imatinib mesylate inhibits t-cell proliferation in vitro and delayed-type hypersensitivity in vivo

doi:10.1182/blood-2003-12-4266Prepublished online April 20, 2004;

Allan B Dietz, Lina Souan, Gaylord J Knutson, Peggy A Bulur, Mark R Litzow and Stanimir Vuk-Pavlovic

in vivohypersensitivity and delayed-typein vitroImatinib mesylate inhibits T cell proliferation

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DIETZ et al IMATINIB MESYLATE SUPPRESSES T CELL FUNCTION

IMMUNOBIOLOGY

Imatinib mesylate inhibits T cell proliferation in vitro and delayed-type

hypersensitivity in vivo

Allan B. Dietz, Lina Souan, Gaylord J. Knutson, Peggy A. Bulur, Mark R. Litzow, and

Stanimir Vuk-Pavlovic

From the Stem Cell Laboratory, Mayo Clinic Cancer Center; Division of Hematology,

Department of Internal Medicine; Division of Transfusion Medicine, Department of

Laboratory Medicine and Pathology, Mayo Clinic; and Department of Biochemistry and

Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN

Supported by grants from Mrs. Adelyn L. Luther, Singer Island, FL; Commonwealth

Cancer Foundation for Research, Richmond, VA and Mayo Clinic Comprehensive

Cancer Center Support Grant CA15083.

Reprints: Allan B. Dietz ([email protected]) or S. Vuk-Pavlovic ([email protected]),

both at Mayo Clinic, 200 First Street SW, Rochester, MN 55905.

Blood First Edition Paper, prepublished online April 20, 2004; DOI 10.1182/blood-2003-12-4266

Copyright (c) 2004 American Society of Hematology

For personal use only.on March 27, 2014. by guest bloodjournal.hematologylibrary.orgFrom




2

Abstract

Imatinib mesylate (STI-571, imatinib, Gleevec®) inhibited DNA synthesis in primary human T

cells stimulated with allogeneic mature dendritic cells or phytohemagglutinin (PHA), but did not

induce apoptosis. The IC50 values for T cell proliferation stimulated by dendritic cells and PHA

were 3.9 µM and 2.9 µM, respectively, i.e., within the concentration range found in

imatinib-treated patients. Interestingly, imatinib did not inhibit expression of T cell activation

markers CD25 and CD69, although it reduced the levels of activated NF-κB and changed

phosphorylation and/or protein levels of Lck, ERK1/2, retinoblastoma protein and cyclin D3.

When T cells were washed free of imatinib, they proliferated in response to PHA demonstrating

that inhibition is reversible. Imatinib treatment led to accumulation of the cells in G0/G1 phase of

the cell cycle. The in vitro observations were confirmed in vivo in a murine model of delayed-

type hypersensitivity (DTH). In imatinib treated mice, DTH was reduced in comparison to sham

injected controls. However, the number of splenic T cells was not reduced showing that,

similarly to in vitro observations, imatinib inhibited T cell response, but did not cause apoptosis.

These findings indicate that long-term administration of high dose imatinib might affect

immunity.





3

Introduction

Imatinib mesylate (imatinib, STI-571, Gleevec®, Novartis, Basel, Switzerland) is a reversible

tyrosine-kinase inhibitor effective in treatment of chronic myelogenous leukemia1 (CML),

gastrointestinal stromal tumors2,3, eosinophilic disorders4 and systemic mast cell disease5 and

has been tried in several other diseases6,7. The drug binds preferentially to ATP-binding sites of

the c-kit protooncogene product, platelet–derived growth factor receptor (PDGF-R), and abelson

kinase (c-ABL)8-10 impeding the ensuing signal transduction (reviewed in ref. 11). Inhibition of

the ABL portion of the BCR-ABL fusion protein, pathognomonic of CML12, is the basis of

imatinib efficacy in inducing hematological and cytogenetic remissions in CML1,13. Inhibition

of c-KIT is considered critical for imatinib effects in gastrointestinal stromal tumors14 and mast

cell disease5.

In our efforts to develop immunotherapy for CML15, we studied imatinib effects on the

development of CML-specific immunity upon administration of autologous mature dendritic

cells (DC)16. Consequently, we measured the effects of imatinib on in vitro correlates of

immunity17. At concentrations of 5 µM, achieved in the serum of patients on the standard

therapeutic dose of 400 mg per day1, the drug was cytostatic, it arrested transit into the S phase

of the cell cycle and impeded in vitro proliferation of human normal and leukemic T cells. We

analyzed imatinib effects on the activity of intracellular signal transduction molecules associated

with the control of T cell function. The drug diminished phosphorylation of Lck, ERK 1/2 and

Rb proteins, reduced the levels of cyclin D3 and of activated the nuclear transcription factor κB

(NF-κB). In addition, imatinib inhibited delayed-type hypersensitivity in mice without affecting

T cell numbers indicating that the drug inhibits T cell response, but does not induce apoptosis.





4

Materials and Methods

Cells and Reagents

T cells were isolated by negative immunoadsorption (Pan T kit; Miltenyi Biotec, Auburn, CA),

pooled from three or more normal donors and cryopreserved until use. Upon thawing, the cells

were incubated in X-VIVO 15 medium (BioWhittaker, Walkersville, MD) supplemented with

1.0 percent human AB serum (Sigma, St. Louis, MO), in a humidified atmosphere of 5.0 percent

CO2 at 37 �C. For experiments longer than one day, the medium contained 1.0 percent

penicillin/streptomycin solution (Sigma, St. Louis, MO) as well. Human acute T cell leukemia

cells Jurkat (ATCC TIB-152), acute T lymphoblastic leukemia cells CCRF-CEM (ATCC

CCL-119) and hematopoietic malignant K-562 cells (ATCC CCL-243), were obtained from

American Type Culture Collection (Manassas, VA) and cultured under the same conditions.

Allogeneic dendritic cells were derived from CD14-positive cells and cultured with T cells as

previously described16. Imatinib (Novartis, Basel, Switzerland) was dissolved in DMSO to a

final concentration of 10 mM. The stock solution was stored at –20�C until use, diluted to the

tenfold final concentration in X-VIVO 15 medium and added to cells immediately.

Cell Proliferation Assays

T cells, 1×105 in 200 µL per well of 96 well microtiter plates (Corning Glass Works, Corning,

NY), were stimulated with dendritic cells or phytohemogglutanin M (PHA-M; 10 µg/mL,

Sigma) and/or platelet-derived growth factor (R&D Systems, Minneapolis, MN) in a final

volume of 0.2 mL per well. The cells were incubated with graded concentrations of imatinib,

incubated for four days, pulsed with 2.0 µCi tritiated thymidine (Amersham, Arlington Heights,

IL) per well, incubated for further 12 hours when they were harvested and incorporated

radioactivity was quantified16.





5

Flow cytometry

We stained T cells with CD25-specific monoclonal antibody conjugated to fluorescein (Clone

M-A251; Pharmingen, San Diego, CA) or CD69-specific monoclonal antibody conjugated to

phycoerythrin (Clone HP-4B3; Ancell, Bayport, MN) and analyzed them by flow cytometry.

Cell cycle distribution was measured by flow cytometry in propidium–iodide stained T cells18

and analyzed by ModFit LT software (Verity Software, Topsham, ME). Apoptosis was

quantified by Anexin-V binding (Annexin-V-FLUOS staining kit; Roche Diagnostics,

Indianapolis, IN).

Caspase 3 activity

The cells were suspended at 1.0×106 cells per mL in 10.0 percent fetal bovine serum (FBS;

Cellgro, Herndon, VA) and 1.0 percent penicillin/streptomycin (Sigma). One-mL aliquots were

placed in 24-well flat bottom cell culture plates (Corning, Corning, NY) and incubated at 37 °C

in 5.0 percent humidified carbon dioxide in the presence or absence of PHA (10.0 µg/mL) plus

IL-2 (500 U/mL; R&D Systems) and imatinib. After a three-day incubation cells were collected

by centrifugation, washed and treated with the fluorogenic caspase 3 substrate PhiPhiLux–G1D2

(OncoImmunin, Gaithersburg, MD) according to manufacturer's instructions. Fluorescence was

quantified by flow cytometry (excitation at 488 nm) and the relative amounts of activated

caspase 3 determined from a calibration curve.

Protein phosphorylation analysis by Western blotting

T-cells (2×106/mL in X-VIVO 15 medium with 1.0 percent human AB serum) were incubated

with imatinib, 10 µM, or an equivalent volume of DMSO at 37 °C for one hour. Then PHA was

added to a final concentration of 10 µg/mL. Six mL of the cell suspension per well were plated in

six-well tissue culture plates. At various intervals following plating, cells from one well were

collected, centrifuged and lysed in 0.5 mL of Laemmli sample buffer followed by heating at 100

�C for five min19. Lysate aliquots, each equivalent to 0.6×106 cells, were resolved by





6

electrophoresis on 7.5 percent or 10 percent polyacrylamide gels. Electrophoresed proteins were

electroblotted onto Trans-Blot nitrocellulose transfer membranes (Bio-Rad Laboratories,

Hercules, CA) in 25 mM Tris, 192 mM glycine buffer, pH 8.3, at 125 V and 4 �C for 2 hours.

Then the membrane was incubated in 5.0 percent nonfat powdered milk in 0.1 M NaCl, 20 mM

Tris, 0.1 percent Tween-20, pH 7.4, (TBS/T) for one hour. Following incubation, the membrane

was washed with TBS/T and incubated with the primary antibody at 4 �C overnight. (All washes

included triplicate five-minute incubations in TBS/T with agitation. All antibodies were used at

dilutions recommended by manufacturers.) The membrane was washed, incubated with the

secondary antibody for one hour at room temperature, and washed again. Thereafter the

membrane was incubated in the SuperSignal West Pico chemiluminescent HRP substrate (Pierce

Biotechnology, Rockford, IL) for five minutes, exposed to X-OMAT AR film (Eastman Kodak,

Rochester, NY) that was developed by an X-OMAT developer (Eastman Kodak). Optical density

of individual bands on the film was registered by a ChemiDoc image acquisition system (Bio-

Rad Laboratories) and quantified by Quantity One 1-D analysis software (Bio-Rad Laboratories).

For detection of proteins and phosphoproteins we used monoclonal antibodies specific

for β-actin (as control of protein loading; Novus Biologicals, Littleton, CO), cyclin D3 (Cell

Signaling Technology, Beverly, MA) and abelson kinase (BD Biosciences, San Diego, CA). In

addition, from Cell Signaling Technology we obtained immunopurified polyclonal rabbit

antibodies specific for c-Kit, c-Kit Y719-PO4, Lck, Lck Y505-PO4, Erk1/2 T202-PO4/Y204-PO4,

Rb S780 and Rb S807/811-PO4. Polyclonal goat antibodies specific for rabbit IgG and mouse

IgG, both coupled to horseradish peroxidase, were from Santa Cruz Biotechnology (Santa Cruz,

CA).

Measuring NF-κB activation by ELISA

T cells were suspended in serum-free medium at 6.0×106 cells/mL, plated in six-well plates and

rested overnight. Following a one-hour incubation at 37 �C, imatinib was added followed by the

addition of PHA (to the final concentration of 10 µg/mL) one hour later. Thereafter, the cells





7

were incubated for additional four hours, resuspended by pipetting and collected by

centrifugation. Nuclear extracts were prepared according to ref. 20. The level of activated NF-κB

in extracts was assessed in a chemiluminescence-detected enzyme-linked immunosorbent assay

according to manufacturer’s (Oxford Biomedical Research, Oxford, MI) directions.

Luminescence was quantified by a ChemiDoc system (Bio-Rad Laboratories). Protein

concentration was measured by the Bio-Rad DC Assay (Bio-Rad Laboratories).

Delayed-type hypersensitivity

Imatinib effect on delayed-type hypersensitivity was quantified with the permission of the Mayo

Clinic Institutional Animal Care and Use Committee and performed as described in refs. 21-23.

Groups of five female B6AF1 mice (Jackson Laboratories, Bar Harbor, ME), 8 to 12 weeks old,

received intraperitoneal injections of imatinib, 50 mg/kg in 10 percent DMSO, for 21

consecutive days. In addition, groups of similar mice received equivalent amount of DMSO

alone (sham injected). Mice were primed on day 14 by a subcutaneous injection in the flank of

50 µL of 4-hydroxy-3-nitrophenyl acetyl O-succinimide ester (NP-O-Su; Biosearch

Technologies, Novato, CA) dissolved in PBS, pH 7.8, at 7.0 g/100 mL. Six days after priming,

mice were injected with the challenge dose of 25 µL NP-O-Su (2.0 g/100 mL) into the right

footpad and with 25 µL of PBS into the left. Footpad thickness was measured 24 hours later by a

digital micrometer.

We prepared single cell suspensions from spleens of these mice by meshing individual

spleens through metal sieves and lysing red blood cells in lysis buffer (1.0 mL per spleen;

Sigma) for five minutes at room temperature. Thereafter, cells were incubated with anti-CD3 and

anti-CD8 antibody (BD Biosciences, Mountain View, CA) for 20 minutes at 4 ºC, washed and

fixed with 1.0 percent paraformaldehyde. Flow cytometry was performed with a FACScan flow

cytometer and CellQuest software (both BD Biosciences).





8

Statistics

All experiments were repeated two to five times with qualitatively same outcome. The

probability that the mean values of two experimental groups were identical was tested by

two-tailed Student’s t-test for paired samples. The level of significance was set at p=0.05. Where

applicable, data are reported as the mean ± standard deviation.

Results

Imatinib inhibits proliferation of human primary T cells and T cell lines

To determine if imatinib affects T cell proliferation, we stimulated the cells with allogeneic

mature dendritic cells or PHA in the presence of imatinib. The drug inhibited T cell proliferation

as a function of concentration (Fig. 1A). The effects were significant at 0.5 µM imatinib for the

cells stimulated by dendritic cells (p=0.002) and at 1.0 µM imatinib for the cells stimulated with

PHA (p=3.93×10-5). The IC50 values for imatinib-inhibited T cell proliferation stimulated by DC

and PHA were 3.9 µM and 2.9 µM, respectively. Thus, imatinib arrested T cell proliferation in a

dose–dependent manner at concentrations akin to those achieved in the serum of patients on

standard imatinib therapy of 400 mg daily1. Because the effects of PHA and dendritic cells were

similar, for further experiments we selected PHA to simplify interpretation of the results

obtained by electrophoresis and Western blotting.

We quantified imatinib effects on proliferation of transformed human CD3-positive T

cell derived lymphoblastoid line CCL119, T cell leukemia Jurkat line and the BCR-ABL–

positive chronic myelogenous leukemia cell line K562. We incubated the cells with imatinib for

18 hours when we measured the extent of thymidine incorporation. Imatinib inhibited DNA

synthesis of all three transformed cell lines in a dose-dependent fashion, but was less potent than

in normal cells (Fig. 1B). After 18 hours with imatinib none of the cell lines lost viability (as

determined by trypan blue exclusion) and/or increased binding of annexin V (data not shown).

After 48-hours, however, at 1.0 µM imatinib only 28±4 percent of K562 (p=0.005 relative to

imatinib-free control) cells were viable compared to 86±4 percent for CCL-119 cells (p=0.44)





9

and 99±8 percent for Jurkat cells (p=0.92). A quantitative comparison of imatinib effects in

normal T cells and cell lines must await a detailed kinetic analysis of the cell cycle, but

apparently imatinib does not affect normal T cells only.

Imatinib neither reduces T cell viability nor stimulates their apoptosis

Imatinib toxicity might provide an explanation for inhibited T cell proliferation. Therefore, we

incubated the cells with imatinib for 72 hours and quantified apoptosis by determining the

fraction of cells demonstrating activated caspase 3 (ref. 24) and the fraction which bound

annexin V25,26. Frequency of cells positive for activated caspase-3 and binding of annexin V

were low in the absence of PHA (Fig. 2). Stimulation by PHA increased the frequency of cells

exhibiting either markers (p<0.001 for both). Imatinib-treated and PHA-activated T cells did not

change their annexin V binding (p=0.10), indicating that the drug did not induce apoptosis under

these conditions. Caspase 3 activation can be used as a marker for apoptosis, but the levels of

enzyme are increased in T cell stimulation27,28. PHA treatment activated caspase 3, but imatinib

inhibited this effect (p=0.03), similarly as it inhibited T cell proliferation. Thus, the PHA- and

imatinib-induced changes in caspase 3 activation may reflect changes in T cell stimulation, rather

than enhanced apoptosis. Consequently, under these conditions imatinib was cytostatic, but not

cytotoxic.

Imatinib treated T cells accumulate in the G0/G1 phase of the cell cycle

As imatinib was not cytotoxic, we postulated that the decrease in DNA synthesis (Fig. 1) results

from imatinib effects on cell cycle progression. To test this hypothesis, we stimulated

imatinib-treated cells with PHA alone or with the combination of PHA (10 µg/mL) and IL-2

(500 U/mL) that stimulates powerfully T cell proliferation29. We measured DNA content 72

hours later. Data in Fig. 3A show that essentially all unstimulated (control) T cells were in the

G0/G1 phase of the cycle. PHA stimulated DNA synthesis and progression into the S phase

(p<0.001 relative to resting cells), an effect inhibited by imatinib (p<0.001 relative to PHA-





10

stimulated cells). Imatinib inhibited T cell cycle progression even in the presence of PHA plus

IL-2 (p<0.01 relative to the cells stimulated by both PHA and IL-2).

A prerequisite for entry of cells into the S phase is phosphorylation of the retinoblastoma

protein (Rb) that is regulated by the cyclin D3/cdk4 complex30,31 (also reviewed in ref. 32). We

quantified the levels of cyclin D3 protein and Rb phosphorylation in the presence of imatinib by

Western blotting. Twelve, 16 and 24 hours after initiation of a typical experiment the amount of

Rb phosphoprotein was reduced to 19, 39 and 77 percent of control, respectively; the amount of

cyclin D was reduced to 28, 32 and 59 percent of control, respectively (data not shown; effects at

later times might have been artificially reduced by the film saturation at the darker control

bands). These effects are consistent with imatinib-induced arrest of the cell cycle before the S

phase.

Imatinib does not inhibit expression of T cell activation markers CD25 and CD69

Membrane molecules CD25 and CD69 are expressed as a result of T cell stimulation, but their

expression is not coupled to proliferation33. Hence, these molecules can be viewed as evidence

of early T cell activation33. We measured the levels of CD25 and CD69 in imatinib-treated T

cells activated by PHA. After an 18-hour incubation with PHA, the frequency of cells expressing

CD25 and CD69 markedly increased (Fig. 3B). Imatinib-treated T cells demonstrated similar

increase in CD25 and CD 69 levels characteristic of activated T cells, rather than of resting T

cells. Thus, the combined data in Figs. 1, 2 and 3 show that imatinib selectively inhibits cellular

targets leading to proliferation without effecting T cell activation or apoptosis.

Imatinib can terminate already initiated proliferation signals, but its effects are reversible

Because the mechanisms initiating T cell proliferation and activation are uncoupled33, in

agreement with the data in Figs. 3A and 3B, it is of interest to determine if imatinib can

terminate proliferation signals after they had been initiated. We plated cells without imatinib in

the presence of PHA as in Fig. 2. Imatinib was added at increasing intervals from initiation of





11

culture (Fig. 4A) and determined DNA synthesis. We found that imatinib could inhibit

proliferation of T cells stimulated even forty-eight hours before introduction of the drug (Fig.

4A). Thus, imatinib could terminate already initiated intracellular signaling in the pathways

leading to proliferation.

If imatinib inhibits proliferation but does not induce apoptosis, the cells will resume

proliferation upon removal of the drug. To test this hypothesis, we incubated T cells with

imatinib, stimulated one half of the wells with PHA and incubated all cells for 24 hours. Then we

washed the cells, treated one half of the wells from each sample with PHA and incubated all for

additional 96 hours when we measured thymidine incorporation. We found the cells

unstimulated in both incubations did not proliferate (Fig. 4B). The cells stimulated by PHA in

the first incubation proliferated without it in the second, probably due to residual PHA. The cells

stimulated in the second incubation proliferated to the same extent irrespective whether they

were stimulated in the first. These observations demonstrate that imatinib did not impact the

proliferation potential of T cells and that the effects of the drug are reversible.

Imatinib reduces levels of phosphorylated Lck and ERK1/2 and of activated NF-κB

Abelson kinase (and its constitutively activated mutant BCR-ABL), c-KIT protein and PDGF-R

are well-documented cellular targets of imatinib10. PDGF, common in human serum, suppresses

T cell activation34. Monoclonal antibodies specific for the extracellular component of c-KIT did

not recognize T cells (data not shown), suggesting that the full-length c-KIT is absent in T cells.

Indeed, we detected no c-KIT and no imatinib-associated change in the levels of c-ABL

phosphorylation by Western blotting (data not shown). Similarly, by functional assays we found

that exogenous PDGF did not affect T cells (data not shown). Apparently c-ABL, c-KIT and

PDGF-R do not play a manifest role in inhibition of T cell proliferation by imatinib. Thus, T

cells must contain other imatinib target(s). In an attempt to identify some imatinib-sensitive

intracellular signaling pathways we measured the levels of phosphorylation of Lck molecule and





12

ERK1/2, both associated with TCR–mediated signaling35 and of activated NF-κB, a

transcription factor activated by numerous pathways including the one initiated at the TCR35.

We exposed PHA-stimulated T cells to imatinib and determined the relative levels of Lck

protein and phosphorylation at its Tyr-505 as a function of time. We found no apparent

difference between drug-treated and control cells in the levels of Lck protein, but the drug

reduced Tyr-505 phosphorylation (Fig. 5). This effect was apparent at the first time point

measured at 15 minutes after stimulation. Similarly, we noticed that imatinib inhibited

phosphorylation of ERK1/2 (Fig. 5). In addition, we determined the level of activated NF-κB in

nuclear extracts of T cells activated by PHA in the presence of increasing imatinib

concentrations and expressed the results as percentage of PHA-stimulated increase in levels of

activated NK-κB. At 4.4 µM imatinib, the amount of activated NK-κB was reduced to 58.7±1.1

percent (p=0.003) and decreased to 38.4±0.9 percent at 17.6 µM. These imatinib effects on

phosphorylation of Lck and ERK1/2 and activation of NF-κB are compatible with the

concentration dependence of the observed inhibition of T cell proliferation and function.

Imatinib inhibits delayed-type hypersensitivity in mice

To determine if imatinib affects T cells in vivo similarly to the effects in vitro, we treated mice

with the drug daily throughout the experiment36,37. After 10 days of treatment, we immunized

mice with NP-O-Su and challenged six days later with a subcutaneous injection of the same

agent into one footpad and of PBS into the other. Twenty-four hours later we quantified the

extent of delayed-type sensitivity by measuring the thickness of the footpads. Footpads of control

animals (no imatinib) thickened considerably upon the challenge with NP-O-Su (Table 1).

Systemic treatment with imatinib abolished this effect (Table 1). In contrast, imatinib had no

effect on the total splenocyte number (p=0.25) and numbers of CD3+ cells (p=0.52) and CD8+

cells (p=0.16; data not shown). Taken together, these data show that the reduced DTH might

result from a systemic inhibition of the T cell response, rather than from the diminished number

of T cells.





13

Discussion

We found that imatinib inhibits T cell proliferation at concentrations similar to those found in

patients treated for CML and GIST3. The effect is manifest both in T cells nonspecifically

stimulated by PHA and in those stimulated by dendritic cells. Imatinib neither prevented

activation of T cells nor killed them, but it inhibited cell cycle progression. In an attempt to

identify the pertinent intracellular pathways targeted by imatinib, we found reduced

phosphorylation and/or protein levels of all molecules we selected for study. This observation

could explain the attenuation of T cell function, but it was not helpful in identification of critical

imatinib targets. Possibly, T cell function is impaired by the cumulative effect of partial

inhibition of numerous phosphonucleotide-binding molecules rather than by the definitive

inhibition of few key molecules (e.g., BCR-ABL or c-KIT). A similar conclusion has been

reached recently in a study of differentiation of CD34-positive hematopoietic cells into dendritic

cells where imatinib suppressed phosphorylation of molecules participating in numerous

intracellular signaling pathways that are independent of c-ABL, c-KIT and PDGF receptor38.

We did detect the presence of c-ABL in T cells and cannot thus completely rule out its

role in imatinib effects. However, there is little evidence for such a role of c-ABL in signal

transduction leading to T cell cycle triggering, although the molecule generally does participate

in regulation of later stages of cell cycle progression39. Our data are compatible with such role

for c-ABL as imatinib inhibited T cell cycle progression, but did not affect T cell activation. This

observation is fully in line with the recent evidence for such uncoupling of activation and cell

cycle progression in human T cells33. In addition, these data demonstrate that imatinib can be

used as a tool in studies of (un)coupling of T cell activation and proliferation.

We found that imatinib was immunosuppressive in an in vivo model of delayed type

hypersensitivity. The clinical relevance of this finding is unclear. As the effects of the drug are

rapidly reversed upon its removal, these effects are likely to be sensitive to imatinib

pharmacokinetics. It is possible, in fact, that the key determinant of the extent of





14

immunosuppression is the nadir in fluctuations of imatinib levels. Thus, sustaining

therapeutically adequate imatinib levels may impede acute inflammatory responses and/or

undermine control of subclinical infections. Nonetheless, information about imatinib effects on

immunity is scarce and circumstantial. Several mostly anecdotal observations of secondary

effects of imatinib in CML patients support the notion that imatinib suppresses immunity in vivo.

For example, imatinib-treated patients experienced a higher incidence of herpes zoster outbursts,

which diminished upon treatment with antiviral drugs40. One patient developed an EBV-positive

B-cell lymphoma41

and another developed pulmonary alveolar proteinosis42

; both complications

receded after imatinib dose reduction or complete discontinuation. Similarly, imatinib-borne

immunosuppression could explain the surprising remission of rheumatoid arthritis in a CML

patient two months upon initiation of imatinib therapy43

. Recent attempts to overcome resistance

to imatinib by higher doses of the drug may provide data for further unraveling of the

relationship of imatinib and immunity. A more definite establishment of immunosuppressive

effects of imatinib may add this well tolerated drug to the list of clinically useful agents for

control of T cell malignancies and autoimmunity.

Acknowledgements

We thank Mr. Troy Voeltz for technical help and Dr. Frank Prendergast for continuing interest

and support.





15

Table 1. Imatinib suppresses delayed-type hypersensitivity in mice

Group Footpad thickness

Mean ± SD (mm)

1. Sham injected, not primed 2.74±0.11

2. Sham injected, primed 3.55±0.13

3. Imatinib-treated, not primed 2.80±0.33

4. Imatinib-treated, primed 2.88±0.29

The mean ± standard deviation (SD) values refer to the thickness of the footpad of mice, which

received (Groups 1 and 2) or did not receive imatinib (3 and 4) and were primed (i.e.,

immunized; 2 and 4) or not primed by NP-O-Su. All groups were challenged by NP-O-Su one

day before measurement of the footpad thickness. A two-tailed t-test comparison for

independent groups yielded p1:2=6×10-5, p3:4=0.21, p1:3=0.702 and p2:4=0.004.





16

Figure Legends

Figure 1. Imatinib inhibits proliferation of primary human T cells and T cell lines. (A)

Proliferation of 1.5×107 T cells/2.5 mL was stimulated with allogeneic mature dendritic cells

(open circles) or PHA (full circles) and measured as a function of imatinib concentration. The

cells were incubated with imatinib for 96 hours. Then tritiated thymidine was added for 12 hours

when the cells were harvested and incorporated radioactivity was measured. Differences

between positive controls and imatinib–treated cells were apparent at 0.5 µM for the cells

stimulated with DC (p=0.002) and at 1.0 µM for cells stimulated with PHA (p=3.93×10-5). (B)

Human T cell lines CCRF-CEM (circles) and Jurkat (squares) and chronic myelogenous

leukemia cell line K562 (diamonds) were incubated in graded concentrations of imatinib for 18

hours and evaluated as in panel A. The results are expressed as stimulation indices (mean

value ± standard deviation of radioactivity incorporated in the presence of the drug divided by

the mean value ± standard deviation of radioactivity incorporated in the absence of the drug).

Figure 2. Imatinib neither reduces viability nor stimulates apoptosis in T cells.

Unstimulated T cells and T cells stimulated with PHA were cultured for 72 hours without or with

imatinib (5.0 µM). Then the cells were assayed for activated caspase 3 and for binding of

annexin V. PHA stimulation increased the frequency of cells with activated caspase 3 and cells

binding annexin V (p<0.001 for both). Imatinib induced a decrease of activated caspase 3 levels

(p=0.03), but did not change the frequency of cells binding annexin V (p=0.10).

Figure 3. Imatinib treatment results in T cell accumulation in the G0/G1 phase of the cell

cycle, but does not inhibit T cell activation. (A) Cell cycle distribution of unstimulated T cells

and T cells stimulated with PHA, or the combination of PHA and IL-2 stimulated T cells with and

without imatinib. The cells were stained with propidium iodide 24 hours after initiation of

experiment and analyzed by flow cytometry. Dark areas represent the fraction of cells in G0/G1,

lighter areas stand for the fraction in S phase and white areas correspond to the fraction in G2/M





17

phase. (B) Untreated T cells (dashed line) and T cells activated with PHA (10 µg/mL) in imatinib

absence (solid line) or presence (10 µM; shaded area) were analyzed for expression of CD25 or

CD69 24 hours after initiation of culture.

Figure 4. Imatinib can terminate proliferation signals already initiated, but its effects on T

cells are reversible. (A) Imatinib (10 µM) was added four to forty-eight hours after PHA

stimulation. DNA synthesis was quantified as in Fig. 1. Values measured in the presence of

imatinib at all time points were different from values in imatinib–free cells (p<0.05). (B) First, the

cells were treated with imatinib, one group stimulated by PHA and the other unstimulated. The

cells were incubated for 24 hours, washed free of imatinib and replated without it. Subsequently,

one half of each group was stimulated with PHA and the other half remained unstimulated. After

incubation for an additional 96 hours, DNA synthesis was measured as in Fig. 1. Cells treated

with imatinib and without PHA in the first incubation did not synthesize DNA without PHA in the

second, while those treated with PHA in the first incubation did resume proliferation without

stimulation in the second. However, in the presence of PHA in the second incubation the cells

proliferated similarly (p=0.22) irrespective of whether pretreatment with imatinib took place in

the absence or presence of PHA.

Figure 5. Imatinib inhibits phosphorylation of molecules participating in cellular

signaling. In an attempt to identify imatinib-sensitive intracellular signaling pathways we

measured the levels of phosphorylation of Lck and ERK1/2, both associated with TCR–

mediated signaling. We exposed PHA-stimulated T cells to imatinib (10 µM) and found that the

drug did not affect the levels of Lck protein, but it inhibited Tyr-505 phosphorylation. Similarly,

imatinib inhibited phosphorylation of ERK1/2.





18

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Figure 1A





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Figure 1B





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Figure 2





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Figure 3A





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Figure 3B





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Figure 4A





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Figure 4B





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Figure 5





imatinib mesylate inhibits t-cell proliferation in vitro and delayed-type hypersensitivity in vivo

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