Introduction

Imatinib mesylate (STI571, Gleevec, Novartis) is a highly effective and well-tolerated oral agent that has had a significant impact on the treatment of patients with chronic myeloid leukemia (CML).^{1, 2, 3, 4, 5} By inhibiting the BCR-ABL protein kinase, imatinib targets the molecular event which forms the basis for CML. Although highly specific, imatinib occupies the ATP-binding site of several tyrosine kinase molecules such as ABL,^{2, 6, 7, 8, 9} the stem cell factor receptor c-kit,^{10, 11} the platelet-derived growth factor receptor (PDGFR),^{6, 10, 12} and ARG.¹³

Although imatinib is highly effective in the treatment of CML, long-term follow-up is lacking and allogeneic SCT remains the only proven curative option. Presently, it is unclear how best to incorporate imatinib into studies related to stem cell transplantation. Strategies include postponing transplantation pending some degree of imatinib-induced clearing of leukemic clones, reserving the drug until after marrow transplantation (using imatinib as an 'adjuvant' to marrow transplantation), or administration of the drug in situations of leukemic relapse after allogeneic SCT.

However, little is known about the effects of imatinib on nonmalignant (Philadelphia negative) hematopoietic cells. In the majority of studies, T lymphocytes of CML patients have been found to be Philadelphia negative and/or BCR-ABL negative.^{14, 15, 16} In vitro studies have demonstrated that imatinib specifically inhibits or kills proliferating myeloid cell lines containing BCR-ABL, but is minimally harmful to normal cells.^{6, 7, 17} The agent also reduces the formation of BCR-ABL-positive colony-forming units generated from CML patients in vitro by approximately 95% at imatinib concentrations of 1 M. This selective inhibition of colony formation is observed over a 2-log dose range, with a maximal differential effect at 1 mol/l.⁷ Substantial in vivo inhibition of the enzymatic activity of BCR-ABL at a daily dose of 400 mg has been demonstrated, which correlates well with clinical response and with levels of the drug that kill CML cells in vitro.²

However, several of the intracellular signaling molecules triggered by the ABL kinase are also involved in the activation pathways of immune cells. It has been shown that ABL-/- knockout (KO) mice have thymic and splenic atrophy and a substantial T- and B-cell lymphopenia.^{18, 19} In contrast, the myeloid/macrophage lineages are unaffected. Although the follow-up of CML patients treated with imatinib is short, an increase in the incidence of infections and other malignancies has been reported.^{20, 21} In light of these observations, we were concerned that inhibition of ABL kinases may have a detrimental effect on the immune response, and we sought to identify whether T-cell proliferative responses in vitro were affected by imatinib.

Materials and methods

T-cell isolation

Blood samples were obtained from 30 healthy donors with informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Nycomed Pharma, Oslo, Norway) density gradient centrifugation. The following antibodies were used for the purification of CD3⁺ T lymphocytes from freshly prepared PBMC: anti-CD14, anti-CD16, anti-CD19, anti-CD33, and anti-CD56 (all from Caltag, Towcester, UK). For the purification of CD4⁺ and CD8⁺ T cells, OKT8 (American Type Culture Collection, USA) or anti-CD4 antibodies (BD PharMingen, USA) were added, respectively. All antibodies were used for depletion in combination with sheep anti-mouse dynalbeads (Dynal (UK) Ltd). Purity was confirmed by FACS analysis and was 93.2–99% (data not shown).

Proliferation assays

Cells were cultivated in RPMI 1640 medium with 10% human serum (HS), L-glutamine, penicillin, and streptomycin (Life Technologies, UK). T-cell proliferation was assessed by ³H-thymidine (³H-TdR) (Amersham Pharmacia) incorporation added after 2–5 days and measured 18 h later.^{22, 23} The effect of imatinib (a gift from Novartis Pharma, Basel, Switzerland) on T-cell stimulation was assessed as described previously for other drugs.^{24, 25, 26} Where indicated, imatinib was added to T-cell cultures at serial concentrations (0, 1, 2, or 5 mol/l).

Mixed lymphocyte reaction (MLR)

Briefly, purified cord blood-derived naïve (CD3⁺ RA⁺) T cells (5 10⁴/well) were stimulated with decreasing numbers (3 10³-1 10⁵/well) of irradiated unfractionated PBMC from an MHC mismatched healthy donor in 96 U-bottomed well plates (Nunc). After 5 days ³H-TdR incorporation was measured.

T-cell response to phytohemagglutinin (PHA) or CD3/CD28 stimulation

Proliferation of PBMC (1 10⁵/well) or purified T-lymphocyte subsets (5 10⁴ T cells/well) in response to PHA (2 g/ml)²³ or anti-CD3/D28 Dynabeads (Dynal Biotech, Oslo, Norway)²² was assessed in the presence or absence of imatinib after 72 h. Supernatants were harvested for cytokine analysis from proliferation assays, involving purified CD8⁺ or CD4⁺ lymphocytes, stimulated with anti-CD3/D28 Dynabeads prior to the addition of ³H-TdR.

PPD-specific CD4⁺ T-cell clones

Tuberculin purified protein derivative (PPD)-specific T-cell clones were derived from an individual by culturing PBMC with 300 U/ml PPD (EVANS Vaccine Limited, Liverpool, UK) in RPMI/10% HS. PPD-specific T-cell lines were cloned by limiting dilution in U-bottomed 96-well plates (Nunc) in the presence of irradiated PBMC previously pulsed with 10 g/ml of PHA (Murex Biotech, Dartford, UK) and rIL-2 (10 U/ml) (Roche, Mannheim, Germany). T-cell clones were stimulated every 7 days with irradiated PBMC previously pulsed with PPD (300 U/ml) in RPMI/10% HS and rIL-2. T-cell clones were used for proliferative tests 1 week from their last stimulation. For these experiments, PBMC were pulsed overnight with PPD (300 U/ml) or the irrelevant antigen, 8 10^-2 U/ml tetanus toxoid (TT) (EVANS Vaccine Limited, Liverpool, UK), in the absence of imatinib. The following day, T-cell clones (1 10⁴/well) were co-cultured with irradiated PBMC (1 10⁴/well) in 96 U-bottomed well plates (200 l total volume/well) with serial concentrations (0, 1, 2, or 5 mol/l) of imatinib. After 72 h incubation, ³HTdR-incorporation was measured.

Measurement of cytokine production

Supernatants for cytokine analysis were harvested after 24, 48, 72, and 96 h from wells in which purified CD4⁺ or CD8⁺ T lymphocytes (5 10⁴ T cells/well) were stimulated with anti-CD3/D28 Dynabeads in the presence (1–5 uM) or absence of imatinib. Samples were assayed for IFN- (MS Biotechnology, UK), IL-5 (Biosource International, California, USA), and IL-10 (Pharmingen, USA) production by double antibody sandwich ELISA using a purified mAb, paired with a biotinylated mAb as previously described.²⁷ Absorbance values were measured using a Labsystems Multiscan Plus spectrophotometer at 450 nm and levels of cytokine estimated using the curve obtained from the set of standards. The data were transferred to a computer for analysis using the Cytafzal programme.

Measurement of cell viability and apoptosis

PBMCs stimulated for 24–72 h with PHA or anti-CD3/D28 Dynabeads at various imatinib concentrations were labeled with FITC-conjugated annexin-V (BD PharMingen, USA) and Via-Probe 7-aminoactinomycin D (7-AAD; BD PharMingen). CD3⁺ T lymphocytes were analyzed using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA, USA).²³ Apoptotic cells were defined as annexin V⁺/7-AAD^-. Positive controls were ultraviolet B (UVB312 nm) irradiated PBMCs (800 mJ/cm²).

Measurement of T-cell activation

T-cell activation following 72 h stimulation with anti-CD3/D28, in the presence or absence of imatinib, was assessed by quantifying the number of CD3⁺ T cells expressing the activation markers CD25, CD69, and HLA-DR. Briefly, T cells (1 10⁵) were incubated with the appropriate directly conjugated antibody for 30 min on ice. Cells were washed twice with ice-cold FACS buffer (PBS, 1% fetal calf serum, 0.05% sodium azide) and analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using CellQuest software. Dead cells and debris were excluded by forward and side scatter gating.

Statistical analysis

Student's t-test analysis was used to compare different groups. P-values <0.05 were considered statistically significant.

Results

Inhibition of polyclonal mitogen-induced stimulation of T lymphocytes

Imatinib inhibited T-cell proliferation in the MLR in a dose-dependent fashion (Figure 1) at all stimulator (PBMC) numbers analysed. Maximum inhibition was noted at the maximum imatinib concentration assessed: 5 mol/l (median 53%, range 46–55%). The median inhibition was 16.4% (range 8–18.1%) and 28.4% (range 22.5–31%) at 1 and 2 mol/l, respectively. The inhibitory effect was not increased if responder PBMCs were incubated with imatinib for 24 h prior to use in the MLR (data not shown).

Figure 1.

Imatinib inhibits T-lymphocyte proliferation induced by allogeneic PBMC in a dose-dependent manner. Naïve CD4⁺ T cells (5 10⁴/well) were cultured with irradiated unfractionated PBMC (3 10³-1 10⁵/well) from healthy individuals. After 5 days, ³H-TdR was added to cultures and 18 h later cells were harvested and measured for ³H-TdR incorporation. The data from four experiments are expressed as mean values of the % maximal responsestandard deviations (s.d.) at each imatinib concentration. The mean ³H-TdR-incorporation was 90 000 (range 71 000–111 000) in the absence of imatinib.

Full figure and legend (86K)

Target population

T-cell proliferation in MLR is dependent on the presence of antigen-presenting cells (APC). To investigate the possibility that imatinib specifically targeted APC, we compared its effect on PHA- and CD3/CD28-induced stimulations, which are APC-dependent and APC-independent, respectively. No difference was observed (Figure 2a). At 1 mol/l imatinib the mean inhibitions of PBMC were 21 and 29% with PHA and CD3/CD28, respectively, at 2 mol/l imatinib the inhibitions were 38 and 47%, and at 5 mol/l imatinib the inhibitions were 66 and 72%, respectively (Figure 2a). The lack of requirement of APC for imatinib to exert its inhibitory effect on T cells was also supported by the observation that PPD-specific CD4⁺ T-cell clones, in which APC were pulsed with the cognate antigen (PPD) in the absence of imatinib, were similarly inhibited (Figure 2b). Mean inhibitions of 13, 27, and 55% were observed with imatinib at 1, 2, and 5 mol/l, respectively. The PPD-specific T-cell clones did not proliferate in response to PBMC pulsed with irrelevant antigen (TT).

Figure 2.

(a) Imatinib inhibits PHA- and anti-CD3/CD28-induced stimulation of PBMC in a dose-dependent manner. Proliferation of PBMC to PHA (2 g/ml) (top panel) or anti-CD3/D28 Dynabeads (lower panel) was assessed in the absence or the presence of increasing concentrations of imatinib (0–5 mol/l) 72 h after the initiation of the cultures. After 2 days ³H-TdR was added and its incorporation measured after 18 h. The data from eight different responders are expressed as mean values of the % maximal responses.d. at each imatinib concentration. The mean ³H-TdR incorporations were 216 430 (range 13 186–296 399) and 86 220 (range 55 445–108 359) with PHA and anti-CD3/D28, respectively, in the absence of imatinib. (b) Imatinib inhibits anti-CD3/D28-induced proliferation of CD8⁺and CD4⁺T lymphocytes and antigen-induced proliferation of PPD-specific CD4⁺ T-cell clones. Escalating doses of imatinib were added to cultures in which purified CD8⁺ (top panel) and CD4⁺ (middle panel) T cells (5 10⁴ T cells/well) were stimulated with anti-CD3/CD28. The data from six and four different responders, respectively, were expressed as mean values of the % maximal responses.d. at each imatinib concentration. The mean ³H-TdR incorporations of CD8⁺ and CD4⁺ T cells, respectively, were 32 450 (range 25 577–44 960) and 93 545 (range 32 210–121 897) in the absence of imatinib. PBMC were pulsed overnight with whole PPD (300 U/ml) or an irrelevant antigen (8 10^-2 U/ml TT) in the absence of imatinib. The next day, PBMCs (1 10⁵/well) were cultured in the presence of PPD-specific CD4⁺ T-cell clones (1 10⁴ T cells/well) and escalating doses of imatinib (0–5 mol/l). After 2 days ³HTdR was added and ³HTdR incorporation was measured after 18 h. The data from three different PPD-specific CD4⁺ T-cell clones are expressed as mean values of the % maximal responses.d. at each imatinib concentration (lower panel). The mean ³H-TdR-incorporation was 82 740 (range 62 748–93539) in the absence of imatinib.

Full figure and legend (56K)

Figure 2.

(a) Imatinib inhibits PHA- and anti-CD3/CD28-induced stimulation of PBMC in a dose-dependent manner. Proliferation of PBMC to PHA (2 g/ml) (top panel) or anti-CD3/D28 Dynabeads (lower panel) was assessed in the absence or the presence of increasing concentrations of imatinib (0–5 mol/l) 72 h after the initiation of the cultures. After 2 days ³H-TdR was added and its incorporation measured after 18 h. The data from eight different responders are expressed as mean values of the % maximal responses.d. at each imatinib concentration. The mean ³H-TdR incorporations were 216 430 (range 13 186–296 399) and 86 220 (range 55 445–108 359) with PHA and anti-CD3/D28, respectively, in the absence of imatinib. (b) Imatinib inhibits anti-CD3/D28-induced proliferation of CD8⁺and CD4⁺T lymphocytes and antigen-induced proliferation of PPD-specific CD4⁺ T-cell clones. Escalating doses of imatinib were added to cultures in which purified CD8⁺ (top panel) and CD4⁺ (middle panel) T cells (5 10⁴ T cells/well) were stimulated with anti-CD3/CD28. The data from six and four different responders, respectively, were expressed as mean values of the % maximal responses.d. at each imatinib concentration. The mean ³H-TdR incorporations of CD8⁺ and CD4⁺ T cells, respectively, were 32 450 (range 25 577–44 960) and 93 545 (range 32 210–121 897) in the absence of imatinib. PBMC were pulsed overnight with whole PPD (300 U/ml) or an irrelevant antigen (8 10^-2 U/ml TT) in the absence of imatinib. The next day, PBMCs (1 10⁵/well) were cultured in the presence of PPD-specific CD4⁺ T-cell clones (1 10⁴ T cells/well) and escalating doses of imatinib (0–5 mol/l). After 2 days ³HTdR was added and ³HTdR incorporation was measured after 18 h. The data from three different PPD-specific CD4⁺ T-cell clones are expressed as mean values of the % maximal responses.d. at each imatinib concentration (lower panel). The mean ³H-TdR-incorporation was 82 740 (range 62 748–93539) in the absence of imatinib.

Full figure and legend (81K)

Having excluded the hypothesis that APC are the targets of imatinib-induced inhibition of T-cell proliferation, we asked whether different T-cell subsets were predominantly affected. The proliferative response of purified CD8⁺ and CD4⁺ T lymphocytes to CD3/CD28 antibodies was inhibited similarly by imatinib in a dose-dependent manner (Figure 2b). At 1 mol/l imatinib the inhibition of CD8⁺ T cells was 31%, at 2 mol/L imatinib the inhibition was 49%, and at 5 mol/L imatinib the inhibition was 69%. The inhibitions of CD4⁺ T cells were 29, 46, and 80% at 1, 2, and 5 mol/l, respectively. Furthermore, no difference in the magnitude of inhibition of anti-CD3/28-induced proliferation of purified CD4CD45RA⁺ (naïve) and CD4CD45RO⁺ (memory) T cells was observed (data not shown).

Imatinib inhibits the production of IFN- from CD8⁺ and CD4⁺ T cells

Cytokine analysis was performed to determine whether the Th phenotype of the purified CD8⁺ or CD4⁺ cells differed as a consequence of the presence of imatinib in the cultures. Imatinib resulted in reduction in IFN- production by both CD8⁺ and CD4⁺ T cells after 48–96 h anti-CD3/CD28 antibody stimulation (Figure 3). In contrast, there was no reproducible effect on the production of either IL-4 or IL-10 (data not shown).

Figure 3.

Imatinib inhibits production of IFN- after anti-CD3/28-induced proliferation of CD8⁺and CD4⁺T lymphocytes. Increasing concentrations of imatinib (0–5 mol/l) were added to cultures in which purified CD8⁺ (top panel) and CD4⁺ (lower panel) T cells (5 10⁴ T cells/well) were stimulated with anti-CD3/CD28. Supernatants were taken at 72 and 48 h, respectively, and IFN- was measured in triplicate by ELISA. Results from four different responders (a–d) in one experiments.d. at each imatinib concentration are shown. Data are representative of three experiments with similar results.

Full figure and legend (119K)

Imatinib mesylate does not increase apoptosis

It has been described that imatinib induces apoptosis and reduces proliferation of BCR-ABL+ cells.^{7, 8} Therefore, the inhibitory effect observed in our T-cell cultures might result from apoptosis rather than functional inactivation. However, the fraction of apoptotic cells in the CD3⁺ population after CD3/CD28 stimulation in the presence of imatinib did not differ from controls without imatinib (Figure 4). The fraction of apoptotic cells remained between 15 and 18% after incubation of PBMC with imatinib at concentrations of 1–5 mol/l for 24–72 h.

Figure 4.

Imatinib-induced T-cell inhibition is not the result of a higher rate of apoptosis. PBMCs were stimulated by PHA or anti-CD3/28 Dynabeads in the absence or presence of various concentrations of imatinib (0, 1, 2, or 5 mol/l). After 72 h, cells were harvested and CD3⁺'s were stained for annexin-V-FITC and Via-Probe 7-AAD. Apoptotic CD3⁺ cells (annexinV⁺/7-AAD^-) were analyzed by flow cytometry. The data from five different experiments are expressed as mean values of the percentage of apoptotic cellss.d. at each imatinib concentration.

Full figure and legend (30K)

A mechanism of suppression of T-cell proliferation by imatinib mesylate is decreased T-cell activation

When we analyzed the expression of T-cell activation markers on CD3⁺ T lymphocytes isolated from five different donors following 72 h CD3/CD28 stimulation, a reduction in both the percentage and mean fluorescence intensity of CD25, CD69, and HLA-DR was observed (Figure 5). This effect was most marked at the highest imatinib concentration assessed (5 mol/l). The percentage expression of CD25 and HLA-DR molecules on CD3⁺ T lymphocytes was significantly reduced (P<0.05) at 1 mol/l imatinib. Significant reduction in CD69 expression was observed at 5 mol/l imatinib.

Figure 5.

Reduced expression of activation markers in the presence of imatinib. CD3⁺ T lymphocytes were stimulated with anti-CD3/CD28 for 72 h. Cell surface expression of activation markers was assessed by immunofluorescence staining. Cells (1 10⁵) were incubated with FITC-conjugated mAbs specific for CD25, CD69, HLA-DR, and isotype-matched mAbs. Cell surface expression was analyzed using a Becton Dickinson FACSCalibur flow cytometer. The percentages of CD3⁺ T lymphocytes expressing the relevant marker are shown. Data shown are representative of five experiments.

Full figure and legend (180K)

Inhibition of T-cell proliferation is reversible

In order to assess the duration of the inhibition, imatinib was removed from T-cell cultures and, after 48 h resting, T cells were re-stimulated with anti-CD3/CD28 Dynabeads. T-cell proliferation could be completely restored, thus demonstrating that the inhibitory effect is transient (Figure 6).

Figure 6.

Imatinib-induced T-cell inhibition is reversible. Proliferation of CD4⁺ T lymphocytes to anti-CD3/28 Dynabeads was measured after 72 h in the absence or presence of increasing concentrations of imatinib (0, 1, 2, or 5 mol/l). Imatinib was then removed from the cultures and, after 48 h resting, T cells were restimulated with anti-CD3/CD28. Data are representative of four experiments with similar results.

Full figure and legend (66K)

Discussion

We have demonstrated that imatinib inhibits the proliferation and activation of T cells at in vitro concentrations representative of the pharmacological doses used therapeutically in vivo. This effect is reversible, APC-independent, and does not involve induction of apoptosis.

The effect of imatinib on T-cell proliferation in vitro was assessed using an array of T-cell proliferative tests. The stimuli to induce T-cell proliferation in vitro included: histoincompatibility between cell populations (primary mixed lymphocyte reaction: MLR), nonspecific mitogens (PHA), direct T-cell activation (anti-CD3/CD28 antibodies), or cognate peptide presented in conjunction with the relevant MHC molecule (PPD-specific T-cell clones). T-cell populations in vivo are affected by the cytokine milieu, cell:cell interactions, and stromal influences. Although these assays underestimate the complexity of the immune system in vivo, they do assess the ability of T lymphocytes to respond to a range of stimuli.

The inhibitory effect on T-cell proliferation appears to be nonspecific since all proliferative assays were inhibited by imatinib concentrations 1 mol/l. Inhibition of proliferation was also associated with inhibition of IFN- production by both CD8⁺ and CD4⁺ T lymphocytes. Although cytokines were assayed every 24 h over 4 days, there was no clear trend observed for IL-4 and IL-10 production by either CD8⁺ or CD4⁺ T lymphocytes. Thus, it appears that imatinib does not alter the Th phenotype of the T cells in culture. Reduction in production of IFN- suggests that the effector function of T lymphocytes may be reduced in the presence of imatinib. A number of studies have demonstrated abnormal cytokine profiles in T lymphocytes stimulated ex vivo as part of mononuclear fractions isolated from untreated CML patients. A reduced capacity to produce Th1 cytokines and a shift toward a Th2 phenotype is well described in CML patients.^{28, 29, 30} Restoration of Th1 cytokine synthesis by T cells of CML patients who had achieved durable complete cytogenetic remission for >2 years in response to IFN-, but who had discontinued the drug, has been described.³¹ The mechanisms underlying this abnormal cytokine profile are unclear, but recent evidence suggests that such T-cell abnormalities may be mediated indirectly by leukemia or APCs present in these fractions. Leukemic cells from CML patients have been shown to secrete the Th2 cytokine IL-10.³² IL-10 can suppress the production of IL-2 and IFN- and can affect the quality and magnitude of the immune response.³³ We have recently described abnormal function in vitro of dendritic cells (DCs) generated from PBMC of CML patients.³⁴ Further support is provided by the demonstration that the ex vivo cytokine-producing capacity of isolated CD4⁺ T cells, depleted of leukemic and accessory cells, from CML patients in response to anti-CD3/CD28, was normal.³⁵

Another potential confounding factor when assessing the effect of imatinib in vivo is that imatinib affects APC development and function in vitro.³⁶ Appel et al³⁶ have reported that imatinib affects the differentiation of DCs generated from CD34⁺ peripheral blood progenitor cells. This was associated with a reduced ability of DCs to induce primary cytotoxic T-lymphocyte (CTL) responses.

Other interactions than those proposed may be involved, and thus a study to analyze the effect of imatinib in vivo requires careful design. Lymphocytes should be stored from individuals before starting imatinib and taken at time intervals. Recent data suggest differences in the effect of imatinib on the function of B lymphocytes³⁷ and plasmacytoid DC³⁸ isolated from CML patients treated with imatinib in vivo, depending on their response to imatinib. Thus, such studies must involve comparisons of T-cell function between normal individuals and CML patients before and during treatment with imatinib.

There is some evidence that the inhibition of T-cell proliferation we have observed in vitro may be associated with a clinical effect. Although the follow-up for patients receiving imatinib is relatively short, an increase in the incidence of infections and other malignancies has been reported and this may be a result of immune dysfunction.^{20, 21} Many studies show that T lymphocytes are not part of the Philadelphia-positive clone in CML patients,^{15, 39} and that the majority of T cells are BCR-ABL negative.^{14, 16}

B-cell dysfunction has been suggested by the recent observation of hypogammaglobulinemia in CML patients (previously resistant or intolerant of IFN-) who have been treated with imatinib.³⁷ It is unlikely that this was related to previous treatment with IFN-, since this has never been reported, and levels of IgG were normal in all patients before imatinib therapy. Studies assessing B-cell function in imatinib-treated patients unexposed to IFN- have not been published to date.

Our results raise questions that are central to the treatment of CML, especially in situations of minimal leukemic burden and normal APCs. After myeloablative allogeneic SCT, engraftment of normal donor dendritic cells is rapid.⁴⁰ Imatinib is increasingly used as a selective leukemia de-bulking agent prior to allografting,⁴¹ or in treatment of relapse with or without donor lymphocyte infusions.^{42, 43, 44} Our observations suggest that imatinib may influence T-cell immune responses in vivo. An adverse effect of IFN- on transplantation outcome has been described previously, and this association was unexpected.⁴⁵ The reversibility of the imatinib-induced inhibition is in agreement with the lack of significant clinical impact and makes further investigation ex vivo of T-lymphocyte function in patients unlikely to be informative. However, it is possible that imatinib interferes with the expansion of graft-versus-leukemia effector T cells, thus impending or at least reducing the therapeutic efficacy of transplant or DLI. The effect of imatinib on immune responses in vivo warrants further investigation.

References

1. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM et al. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001; 344: 1038–1042. | Article | PubMed | ISI | ChemPort |
2. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: 1031–1037. | Article | PubMed | ISI | ChemPort |
3. O'Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348: 994–1004. | Article | PubMed | ISI | ChemPort |
4. Sawyers CL, Hochhaus A, Feldman E, Goldman JM, Miller CB, Ottmann OG et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood 2002; 99: 3530–3539. | Article | PubMed | ISI | ChemPort |
5. Talpaz M, Silver RT, Druker BJ, Goldman JM, Gambacorti-Passerini C, Guilhot F et al. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood 2002; 99: 1928–1937. | Article | PubMed | ISI | ChemPort |
6. Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996; 2: 561–566. | Article | PubMed | ISI | ChemPort |
7. Deininger MW, Goldman JM, Lydon N, Melo JV. The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells. Blood 1997; 90: 3691–3698. | PubMed | ISI | ChemPort |
8. Gambacorti-Passerini C, le Coutre P, Mologni L, Fanelli M, Bertazzoli C, Marchesi E et al. Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis. Blood Cells Mol Dis 1997; 23: 380–394. | Article | PubMed | ChemPort |
9. Druker BJ, Lydon NB. Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 2000; 105: 3–7. | PubMed | ISI | ChemPort |
10. Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000; 295: 139–145. | PubMed | ISI | ChemPort |
11. Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ. Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood 2000; 96: 925–932. | PubMed | ISI | ChemPort |
12. Apperley JF, Gardembas M, Melo JV, Russell-Jones R, Bain BJ, Baxter EJ et al. Response to imatinib mesylate in patients with chronic myeloproliferative diseases with rearrangements of the platelet-derived growth factor receptor beta. N Engl J Med 2002; 347: 481–487. | Article | PubMed | ISI | ChemPort |
13. Okuda K, Weisberg E, Gilliland DG, Griffin JD. ARG tyrosine kinase activity is inhibited by STI571. Blood 2001; 97: 2440–2448. | Article | PubMed | ISI | ChemPort |
14. Takahashi N, Miura I, Saitoh K, Miura AB. Lineage involvement of stem cells bearing the philadelphia chromosome in chronic myeloid leukemia in the chronic phase as shown by a combination of fluorescence-activated cell sorting and fluorescence in situ hybridization. Blood 1998; 92: 4758–4763. | PubMed | ISI | ChemPort |
15. Garicochea B, Chase A, Lazaridou A, Goldman JM. T lymphocytes in chronic myelogenous leukaemia (CML): no evidence of the BCR/ABL fusion gene detected by fluorescence in situ hybridization in 14 patients. Leukemia 1994; 8: 1197–1201. | PubMed |
16. Cho EK, Heo DS, Seol JG, Seo EJ, Chi HS, Kim ES et al. Ontogeny of natural killer cells and T cells by analysis of BCR-ABL rearrangement from patients with chronic myelogenous leukaemia. Br J Haematol 2000; 111: 216–222. | Article | PubMed |
17. Holtz MS, Slovak ML, Zhang F, Sawyers CL, Forman SJ, Bhatia R. Imatinib mesylate (STI571) inhibits growth of primitive malignant progenitors in chronic myelogenous leukemia through reversal of abnormally increased proliferation. Blood 2002; 99: 3792–3800. | Article | PubMed | ISI | ChemPort |
18. Tybulewicz VL, Crawford CE, Jackson PK, Bronson RT, Mulligan RC. Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 1991; 65: 1153–1163. | Article | PubMed | ISI | ChemPort |
19. Schwartzberg PL, Stall AM, Hardin JD, Bowdish KS, Humaran T, Boast S et al. Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations. Cell 1991; 65: 1165–1175. | Article | PubMed | ISI | ChemPort |
20. Baskaynak G, Kreuzer KA, Schwarz M, Zuber J, Audring H, Riess H et al. Squamous cutaneous epithelial cell carcinoma in two CML patients with progressive disease under imatinib treatment. Eur J Haematol 2003; 70: 231–234. | Article | PubMed |
21. Mattiuzzi GN, Cortes JE, Talpaz M, Reuben J, Rios MB, Shan J et al. Development of Varicella–Zoster virus infection in patients with chronic myelogenous leukemia treated with imatinib mesylate. Clin Cancer Res 2003; 9: 976–980. | PubMed | ISI | ChemPort |
22. Jiang S, Camara N, Lombardi G, Lechler RI. Induction of allopeptide-specific human CD4+CD25+ regulatory T cells ex-vivo. Blood 2003; 102: 2180–2186. | Article | PubMed | ISI | ChemPort |
23. Ng WF, Duggan PJ, Ponchel F, Matarese G, Lombardi G, Edwards AD et al. Human CD4(+)CD25(+) cells: a naturally occurring population of regulatory T cells. Blood 2001; 98: 2736–2744. | Article | PubMed | ISI | ChemPort |
24. Ferraresso M, Rigotti P, Stepkowski SM, Chou TC, Kahan BD. Immunosuppressive effects of defibrotide. Transplantation 1993; 56: 928–933. | PubMed |
25. Ferron GM, Jusko WJ. Species- and gender-related differences in cyclosporine/prednisolone/sirolimus interactions in whole blood lymphocyte proliferation assays. J Pharmacol Exp Ther 1998; 286: 191–200. | PubMed | ISI | ChemPort |
26. Kahan BD, Gibbons S, Tejpal N, Stepkowski SM, Chou TC. Synergistic interactions of cyclosporine and rapamycin to inhibit immune performances of normal human peripheral blood lymphocytes in vitro. Transplantation 1991; 51: 232–239. | PubMed | ISI | ChemPort |
27. Jordan WJ, Ritter MA. Optimal analysis of composite cytokine responses during alloreactivity. J Immunol Methods 2002; 260: 1–14. | Article | PubMed | ISI | ChemPort |
28. Tsuda H, Yamasaki H. Type I and type II T cell profiles in chronic myelogenous leukemia. Acta Haematol 2000; 103: 96–101. | Article | PubMed |
29. Guarini A, Breccia M, Montefusco E, Petti MC, Zepparoni A, Vitale A et al. Phenotypic and functional characterization of the host immune compartment of chronic myeloid leukaemia patients in complete haematological remission. Br J Haematol 2001; 113: 136–142. | Article | PubMed |
30. Aswald JM, Lipton JH, Messner HA. Intracellular cytokine analysis of interferon-gamma in T cells of patients with chronic myeloid leukemia. Cytokines Cell Mol Ther 2002; 7: 75–82. | Article | PubMed |
31. Reuben JM, Lee BN, Johnson H, Fritsche H, Kantarjian HM, Talpaz M. Restoration of Th1 cytokine synthesis by T cells of patients with chronic myelogenous leukemia in cytogenetic and hematologic remission with interferon-alpha. Clin Cancer Res 2000; 6: 1671–1677. | PubMed | ISI | ChemPort |
32. Pawelec G, Rehbein A, Schlotz E, da Silva P. Cellular immune responses to autologous chronic myelogenous leukaemia cells in vitro. Cancer Immunol Immunother 1996; 42: 193–199. | Article | PubMed |
33. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001; 19: 683–765. | Article | PubMed | ISI | ChemPort |
34. Dong R, Cwynarski K, Entwistle A, Marelli-Berg F, Dazzi F, Simpson E et al. Dendritic cells from CML patients have altered actin organization, reduced antigen processing, and impaired migration. Blood 2003; 101: 3560–3567. | Article | PubMed | ISI | ChemPort |
35. Kiani A, Habermann I, Schake K, Neubauer A, Rogge L, Ehninger G. Normal intrinsic Th1/Th2 balance in patients with chronic phase chronic myeloid leukemia not treated with interferon-alpha or imatinib. Haematologica 2003; 88: 754–761. | PubMed |
36. Appel S, Boehmler AM, Grunebach F, Muller MR, Rupf A, Weck MM et al. Imatinib mesylate affects the development and function of dendritic cells generated from CD34+ peripheral blood progenitor cells. Blood 2004; 103: 538–544. | Article | PubMed | ISI | ChemPort |
37. Steegmann JL, Moreno G, Alaez C, Osorio S, Granda A, de la Camara R et al. Chronic myeloid leukemia patients resistant to or intolerant of interferon alpha and subsequently treated with imatinib show reduced immunoglobulin levels and hypogammaglobulinemia. Haematologica 2003; 88: 762–768. | PubMed | ISI | ChemPort |
38. Mohty M, Jourdan E, Ben Mami N, Vey N, Damaj G, Blaise D et al. Imatinib and plasmacytoid dendritic cell function in chronic myeloid leukemia patients. Blood 2004, (Epub ahead of print).
39. Jonas D, Lubbert M, Kawasaki ES, Henke M, Bross KJ, Mertelsmann R et al. Clonal analysis of bcr-abl rearrangement in T lymphocytes from patients with chronic myelogenous leukemia. Blood 1992; 79: 1017–1023. | PubMed | ISI | ChemPort |
40. Auffermann-Gretzinger S, Lossos IS, Vayntrub TA, Leong W, Grumet FC, Blume KG et al. Rapid establishment of dendritic cell chimerism in allogeneic hematopoietic cell transplant recipients. Blood 2002; 99: 1442–1448. | Article | PubMed | ISI | ChemPort |
41. Wassmann B, Pfeifer H, Scheuring U, Klein SA, Gokbuget N, Binckebanck A et al. Therapy with imatinib mesylate (Glivec) preceding allogeneic stem cell transplantation (SCT) in relapsed or refractory Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL). Leukemia 2002; 16: 2358–2365. | Article | PubMed | ISI | ChemPort |
42. Kantarjian HM, O'Brien S, Cortes JE, Giralt SA, Rios MB, Shan J et al. Imatinib mesylate therapy for relapse after allogeneic stem cell transplantation for chronic myelogenous leukemia. Blood 2002; 100: 1590–1595. | PubMed | ISI | ChemPort |
43. Olavarria E, Craddock C, Dazzi F, Marin D, Marktel S, Apperley JF et al. Imatinib mesylate (STI571) in the treatment of relapse of chronic myeloid leukemia after allogeneic stem cell transplantation. Blood 2002; 99: 3861–3862. | Article | PubMed | ISI | ChemPort |
44. Wassmann B, Scheuring U, Thiede C, Pfeifer H, Bornhauser M, Griesinger F et al. Stable molecular remission induced by imatinib mesylate (STI571) in a patient with CML lymphoid blast crisis relapsing after allogeneic stem cell transplantation. Bone Marrow Transplant 2003; 31: 611–614. | Article | PubMed |
45. Hehlmann R, Hochhaus A, Kolb HJ, Hasford J, Gratwohl A, Heimpel H et al. Interferon-alpha before allogeneic bone marrow transplantation in chronic myelogenous leukemia does not affect outcome adversely, provided it is discontinued at least 90 days before the procedure. Blood 1999; 94: 3668–3677. | PubMed | ISI | ChemPort |