of T-cells to produce IFN-γ in response to ex vivo stimulation was reduced by 60 to 70%. The higher inhibitory effect observed in vivo, as compared to that observed in vitro, may have resulted from chronic exposure to the compound or, potentially, from the in vivo production of active metabolites of the compound, a hypothesis which remains to be tested.
In addition to impaired IFN-γ production upon in vitro or in vivo exposure to the drug, further evidence for impaired T-cell activation was also reflected by a dose-dependent reduction of cell surface activation marker expression in response to in vitro stimulation. CD25 expression was more potently inhibited than CD71 expression by CP-690,550, a fact that is in agreement with existing evidence that IL-2 regulates CD25 expression (37) whereas up-regulation of the CD71 transferrin receptor also requires PI3 kinase signaling (38), an enzyme unaffected by CP-690,550 (8). Expression of both CD25 and CD71 molecules was significantly down-regulated in both transplanted and nontransplanted animals dosed with the drug. In the transplanted animals, inhibitory effects on the expression of lymphocyte activation markers were no longer significant at the later time points tested. We have no clear explanation for this phenomenon. Drug blood trough levels were not significantly reduced over time posttransplantation even though dosing was progressively tapered. It is likely that the incremental expression of T cell surface activation markers resulted from insufficient immunosuppression and forecasted upcoming rejection, thereby displaying a key feature of relevant pharmacodynamic markers (39). Lymphocyte proliferation, reflected by the percentage of lymphocytes in the S/G2M phase expressing the PCNA antigen (18, 28) in transplanted and non transplanted dosed animals was also significantly reduced to 20-30% of the levels observed prior to introduction of the drug. This finding was consistent with previous demonstrations that CP-690,550 affects cell proliferation and potently inhibited mixed leukocyte reactions set up with murine, nonhuman primate or human cells (8).
Because a peculiar immune cell phenotype results from JAK3 deficiency, we studied the effects of JAK3 inhibition with CP-690,550 on immune cell populations. The T−B+NK− SCID phenotype observed in JAK3 deficient patients and mice can be readily explained by accompanying alterations of cytokine signaling (41, 42). The NK cell defects reported in JAK3−/− mice have been attributed to failure of signaling from the IL-15 receptor (15, 43, 44) which is required for mature NK cell survival in vivo (45). In fact, mice chronically dosed with CP-690,550 demonstrated a 96% reduction in splenic NK1.1+TCRβ− cell numbers following 21 days of treatment (9). In the current study as well, a striking reduction of NK cell numbers was observed in animals dosed with CP-690,550. This was particularly evident in transplanted animals that, at all time points tested after drug treatment, displayed reduced NK cell percentages that averaged 10% of pretransplantation levels. Also in agreement with studies conducted in rodents, significant reductions of both CD4+ and CD8+ numbers were observed in transplanted animals in the current study. This mirrored the recent report of a reversible significant reduction of both NK and CD8+—but not CD4+—cell numbers in naïve cynomolgus monkeys dosed for three weeks with CP-690,550 (30). Consistent with the phenotype of JAK3 mutation(s) in human patients (46), chronic exposure to CP-690,550 in transplanted animals did not significantly affect B-cell numbers.
As IL-7 and IL-15 are both critical for the proliferation of CD8+ memory T-cells (44, 47, 48), we were lead to ascertain the effects of chronic JAK3 inhibition on the development of memory cell populations. Our studies were further motivated by a previous report that JAK3−/− SCID mice exposed to influenza virus die of infection whereas 90% of mice reconstituted with JAK3+/+ cells survive and have evidence of reconstituted long term T-cell memory (49) and by the fact that chronic exposure of naïve untransplanted cynomolgus monkeys to CP-690,550 resulted in a significant reduction of the number of CD8 effector memory cells (30). The threat that memory cells present to allograft survival (reviewed in (50)) is supported by the results of studies suggesting that not only persistence of memory T-cells hinders long-term allograft survival in humans (51) but also that memory T-cells may be resistant to the effects of currently used therapies. Contrary to what was previously reported in naïve animals dosed with CP-690,550 (30), evaluation of CD8+ memory cell population in a limited set of transplanted animals in the current series did not find a reduction in CD8+ effector memory T-cell populations. As placement of an allograft is expected to stimulate the development of memory populations, it is unknown if, in transplanted animals, this phenomenon might have counterbalanced the inhibitory effects of the drug observed in naïve animals in the absence of stimuli. We, furthermore, observed a significant increase in central memory cell populations. Those apparently discordant effects on memory populations might in fact be explained by different requirements of memory cell populations (52), by the results of studies suggesting that central and effector memory cell populations are largely independent subpopulations (53), and by additional data as we expand our understanding of memory cell populations (47). We caution that the current data on CD8+ memory T-cell populations was generated from a limited set of observations and moreover refers solely to normal memory phenotype cells, not to memory cells putatively specific for defined alloantigens. Because of their far-reaching implications, the effects of JAK3 inhibition on memory T cell populations will indeed need to be critically characterized. Extensive T-cell proliferation can occur under conditions of lymphopenia, a process termed homeostatic proliferation, which may induce acquisition of functional memory T-cells (54) that show dominant resistance to tolerance (55). A combination of lymphocyte depleting induction therapy with JAK3 inhibition might, on these grounds, prove an attractive immunosuppressive combination.
In conclusion, we have presented here some of the features of JAK3 inhibition on immune functions of NHP recipients of life-supporting allografts immunosuppressed with CP-690,550. Treatment with CP-690,550 was responsible for significant reduction of lymphocyte activation, IFN-γ production and proliferation, and was further responsible for an important reduction of NK and T-cell numbers. Current evidence suggests the likely development of JAK3 inhibition as a major modality of achieving immunosuppression to prevent organ allograft rejection (12–14). Results of pharmacodynamic assays performed in the current study support the concept of considered use of JAK3 inhibition for the treatment of other diseases that involve the activation of Type I cytokine receptors—such as T-cell leukemia/lymphomas, rheumatoid arthritis, and inflammatory bowel disease (reviewed in (56))—or for that of diseases in which NK T-cell derived IFN-γ plays a central role, such as systemic lupus erythematosus (57).
ACKNOWLEDGMENTS
The authors would like to thank Ms. Kathy Richards for her editorial assistance in the preparation of the manuscript.
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Keywords:
Transplantation; Immunosuppression; Pharmacodynamics; Primates; JAK3; JAK/STAT; CP-690,550
© 2005 Lippincott Williams & Wilkins, Inc.