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The Journal of Immunology, 2004, 173: 6745-6752.
Copyright © 2004 by The American Association of Immunologists

Antiapoptotic Microenvironment of Acute Myeloid Leukemia

Dragana Milojkovic1, Stephen Devereux, Nigel B. Westwood, Ghulam J. Mufti, N. Shaun B. Thomas2 and Andrea G. S. Buggins2

Department of Haematological Medicine, Leukaemia Sciences, Guy’s, King’s and St. Thomas’ School of Medicine, Rayne Institute, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We showed previously that tumor-derived supernatant (TSN) from acute myeloid leukemia (AML) myeloblasts inhibits peripheral blood T cell activation and proliferation, rendering the T cells functionally incompetent. We show here that the AML TSN also significantly delays apoptosis of both resting and stimulated T cells, as judged by reduction in annexin V/propidium iodide staining. In addition, we show that this is not unique to T cells and that AML TSN inhibits apoptosis of peripheral B cells, neutrophils, and monocytes. Furthermore, it also enhances the survival of other AML myeloblasts with lower viability. Investigations into the mechanism demonstrate a reduction in the cleavage of procaspase-3, -8, and -9 and the caspase substrate, poly(ADP-ribose)polymerase (PARP). This may be due to Bcl-2, which is normally down-regulated in CD3/CD28-stimulated T cells, but is maintained in the presence of AML TSN. We conclude that AML cells generate an antiapoptotic microenvironment that favors the survival of malignant cells, but also inhibits apoptosis of other normal hemopoietic cells. Reversal of these immunosuppressive effects and restoration of normal immune responses in patients with AML would improve the success of immunotherapy protocols.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leukemia immunotherapy has limited success as a treatment strategy for acute myeloid leukemia (AML).3 Our previous work showed that tumor-derived supernatant (TSN) from AML myeloblasts prevents peripheral T cell activation (1) and we suggested that this may account for the immunosuppression observed in patients with AML. Inhibition of T cell responses by tumor cells would jeopardize immunotherapy protocols targeted to eradicate minimal residual disease. During the course of our previous study, we observed that although T cells failed to become activated when stimulated with CD3/CD28 or PMA/ionomycin in the presence of AML TSN, their viability appeared better than those that were activated alone.

The numbers of T cells in the body are regulated in part by apoptosis. Clonal deletion of thymic T cells occurs by apoptosis upon ligation of the CD3/TCR complex and is referred to as activation-induced cell death (AICD) (2, 3). Mature T cells in peripheral blood also die upon CD3 activation by means of peripheral deletion. In response to an antigenic stimulus, peripheral T cells first enter the cell cycle and proliferate, then decrease in number as they are deleted by apoptosis (4, 5, 6). AICD occurs when T cells, which are proliferating in the presence of IL-2, are reactivated. This mechanism is thought to limit the immune response to Ag and prevents the continuous proliferation of a single T cell clone (7). It has been suggested that the dominant role of IL-2 in vivo is to terminate T cell responses by making T cells susceptible to AICD (8, 9). In addition, it plays a role in maintaining peripheral tolerance to self-Ags that are presented continuously (8). During AICD, phosphatidylserine is translocated from the inner to the outer surface of the plasma membrane, the T cells shrink, and chromatin is condensed and cleaved. Many of the nuclear and cytoplasmic changes are dependent on activation of intracellular cysteine containing aspartic acid proteases (caspases). Indeed, blocking caspase activity can prevent the apoptotic process (10). T cells activated via the TCR can be saved from AICD by a second signal from costimulatory molecules, such as CD28. Costimulation enhances T cell viability not only by inducing IL-2 but also by up-regulating the antiapoptotic proteins Bcl-xL (11, 12, 13) and to a lesser extent Bcl-2 (14, 15, 16). Indeed, deprivation of IL-2 from stimulated T cells causes the up-regulation of proapoptotic proteins and induces apoptosis (17). Thus, CD28 costimulation enhances the proliferative expansion of T cells activated through the TCR and also increases T cell survival by inducing Bcl-xL and Bcl-2.

A second set of apoptotic regulators consists of members of the Bcl-2 family. These are classified into two functional groups. The first group, which contains Bcl-2 and Bcl-xL, possesses antiapoptotic activity and acts by preserving mitochondrial integrity and preventing the release of proapoptotic polypeptides (18). The second group, comprised of proteins such as Bax and Bim, promotes cell death. The key function of the Bcl-2 family members appears to be regulating the release of proapoptotic factors, in particular cytochrome c, from the mitochondrial intermembrane compartment into the cytosol (19, 20). Proapoptotic and antiapoptotic Bcl-2 family members meet at the surface of mitochondria where they compete to regulate cytochrome c exit. Cytochrome c coordinates the assembly of a complex involving Apaf-1 with caspase-9, a caspase-activating recruitment domain initiator caspase, which then initiates a downstream cascade of effector caspases, such as caspase-3, -6, and -7. The death receptor and mitochondrial pathways then converge at the level of caspase-3, ultimately leading to cell death (21, 22).

Entry into the cell cycle in response to CD3/CD28 costimulation is dependent on inactivation of the retinoblastoma protein (pRb) family members pRb and p130, which occurs by phosphorylation by cyclin-dependent kinases (23). In addition to regulating proliferation, pRb induces differentiation of some cell types and is also an inhibitor of apoptosis (23).

The c-Myc protein is also a central regulator of cell proliferation, but can independently sensitize cells to apoptosis (24) and apoptosis driven by c-Myc can be suppressed by antiapoptotic members of the Bcl-2 family (25, 26).

We investigated whether AML cells produce a microenvironment that is antiapoptotic in resting T cells, B cells, neutrophils, and monocytes and also whether it enhances the survival of other AML myeloblasts with low viability. We then examined whether AML TSN also prevents peripheral T cell AICD and show here that this is the case. AML TSN delays apoptosis of all of the hemopoietic cells we tested as judged by annexin V/PI staining. Investigation into the mechanism of inhibition in T cells demonstrated reduction in cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3, -8, and -9, which may be due to maintenance of Bcl-2. This is independent of their ability to inhibit induction of IL-2 and c-Myc as well as the phosphorylation of pRb. Our data provide proof of principle that myeloid diseases produce a microenvironment that is antiapoptotic.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of primary hemopoietic cells and culture model

Normal primary peripheral blood mononuclear cells (PBMNC) were obtained from normal donors by density gradient separation as described previously (1), after which T cells were negatively selected using immunomagnetic beads conjugated to anti-CD14, anti-CD19, anti-CD16, and anti-CD56 (Dynal, Oslo, Norway) as described before (1). Primary neutrophils were isolated from peripheral blood as described previously (1, 27). Briefly, a 45-ml sample of blood was anticoagulated with 10 mmol/L EDTA (pH 7.4) and centrifuged at 150 x g at 20°C for 10 min to separate platelet-rich plasma, after which the volume was restored to 50 ml by the addition of RPMI 1640 containing 10 mmol/L EDTA and 2% (v/v) FCS. Erythrocytes were then sedimented with dextran 500 (0.55% v/v; Sigma-Aldrich, Dorset, U. K.), after which the leukocyte-rich plasma then underwent density gradient separation (Ficoll-Hypaque) for 20 min at 20°C. The neutrophil pellet was then resuspended in RPMI 1640/EDTA/FCS.

B cells and monocytes were isolated using CD19 and CD14 immunomagnetic beads (Miltenyi Biotec, Surrey, U.K.) on an AutoMACS cell separator (Miltenyi Biotec) per the manufacturer’s instructions. Mononuclear cells were isolated from blood or bone marrow samples of presentation AML patients pretreatment, which were obtained with full informed consent. These patients had >95% type 1 myeloblasts.

Tumor cells were either cultured directly or cryopreserved before use. AML cells were cultured in RPMI 1640/30% FCS. The purified T cells were cultured at 1 x 106/ml for 5–16 h in RPMI 1640/10% FCS either alone, or in the presence of 1 x 106/ml primary AML cells, which were contained in a cell culture insert (pore size, 0.4 µm, Falcon; BD Biosciences, Oxford, U.K.). This Transwell system prevents cell–cell contact between normal hemopoietic cells, such as T cells, and leukemic cells in the same culture. The T cells were activated at 37°C for different time points, by adding either 1 µg/ml anti-CD3 (clone OKT-3; Janssen-Cilag, Buckinghamshire, U.K.) and 1 µg/ml anti-CD28 (clone CD28.2; BD Pharmingen, San Diego, CA), followed by cross-linking using a final dilution of 1/250 rabbit anti-mouse IgG (RAM; DakoCytomation, Carpinteria, CA), or by the addition of PMA (10 ng/ml) and ionomycin (1 µg/ml, both from Sigma-Aldrich).

Flow cytometric analysis of T cell activation

Intracellular IL-2, c-Myc expression, and pRb phosphorylation were detected as described previously (1). Briefly, for intracellular IL-2 detection, PBMNC were activated with PMA and ionomycin overnight in the presence of 1.4 mg/ml sodium monensin (Sigma-Aldrich). Cells were fixed and permeabilized (Fix and Perm; Caltag Laboratories, Burlingame, CA) and then labeled with a FITC-conjugated anti-IL-2 (Serotec, Oxford, U.K.). Flow cytometric analysis was performed using a FACSCalibur (BD Biosciences).

Analysis of t cell activation by Western blot

For analysis of c-Myc expression and pRb phosphorylation, T cells were activated with either anti-CD3 and anti-CD28 or PMA and ionomycin. Whole cell lysates of 1 x 106 cells were prepared on ice by the addition of 30 µl of ice-cold PBS, 7.5 µl of protease inhibitor (containing 4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin; Sigma-Aldrich), 1 µl of phosphatase inhibitor mixture (containing sodium orthovanadate, sodium molybdate, sodium tartrate, and imidazole; Sigma-Aldrich), and 30 µl of 2x SDS-PAGE sample buffer (Novex, San Diego, CA), followed by heating to 100°C for 10 min, then returned to ice, after which 5 µl of reducing agent (Novex) was added. Western blotting was performed using the Novex Powerease system (Novex) and NuPAGE 4–12% bis-Tris gels (Novex) per the manufacturer’s instructions. Membranes were blocked in PBS/5% nonfat dried milk/0.1% Tween 20 and then incubated with Abs to c-Myc (clone 9E10 mAb; Santa Cruz Biotechnology, Santa Cruz, CA), phospho-pRb (S807/811), and pRb (New England Biolabs, Little Chalfont, Hertfordshire, U.K.). Detection was via HRP-conjugated secondary Abs (DakoCytomation) followed by ECL or ECL-plus (Amersham, Hitchin, Buckinghamshire, U.K.).

Flow cytometric analysis of apoptosis

T cell, neutrophil, and myeloblast apoptosis was assessed by annexin V-FITC and propidium iodide (PI) staining according to the manufacturer’s instructions (Sigma-Aldrich). B cells and monocytes were detected with CD19-FITC and CD14-FITC, respectively, and stained with annexin V-PE and 7-aminoactinomycin D (Via-Probe; BD Biosciences). Regular samples were taken and the time points when 50 and 75% apoptosis occurred were noted. For B cells and monocytes, the 50% apoptosis point regularly appeared in-between harvesting points and so data were not sufficiently accurate. It was therefore decided to use the 75% apoptosis point for statistical purposes as accurate time points for this were available for all experiments performed. Neutrophil apoptosis was rapid and therefore the 50% apoptosis demarcation point was easily identifiable and thus used. Forward scatter (FSC) and side scatter (SSC) properties were also assessed in each case. Monocytes autofluoresce and this significantly affected the reliability of annexin V/PI staining. Therefore, the proportion of viable monocytes was assessed by determining the percentage in the characteristic FSC/SSC gate. This method is well established as an alternative means of identifying monocytes in blood mononuclear cells.

Analysis of apoptotic proteins by Western blot

T cell and neutrophil lysates were prepared as previously described (1) and lysed in SDS sample buffer containing 1 µM diisopropylfluorophosphate. Diisopropylfluorophosphate was added to inhibit protein degradation (28). Western blotting was performed using Abs to protein kinase C{delta}(PKC{delta}; C-20), Bag-1(C-16) (all from Santa Cruz Biotechnology), Bcl-2 (clone 124; DakoCytomation), Bim (Affinity BioReagents, Golden, CO), caspase-3 (CPP32), caspase-8 (B9–2), caspase-9 (B40), and PARP (C2–10) (all from BD Pharmingen), Bcl-x (44) (from BD Transduction Laboratories, Lexington, KY). Densitometry was performed using a Genegenius Bioimaging System (Syngene, Cambridge, U.K.). All densitometry figures are arbitrary and for each blot the lowest visible band was given a value of 1.0, all other band quantities are multiples of this. For this reason, densitometry values can only be compared with bands on the same gel. Nonspecific bands that cross-reacted with the Ab were used as internal loading controls and these are shown for all proteins not controlled by the production of cleavage products.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AML TSN delays T cell death

Our preliminary observations were that AML TSN maintained the viability of T cells, as judged by flow cytometric measurements of FSC and SSC (data not shown). To determine whether AML TSN inhibited or delayed T cell death, we cultured both resting and activated primary peripheral blood T cells in the presence and absence of AML cells in a Transwell system and assayed for apoptosis by annexin V/PI staining. This system enabled us to investigate the microenvironment produced by the AML cells and was not dependent on cell–cell contact. Annexin V-FITC was used to quantitatively determine the percentage of T cells that were undergoing apoptosis along with PI to distinguish viable from nonviable cells. Samples of nonactivated and CD3/CD28-activated T cells were analyzed every few days up to 21 days in culture. The AML TSN reduced the number of nonactivated as well as activated T cells that became annexin V/PI positive (Fig. 1, A and B). The time at which >50% annexin V/PI-positive cells were detectable was delayed in the presence of the AML TSN by a mean of 4.3 ± 2.2 days (SD) for nonactivated, 7.5 ±1.5 days for CD3/CD28-activated T cells, and 3.7 ± 1.0 days for PMA/ionomycin-activated T cells (n = 4). T cell death was not significantly delayed when normal PBMNC were present in the Transwell insert instead of AML cells (Fig. 1C).



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  		FIGURE 1. T cell apoptosis is suppressed in the presence of AML cells. T cells from normal donors were incubated, either unstimulated (A) or stimulated (B), with anti-CD3 and anti-CD28 (both at 1 µg/ml), up to 14 days in the presence or absence of AML cells. T cells were harvested at different time points and apoptosis was assayed using dual-color flow cytometry for annexin V and PI. A, After 6 days in culture, the number of dual-positive late (apoptotic cells), unstimulated T cells in the absence of AML cells was more than seven times that of those cultured with AML cells. B, Eight days poststimulation with anti-CD3 and anti-CD28, the proportion of T cells that had undergone apoptosis was halved in the presence of AML cells. A and B, Representative of four independent experiments, and similar results were obtained using PMA and ionomycin. C, To confirm that this inhibition of apoptosis was not caused by normal hemopoietic cells, PBMNC were used in place of the AML cells and tested at equivalent time points. T cell death was not significantly delayed at any time points in the presence of normal PBMNC (n = 4). This figure shows no significant apoptosis inhibition following 10 days of culture.

 
AML cells produce a microenvironment that prevents cell death in other hemopoietic cells

Inhibition of T cell death by tumor cells would seem to be counterintuitive. We surmised that the effect could be attributable to the production of an antiapoptotic microenvironment that promotes the survival of tumor cells and also maintains the viability of other cells in proximity, such as T lymphocytes. This hypothesis was tested by investigating the effect of AML TSN on cell death of other normal hemopoietic cells, namely, neutrophils, monocytes, and B cells. Neutrophils undergo apoptosis rapidly when cultured in vitro. Following culture with AML TSN, the time taken for 50% of neutrophils to undergo apoptosis was delayed by a mean of 33.8 ± 21.2 h as judged by annexin V/PI staining (Fig. 2A). Furthermore, PKC{delta} cleavage, which plays a central role in regulating neutrophil apoptosis and is an early event preceding annexin V detection (29), was also reduced. Full-length PKC{delta} (93 kDa) was degraded to the 53-kDa truncated form by 24 h in the absence of AML TSN but was maintained when the neutrophils were cultured in the presence of AML cells (Fig. 2B). Next, we determined whether AML TSN inhibited apoptosis of other hemopoietic cells. Our data show that 75% apoptosis of B cells (Fig. 2C) and monocytes (Fig. 2D) were also delayed by the presence of AML TSN (by 26.6 ± 19.5 and 13.2 ± 1.9 h, respectively (n = 3)).



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  		FIGURE 2. Neutrophil, B lymphocyte, monocyte, and myeloblast apoptosis is suppressed in the presence of AML cells. A, Neutrophils were selected from normal donors and then incubated for 48 h either alone or in culture with AML cells and their viability was assessed by staining with annexin V and PI. Neutrophil viability was enhanced when they were cultured with AML cells (57% annexin V negative/PI negative) rather than alone (16%). Representative of three experiments. B, In vitro incubation of neutrophils leads to the generation of a C-terminal fragment of PKC{delta}. Neutrophil PKC{delta} is proteolytically cleaved by 24 h of incubation with only the C-terminal fragment (53 kDa) and no full-length protein (93 kDa) was detected. Incubation with AML cells partially inhibits PKC{delta} proteolysis at 24 h and this effect is still maintained at 48 h. Representative of two experiments. C, B cells were selected from normal donors and then cultured for 6 days either alone or with AML cells. B cell viability (annexin V negative/7-aminoactinomycin D negative) was maintained following incubation with AML cells, (10% vs 1%). Representative of three experiments. D, CD14-positive monocytes were cultured for up to 14 days in the presence and absence of AML cells. Viability was assessed by loss of cells from the characteristic FSC/SSC gates. Following 6 days of culture, the percentage of viable monocytes was higher when they were cultured in the presence of AML cells (74%) as opposed to alone (27%). Representative of three experiments. E, Myeloblast apoptosis is also suppressed in the presence of AML cells. AML patient myeloblasts (designated AML1) with poor in vitro viability were incubated for up to 25 days, either alone or in culture with AML cells with a greater viability (designated AML2). Myeloblast viability (AML1) was maintained following incubation with AML cells (AML2) as demonstrated at day 21 (viability (annexin V negative/PI negative) alone (29%) and in the presence of AML cells (55%)). This experiment was also performed in the presence of normal PBMNC as a control and viability was not significantly higher (36%). Representative of three experiments.

 
These data are consistent with the development of an antiapoptotic microenvironment that may promote the survival of AML blasts. To test this hypothesis, we determined whether a sample of AML myeloblasts that remain viable in culture for several weeks (designated AML(2)) would reduce the apoptosis of AML myeloblasts that died by apoptosis when cultured in vitro for a few days (designated AML1). The viability of AML1 myeloblasts was maintained following incubation with AML 2 cells up to 25 days in culture (Fig. 2E). Apoptosis of AML1 was not significantly delayed by the presence of PBMNC used as a control. Taken together, our data indicate that AML blasts generate an antiapoptotic microenvironment that promotes the survival of tumor cells. This environment also promotes the survival of nonmalignant cells such as T and B lymphocytes, neutrophils, and monocytes.

AML TSN delays T cell apoptosis

T cells can die by apoptotic and nonapoptotic mechanisms. Our annexin V/PI data indicate that AML TSN may be inhibiting apoptosis, but to confirm this we assayed its effects on activation of caspases known to be involved in T cell apoptosis. We investigated whether the cleavage of caspase-3, -8, and -9 and their substrate, PARP, were affected in T cells cultured in the presence of AML cells. Each caspase is present as a proenzyme, which is cleaved to produce the active enzyme. Active caspase-3 (17-kDa subunit) is derived from the 32-kDa proenzyme. Caspase-8 is produced as a 55-kDa proenzyme, which is cleaved into two smaller subunits consisting of a 40/36-kDa (doublet) and 23-kDa subunit. The caspase-9 proenzyme is a 46- to 48-kDa protein but the active, cleaved form was not detected in our assay. Over a period of 12 days following mitogenic stimulation, the cleavage of caspase-3, -8, and -9 proenzymes to their active forms were significantly reduced by the presence of AML TSN (Fig. 3, A–C). PARP becomes cleaved by caspase-3, -8, and -9, from the 116-kDa intact nuclear protein to an 85-kDa fragment in cells that have become activated to undergo apoptosis. PARP remained mostly intact in the presence of AML TSN (Fig. 3D), consistent with an inhibition of caspase activation. Therefore, AML TSN delays caspase activation as well as annexin V/PI staining, consistent with an inhibition of peripheral T cell apoptosis. Inhibition of apoptosis occurred for both nonactivated as well as CD3/CD28-activated T cells, indicating that this was not specific to AICD.



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  		FIGURE 3. The cleavage of caspase-3, -8, and -9 and PARP is significantly reduced in T cells when cocultured with AML TSN. A—D, Caspase-3, -8, and -9 are activated by proteolytic cleavage during apoptosis. Caspase cleavage in CD3/CD28-stimulated or unstimulated T cells is much reduced in the presence of AML-TSN. PARP, a substrate of caspase-3 and -8, also remains intact in the presence of AML cells.

 
AML TSN maintains the level of Bcl-2 in T cells, but Bcl-xL, Bag-1, and Bim are regulated normally

Antiapoptotic members of the Bcl-2 family are known to prevent T cell apoptosis. Bcl-2 is present in nonactivated peripheral T cells and was decreased with time in culture (Fig. 4A). However, the level of Bcl-2 was maintained when T cells were cultured in the presence of AML cells (Fig. 4A). The levels of Bcl-2 were similarly maintained in T cells cultured in the presence of AML TSN irrespective of stimulation (Fig. 4A). In contrast to Bcl-2, Bag-1, which enhances antiapoptotic protection by Bcl-2, was unaltered by AML TSN (Fig. 4B). The expression of another antiapoptotic protein, Bcl-xL, was unaffected by the presence of AML TSN (Fig. 4C). Bcl-xL was induced by 3 days after CD3/CD28 stimulation and declined thereafter whether AML cells were present in the Transwell or not. Similarly, the decline in Bcl-xL that occurs in unstimulated cells in culture was unaffected by AML TSN. Next, we analyzed the expression of the proapoptotic protein Bim, a protein that has been implicated in inducing AICD. The expression patterns of this protein in T cells was not affected by the presence of AML TSN (Fig. 4D). Our findings indicate that the reduction of T cell apoptosis by AML TSN may be mediated by maintaining the level of Bcl-2.



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  		FIGURE 4. AML TSN prevents down-regulation of Bcl-2 but not maintenance of the antiapoptotic proteins Bag-1 and Bcl-xL. The levels of proapoptotic BIM is unaffected in T cells stimulated in the presence of AML TSN. Antiapoptotic members of the Bcl-2 family such as Bcl-2, Bcl-xL, and Bag-1 are known to prevent T cell death. A, Analysis of Bcl-2 expression over a period of 10 days shows that it is down-regulated in the absence of AML TSN, but maintained in their presence. This occurs regardless of whether the T cells are stimulated with CD3/CD28 or left unstimulated. Histone staining was performed on each assay. B, After 9 days, Bag-1, which enhances Bcl-2 protection from cell death, is unchanged in the presence of AML TSN in both resting and CD3/CD28-stimulated T cells. C, In unstimulated cells, Bcl-xL levels are unaltered by the presence of AML cells but in the stimulated sample a marginally higher level of Bcl-xL is noted at 6 and 10 days poststimulation. D, Three BIM isoforms (EL, L, and S) are generated by alternative splicing, all of which induce apoptosis. BIM levels in both CD3/CD28-stimulated and unstimulated T cells were unaffected by the presence of AML cells.

 
All of these assays were performed using different combinations of primary AML cells from four patients and eight normal donors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many studies have investigated how gene abnormalities in tumors could give malignant cells a proliferative or antiapoptotic advantage that enables them to survive and expand with respect to their normal counterparts. A few reports have documented that malignant cells of both solid tumors and leukemias produce factors that inhibit cells of the immune system, which may help them evade immune surveillance (Ref.1 and references therein; Ref.30). We showed previously that the TSN of AML blasts inhibits T cell proliferation and activation (1). During that study T cells were noticed to survive better in the presence of AML TSN. We investigated this further and we now show that AML TSN delays T cell apoptosis. The AML cells also delay apoptosis of a number of other normal hemopoietic cells as well as other AML myeloblasts that would normally die more rapidly. This is consistent with the generation of an antiapoptotic microenvironment by AML cells that promotes their own survival and that of other neighboring cells. Our studies indicate that AML cells may flourish by promoting their own survival through generating an antiapoptotic microenvironment and, in some cases, by also rendering T cells functionally incompetent.

We first showed that AML blasts delayed peripheral T cell apoptosis. T cell death was initiated after CD3/CD28 stimulation, which was assayed by annexin V and PI staining. When T cells were cultured with AML TSN, the time taken for 50% of T cells to undergo apoptosis was delayed by 7.5 ± 1.5 days for cells stimulated with CD3/CD28 and 3.7 ± 1.0 days for cells stimulated with PMA/ionomycin. This inhibition was specific to AML TSN as normal PBMNC in the same Transwell system did not protect T cells from apoptosis.

Our data showed that not only did AML TSN inhibit AICD in T cells stimulated with CD3/CD28, it also prevented cell death of resting T cells in culture. This was significant because it implied that inhibition of apoptosis might not be restricted to AICD. Therefore, we investigated whether AML TSN would inhibit apoptosis of other normal hemopoietic cells that are present in the blood or bone marrow. Neutrophils die readily by apoptosis when cultured in vitro and this is dependent on cleavage of PKC{delta} by caspase-3 (29). AML TSN inhibited PKC{delta} cleavage and delayed neutrophil apoptosis by 33.8 ± 21.2 h. Apoptosis of B cells and monocytes was also delayed by AML TSN. Thus, AML TSN inhibition of apoptosis was not restricted to T lymphocytes and might in part account for the survival of AML cells themselves. This was investigated by the analysis of apoptosis of AML myeloblasts from one patient (AML1) that died within a few days in culture in the presence of AML TSN from another patient (AML2) where the myeloblasts were long-lived. The AML2 TSN protected the AML1 myeloblasts from apoptosis and prolonged their survival in culture up to 25 days. We conclude that AML blasts generate an antiapoptotic microenvironment that favors their survival.

We next investigated the mechanism by which TSN inhibits both activation-induced and spontaneous cell death using T lymphocytes as a model. Caspases, which play a central role in the regulation and execution of apoptosis, exist as inactive proenzymes in the cytosol and become activated through proteolysis when cells receive apoptotic signals. The cleavage of caspase-3, -8, and -9 proenzymes were significantly reduced in T cells when cocultured with AML blasts. Accordingly, the DNA repair enzyme PARP, a nuclear substrate of caspase-3 and -8 (31, 32), remained intact in the presence of AML cells. These data are consistent with inhibition of T cell apoptosis rather than death by another mechanism.

Analysis of the antiapoptotic Bcl-2 protein showed that it was down-regulated in T cells during short-term culture but maintained in the presence of AML TSN. In contrast, expression of the antiapoptotic Bcl-xL protein was unaffected by AML TSN. Similarly, Bag-1, which enhances Bcl-2 protection from cell death mediated by IL-2 (33), was also unaffected by AML TSN. Apoptosis can be triggered by members of the Bcl-2 protein family that share only the BH3 domain, such as Bim (34, 35). Indeed, activation of Bim has been implicated in peripheral T cell deletion (36, 37, 38). Bim isoforms generated by alternative splicing (BimEL/L/S) all have greater proapoptotic activity than any other Bcl-2 member, with BimS being the most potent (39, 40). In addition, high levels of Bcl-2 allow stable expression of BIM. In the presence of AML TSN, the levels of Bim protein in T cells were unaffected, which implies that the antiapoptotic effect of AML cells is not mediated through a distinct BH3-only protein. Bcl-2 antiapoptotic properties in T cells are well documented (9, 13) and our findings indicate that the reduction of T cell apoptosis by AML TSN may be mediated by maintaining the levels of Bcl-2.

We showed previously that AML TSN prevents T cell activation and proliferation (1). This was associated with inhibition of IL-2 and IFN-{gamma} production and the induction of c-Myc. c-Myc induces cell cycle progression but it can also cause apoptosis (41, 42). Induction of apoptosis by c-Myc can be delayed significantly by Bcl-2 (25, 26). Therefore, the inhibitory effects of AML TSN on c-Myc may in part explain the delay in T cell apoptosis, especially since the levels of Bcl-2 are also maintained. Consistent with this, TGF-{beta} inhibits AICD and Fas ligand expression, a major inducer of AICD, and this may be mediated by inhibition of c-Myc (42, 43). pRb is another candidate for mediating the antiapoptotic effects of AML TSN. Hypophosphorylated pRb has been shown to inhibit apoptosis (44) and we have previously shown (1) that AML TSN prevents pRb phosphorylation and progression through the recently described G0-G1 restriction point (45). In this study, we showed inhibition of pRb phosphorylation in all cases of AML TSN examined.

In conclusion, AML cells secrete factor(s) with autocrine and paracrine antiapoptotic effects that are mediated in T cells by maintenance of Bcl-2 expression and inhibition of effector caspase activation. Several groups, including our own, have previously shown that disease progression from preleukemia (myelodysplastic syndrome) to AML is associated with a reduction in apoptosis and an increase in the blast cell count (46). Our observation that AML blasts secrete factor(s) with both autocrine and paracrine antiapoptotic effects provides one potential explanation for this phenomenon. Although the advantages of such a microenvironment to the tumor itself are obvious, the relevance of the effects on T cell apoptosis are less clear. It may be that in the tumor microenvironment, inhibition of T cell apoptosis allows the accumulation of quiescent T cells to the exclusion of others. These findings might explain why adoptive immunotherapy, which is so successful in disorders such as chronic myeloid leukemia (47), so frequently fails in patients with AML. Further characterization and identification of these factor(s) will thus be of crucial importance for the further development of future immunotherapeutic strategies.


   Acknowledgments
 
This study is a follow up of work initiated by the late Dr. William. J. R. Hirst.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Dragana Milojkovic, Department of Haematological Medicine, Leukaemia Sciences Laboratories, Guy’s, King’s and St. Thomas’ School of Medicine, Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, U.K. E-mail address: dragana.milojkovic{at}kcl.ac.uk Back

2 N.S.B.T. and A.G.S.B. have made equal contributions to this work. Back

3 Abbreviations used in this paper: AML, acute myeloid leukemia; TSN, tumor-derived supernatant; AICD, activation-induced cell death; pRb, retinoblastoma protein; PARP, poly(ADP-ribose) polymerase; PBMNC, peripheral blood mononuclear cell; PI, propidium iodide; FSC, forward scatter; SSC, side scatter; PKC, protein kinase C. Back

Received for publication April 6, 2004. Accepted for publication September 21, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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