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The Journal of Immunology, 2001, 167: 6021-6030.
Copyright © 2001 by The American Association of Immunologists

Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-{kappa}B, c-Myc, and pRb Pathways

Andrea G. S. Buggins1, Dragana Milojkovic, Matthew J. Arno, Nicholas C. Lea, Ghulam J. Mufti, N. Shaun B. Thomas and William J. R. Hirst

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
 
Tumors produce a variety of immunosuppressive factors which can prevent the proliferation and maturation of a number of normal hemopoietic cell types. We have investigated whether primary acute myeloid leukemia (AML) cells have an effect on normal T cell function and signaling. Tumor cell supernatant (TSN) from AML cells inhibited T cell activation and Th1 cytokine production and also prevented activated T cells from entering the cell cycle. These effects occurred in the absence of AML cell-T cell contact. We have demonstrated that AML TSN contained none of the immunosuppressors described to date, namely gangliosides, nitric oxide, TGF-{beta}, IL-10, vascular endothelial growth factor, or PGs. Furthermore, IL-2 did not overcome the block, despite normal IL-2R expression. However, the effect was overcome by preincubation with inhibitors of protein secretion and abolished by trypsinization, indicating that the active substance includes one or more proteins. To determine the mechanism of inhibition, we have studied many of the major pathways involved in T cell activation and proliferation. We show that nuclear translocation of NFATc and NF-{kappa}B are markedly reduced in T cells activated in the presence of primary AML cells. In contrast, calcium mobilization and activation of other signal transduction pathways, namely extracellular signal-regulated kinase1/2, p38, and STAT5 were unaffected, but activation of c-Jun N-terminal kinase 1/2 was delayed. Phosphorylation of pRb by cyclin-dependent kinase 6/4-cyclin D and of p130 did not occur and c-Myc, cyclin D3, and p107 were not induced, consistent with cell cycle inhibition early during the transition from G0 to G1. Our data indicate that TSN generated by AML cells induces T cell immunosuppression and provides a mechanism by which the leukemic clone could evade T cell-mediated killing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Patients with acute myeloid leukemia (AML)2 are immunosuppressed, and this plays a major role in increasing their morbidity and mortality (1). Although lymphocytes are not generally part of the malignant clone, suppression of their activity is an obstacle to the treatment of this disease because immune mechanisms are thought to be important for the control of minimal residual disease (2). In addition, disease-related immunosuppression has important implications for the development of novel immunotherapeutic strategies such as tumor vaccination or adoptive immunotherapy with allogeneic donor T cells. Many studies have highlighted the difficulty of generating an effective antitumor response despite the presence of tumor-specific Ags, and these findings have rekindled interest in the mechanisms by which tumors evade immune destruction (3). One mechanism adopted by tumors to evade immune rejection is the production of soluble factors that are able to suppress cells involved in immune surveillance. Examples that have been characterized include IL-10, TGF-{beta}, vascular endothelial growth factor (VEGF), NO, PGE2, and gangliosides (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Kiertsher et al. (16) and others (17, 18) have demonstrated that the maturation and function of dendritic cells (DC) is affected when they are in the presence of tumor cell supernatant (TSN) from a number of tumor cell lines and that the effect is not reversed by Abs to known DC immunoregulatory factors such as IL-10, TGF-{beta}, VEGF, or PGE2 (16). T cell abnormalities, including inhibition of Th1 cytokine production (13), cytotoxicity, and proliferation, also occur in patients with cancer (8, 11, 12, 13, 19, 20, 21, 22, 23), as well as in animal models of malignancy (20). This phenomenon is not restricted to tumor-infiltrating or autologous peripheral blood T cells, indicating that it is due to the presence of the tumor rather than an intrinsic T cell abnormality. We hypothesized that supernatant from primary AML cells may have an effect on T cell function similar to the effect TSN from cell lines had on DC (16, 17, 18). We have previously shown that the U937 leukemic cell line generates a TSN that inhibits Ag presentation in an MLR and T cell secretion of IFN-{gamma} and IL-2 (19) within 24 h of coculture. Many of the immune defects observed in patients with leukemia, as well as the disappointing results of some adoptive or active immunotherapy strategies, could be explained by the presence of an immunosuppressive microenvironment generated by the leukemic clone.

Following interaction with an APC, activation and subsequent clonal expansion of mature T cells requires the formation of a large signaling complex at the site of cell-cell contact (24, 25). This triggers a cascade of intracellular signal transduction pathways, briefly summarized below, which leads to activation and transcription of a variety of different genes and entry into the cell cycle (26, 27). Engagement of the TCR causes rapid activation of specific protein tyrosine kinases and these phosphorylate tyrosine motifs in the cytoplasmic domain of the TCR-{zeta} subunit (28), which in turn allows binding and activation of the ZAP-70 kinase (27), followed by calcium mobilization. The transcription factor NF-{kappa}B/Rel is maintained in an inactive form in the cytoplasm of quiescent cells in a complex with I{kappa}B proteins (29), and calcium mobilization leads to the phosphorylation of I{kappa}B, which is then degraded, enabling NF-{kappa}B/Rel (30) to translocate to the nucleus. In addition, the phosphatase calcineurin is activated, leading to dephosphorylation of NFATc (31, 32, 33) and its subsequent nuclear translocation. Concurrently, a number of proteins are phosphorylated on tyrosine residues, including signal transduction adaptor proteins. These events cause the recruitment and assembly of signaling complexes that trigger different signal transduction pathways. The pathways involved include the Janus kinase/STAT (34, 35), protein kinase C (36), as well as mitogen-activated protein kinase (MAPK) pathways, which include extracellular signal-regulated kinases (ERK)1 and 2 (37), c-Jun N-terminal kinase (JNK)1 and 2 (38), and p38 (39). The JNK, ERK, and p38 pathways each can phosphorylate NFATc directly and block its nuclear accumulation (40, 41). One consequence of this cascade of different signals is the induction of c-Myc, a protein that is essential for entrance into the cell cycle (42). In addition, a defined genetic program is initiated that leads to the production of cytokines, which include IL-2 and IFN-{gamma}, and up-regulation of cell-surface receptors, such as CD25 (IL-2R{alpha}) (43, 44, 45). One of the best characterized is the induction of the IL-2 gene, which occurs by coordinated binding of several transcription factors to the IL-2 gene promoter (46), including NF-{kappa}B and NFATc.

T cells are thought to be maintained in a quiescent state by members of the retinoblastoma protein family, pRb and p130 (47). The quiescent T cells contain active, hypophosphorylated forms of pRb and p130 that bind and repress the activity of E2F transcription factors. Mitogenic stimulation causes hyperphosphorylation and inactivation of both pRb and p130 by the cyclin-dependent kinases (cdk), cdk6-cyclin D2 or D3, cdk4-cyclin D2/D3, and cdk2-cyclin E (47, 48). As part of this mechanism, E2F factors are released from inhibition by pRb and p130 and stimulate the transcription of a number of genes that are required for regulating the cell cycle and for DNA synthesis. Such genes are induced before entry into S-phase and include p107 and the proliferating cell nuclear Ag, (PCNA) (49).

We showed previously that TSN from the leukemic cell line U937 inhibits Ag presentation and T cell function (19). The aim of the present study was to determine whether the TSN of primary cells from patients with AML inhibited T cell activation and proliferation. We also investigated the mechanism of action of AML-derived TSN by determining which signaling pathways were affected and which remain intact, and the mechanisms by which the cell cycle is inhibited.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and culture model

PBMC and bone marrow were obtained from normal donors by density-gradient separation. Blood samples were obtained from 18 different patients with AML and cells were purified by the same method. AML cells were either cultured directly or step frozen. Normal nonmalignant T cells were negatively selected from PBMC using immunomagnetic beads conjugated to anti-CD14, anti-CD19, anti-CD16, and anti-CD56 (Dynal, Oslo, Norway) as per the manufacturer’s instructions. The purified T cells or PBMC were cultured for 5–16 h at 37°C in 5% CO2 either alone or in the presence of primary AML cells (5 x 106 cell/ml) contained in a cell culture insert (pore size 0.4 µm), which prevents cell-cell contact between PBMC and leukemic cells. Cells were then activated at 37°C for different time periods, using either 1 µg/ml anti-CD3 (clone OKT-3; Janssen-Cilag, Bucks, 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 (DAKO, Cambridgeshire, U.K.), or by the addition of PMA (10 ng/ml) and ionomycin (1 µg/ml) (both from Sigma-Aldrich, Dorset, U.K.).

Flow cytometric analysis of T activation and proliferation

For intracellular IL-2 and IFN-{gamma} detection, PBMC were activated with PMA and ionomycin overnight in the presence of 1.4 mg/ml sodium monensin (Sigma-Aldrich), which retains cytokines within the cells (50). Labeling with conjugated Abs to cytokines (IL-2-FITC or IFN-{gamma}-FITC; Serotec, Oxford, U.K.) was performed using a commercially available permeabilization kit (Fix and Perm; Caltag Laboratories, Burlingame, CA) (19). Unstimulated cells were used as controls. Flow cytometric analysis was performed using a FACSCalibur (BD Biosciences, Oxford, U.K.).

For assays attempting to reverse the AML inhibitory effect, AML cells were preincubated separately with one of the following: the ganglioside synthesis inhibitor DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PMP) (10 µM overnight; Biomol, Plymouth Meeting, PA), neuraminadase (0.1 U/ml overnight; Boehringer Mannheim, Mannheim, Germany), the nitric oxide inhibitor NG-monomethyl-L-arginine monoacetate salt (10 µg/ml; Calbiochem, Darmstadt, Germany), inhibitors of protein secretion brefeldin A (10 µg/ml; Sigma-Aldrich) or sodium monensin (1.4 mg/ml; Sigma-Aldrich), all for 4 h. For neutralizing assays to known T cell suppressors, agents were used at a concentration of 5 µg/ml anti-TGF-{beta}, 30 ng/ml anti-IL-10, or 10 µM indomethacin (a suppressor of cyclooxygenase that inhibits PG production). For trypsinization experiments, AML cells were cultured in the serum-free medium (AIM V; Life Technologies, Paisley, U.K.) for 48 h. Supernatant was then harvested and incubated for 30 min at 37°C in 5% CO2 either alone or in the presence of 0.5 mg/ml trypsin (tissue-culture grade; Sigma-Aldrich). The reaction was stopped by the addition of 10% FCS and resultant solution incubated with normal PBMCs for 5 h before stimulation as described above.

For intracellular PCNA and activation marker analysis, PBMC were activated for 48 h using PMA and ionomycin. Intracellular PCNA analysis was performed using FITC-conjugated anti-PCNA (Serotec) and a commercially available permeabilization kit (Cytoperm; Serotec) using the methanol modification protocol. Surface labeling for the IL-2R was then performed using PE-conjugated anti-CD25 (BD Biosciences).

For intracellular cyclin D3 and propidium iodide (PI) analysis, PBMC were activated for 24 h with PMA and ionomycin. Intracellular cyclin D3 was detected with FITC-conjugated anti-cyclin D3 (BD PharMingen) following ethanol fixation and Triton X-100 (BDH Laboratories, Poole, U.K.) permeabilization as per the manufacturer’s instructions. Cells were resuspended in 50 µg/ml PI before flow cytometric analysis.

Analysis of T cell signaling

T cells were activated with either anti-CD3 and anti-CD28 or PMA and ionomycin. Activation was stopped by two washes using 1 ml of ice-cold PBS at 4°C and preparation of a cell lysate. Whole cell lysates of 1 x 107 cells were prepared on ice by the addition of 30 µl of ice-cold PBS, 7.5 µl of protease inhibitor (containing 4-AEBSF, 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. Chromatin-bound and unbound preparations were made as previously published (51).

Western blotting was performed using the Novex Powerease system (Novex) and NuPAGE 4–12% Bis Tris gels (Novex) as 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), NF-{kappa}Bp65 (Rel A), NF-{kappa}Bp75(c-Rel) (all from Santa Cruz Biotechnology, Santa Cruz, CA), NF-{kappa}Bp50 (Upstate Biotechnologies, Lake Placid, NY), phospho-JNK1/2 (T185,Y183), JNK1/2, phospho-MAPK (T202,Y204), MAPK, phospho-p38 (T185,Y182), p38, phospho-STAT5 (Y694), STAT5A, phospho-pRb (S807/811), pRb, p130, p107, and phospho-I{kappa}B (S32), and I{kappa}B (all from New England Biolabs, Little Chalfont, Hertfordshire, U.K.). Detection was via HRP-conjugated secondary Abs (DAKO) followed by ECL or ECL-plus (Amersham, Hitchin, Buckinghamshire, U.K.).

Calcium mobilization

T cells were resuspended to 107 cells/ml in RPMI 1640 and labeled with 3 µM of Indo-1 (Molecular Probes, Eugene, OR) for 30 min at 37°C, washed, and resuspended in RPMI 1640 at 106 cells/ml. Kinetic studies of intracellular calcium concentration were performed with a FACSVantage (BD Biosciences) equipped with 488 nm and UV lasers (60 mW). Cells were activated using 1 ng/ml anti-CD3 (clone OKT-3; Janssen-Cilag) and 1 µg/ml anti-CD28 (clone CD28.2; BD PharMingen) and then cross-linked using a final dilution of 1/175 rabbit anti-mouse IgG (DAKO). Calcium concentration was determined by the bound:free Indo-1 ratio (i.e., fluorescence at 480 nm:530 nm) (19) over a 512-s period.

Nuclear translocation of NFATc and NF-{kappa}B by confocal microscopy

T cells were activated for 1 h using PMA and ionomycin. Cytospins were prepared, fixed, blocked, and labeled with Abs to NFATc1 (7A6) or NF-{kappa}Bp65 (F-6) or appropriate isotype-matched controls (all from Santa Cruz Biotechnology) as per the manufacturer’s instructions. Detection was via a FITC-conjugated secondary Ab (DAKO), and slides were analyzed with a confocal microscope (BIO-RAD 1024; Bio-Rad, Hercules, CA).

ELISA for VEGF

An ELISA for levels of VEGF in primary AML cell supernatant was performed using a Quantikine kit as per the manufacturer’s instructions (R&D Systems, Oxon, U.K.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The presence of primary AML cells reduces IL-2 and IFN-{gamma} production by normal PBMC after stimulation

Production of IL-2 and IFN-{gamma} is a hallmark of activated T cells (52). To investigate whether the TSN generated by leukemic cells from AML patients inhibited production of the IFN-{gamma} and IL-2 Th1 cytokines, PBMC from normal individuals were activated in the presence or absence of normal bone marrow or primary AML cells. Stimulation of PBMC induced IFN-{gamma} and IL-2 production alone and in the presence of normal bone marrow, but these were inhibited in the presence of leukemic cells from all of the 12 and 18 patients with AML tested, respectively (Fig. 1Go). The mean IFN-{gamma} production in the presence of AML cells was 25.8% (±5.4% SEM) of that in T cells stimulated alone, with a range of 2.6–55% (paired Student’s t test, p < 0.0001) and mean corresponding figures for IL-2 were 18.2% (±4.7%), with a range of 0–46.5% (paired Student’s t test, p < 0.0001).



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  		FIGURE 1. The presence of primary AML cells reduces the IL-2 and IFN{gamma} production of normal PBMC after stimulation with PMA and ionomycin. PBMC were stimulated with PMA and ionomycin for 16 h in the presence and absence of normal bone marrow (BM) or AML cells, and intracellular IL-2 (A) and IFN-{gamma} (B) were quantified by flow cytometry. The examples shown in the first four panels are representative of 18 (IL-2) and 12 (IFN-{gamma}) independent experiments which are summarized in the fifth panel. A, When PBMC were stimulated in the presence of primary AML cells there was a reduction in numbers producing intracellular IL-2 (stimulated PBMC alone, 33.2% ± 6.5 (mean ± SEM); stimulated PBMC with primary AML cells, 6.3% ± 1.9 (mean ± SEM)). B, In the presence of primary AML cells there was also a reduction in the number of PBMC containing intracellular IFN-{gamma} poststimulation with PMA and ionomycin (stimulated PBMC alone, 25% ± 7.2 (mean ± SEM); stimulated PBMC with primary AML cells, 8% ± 1.7 (mean ± SEM)).

 
The TSN produced by primary AML cells does not contain previously described immunosuppressive factors

To determine the nature of the TSN, AML cells were preincubated with agents to inhibit the production of known inhibitors of T cell activation. Preincubation of AML cells from two different patients with the ganglioside synthesis inhibitors DL-PDMP (53), neuraminadase (54), or the nitric oxide inhibitor NG-monomethyl-L-arginine monoacetate had no effect on the inhibition of T cell activation (Table IGo). In addition, cells from three patients were tested with neutralizing Abs to the known T cell suppressors, TGF-{beta}, IL-10, and indomethacin, which inhibits production of PGs (Table IIGo). None of these agents reversed inhibition by the TSN (Tables IGo and IIGo) and none had any effect on unstimulated or stimulated cells alone (data not shown). An ELISA for VEGF levels in AML cell culture supernatants from three patients showed levels to be below the detectable range of the assay (<31.2 pg/ml; data not shown).


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  		Table I. The immunosuppressive effect of primary AML cells is not reversed by DL-PDMP, neurominadase, or inhibition of NO production1

 

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  		Table II. The immunosuppressive effect of primary AML cells is not reversed by anti-TGF-{beta}, anti-IL-10, indomethacin, or IL-21

 
Preincubation of leukemic cells with either brefeldin A or sodium monensin, which inhibit Golgi function and therefore prevent secretion of glycoproteins (55), overcame the inhibitory effect in all three patients analyzed (Fig. 2GoA). Further evidence indicating that a protein is involved is provided by trypsinization of primary AML cell supernatant. AML cells from the same three patients were cultured in serum-free medium and trypsinization of this supernatant before its addition to the T cells destroyed the inhibitory effect (Fig. 2GoB). The T cells were then activated to the same extent as the control.



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  		FIGURE 2. Preincubation of primary AML cells with inhibitors of protein secretion or trypsinization of AML cell supernatant restores IL-2 production. A, PBMC were left unstimulated (a) or stimulated with PMA and ionomycin for 16 h in the absence (b) or presence (c) of AML cells, and intracellular IL-2 was quantified by flow cytometry. Preincubation of the AML cells for 4 h with brefeldin A (d) or sodium monensin (e) prevented inhibition of T cell IL-2 production. B, Primary AML cells were cultured in serum-free medium and the resultant supernatant was incubated with or without trypsin. The untreated supernatant inhibited intracellular IL-2 production (c), compared with the control (b), but trypsinization abolished the inhibitory effect of the supernatant (d). Unstimulated T cells are shown in a.

 
Calcium mobilization in T cells is not affected by the AML TSN

Next, we investigated the mechanism by which the protein secreted by the AML cells inhibited T cell activation. Calcium mobilization is a very early event in T cell activation that occurs in seconds and is necessary for activation and nuclear translocation of the transcription factors NFATc and NF-{kappa}B. The kinetics and amplitude of the calcium flux generated in T cells stimulated via CD3 and CD28 was identical in the presence or absence of primary AML cells from three patients (Fig. 3Go), indicating that this is not the primary cause of the inhibition.



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  		FIGURE 3. Calcium release in normal T cells is unaffected by the TSN produced by primary AML cells after stimulation with mitogenic anti-CD3 and anti-CD28. T cells were cultured in the presence and absence of AML cells for 16 h, loaded with Indo-1, and stimulated with anti-CD3 and anti-CD28, followed by cross-linking with polyclonal rabbit anti-mouse Ig. Intracellular calcium concentrations are shown (ratio of bound:free) over a 512-s time period, and the arrow represents the point of stimulation. Calcium mobilization occurs rapidly poststimulation and then returns close to resting levels. The top plot is of T cells stimulated alone and in the bottom plot T cells were stimulated in the presence of primary AML cells. This figure is representative of results obtained using AML cells from three different patients. The presence of primary AML cells had no effect on the kinetics or magnitude of calcium mobilization.

 
Nuclear translocation of NFATc and NF-{kappa}B is inhibited by the AML TSN

The effect of AML cells on nuclear translocation of NFATc and NF-{kappa}B in normal T cells was examined by immunocytochemistry. Loss of cytoplasmic NFATc and the p65RelA subunit of NF-{kappa}B occurred when T cells were activated alone, but not when activation was conducted in the presence of primary AML cells (Fig. 4Go, A and B). Inhibition of NF-{kappa}B occurred for all four AML samples tested, and three cases of four blocked NFATc translocation. It should be noted that the anti-NFATc1 Ab used does not detect the nuclear form of the protein (Fig. 4GoB, center panel). These data are consistent with inhibition of nuclear translocation of both these proteins.



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  		FIGURE 4. Nuclear translocation of NFATc and 78 F{kappa}{beta} is inhibited by the TSN generated by primary AML cells. A, NF-{kappa}B levels in unstimulated T cells, and those cultured with and without AML cells for 16 h, then stimulated for 1 h with PMA and ionomycin, were quantified by confocal microscopy. NF-{kappa}B is predominantly cytoplasmic in resting T cells (left panel) but nuclear NF-{kappa}B is clearly detectable upon stimulation (center panel). In the presence of primary AML cells, NF-{kappa}B remains cytoplasmic (right panel). This figure is representative of results obtained using AML cells from four different patients. B, NFATc1 levels in unstimulated and stimulated T cells were analyzed as above. In resting T cells, NFATc1 is cytoplasmic (left panel), and this signal disappears upon stimulation (center panel). The NFATc1 Ab used does not detect NFATc1 in the nucleus. In the presence of primary AML cells NFATc1 remains cytoplasmic (right panel). This figure is representative of results obtained using AML cells from three of four different patients.

 
Active NF-{kappa}B is composed of p65RelA-p50 or p75c-Rel-p50 heterodimers rather than the p50-p50 homodimer. We tested which NF-{kappa}B subunits were affected by the AML TSN by analyzing nuclear DNA binding of each subunit. Chromatin binding of T cell p65RelA was delayed and p75c-Rel was inhibited during the 4-h time period by AML TSN, whereas binding of p50 was less affected (Fig. 5GoA). These data are consistent with inhibition of NF-{kappa}B activation by AML TSN.



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  		FIGURE 5. The presence of primary AML cells suppresses chromatin binding of the p65RelA and p75c-Rel subunit of NF-{kappa}B but does not affect phosphorylation or degradation of I{kappa}B. A, T cells were cultured overnight in the presence or absence of primary AML cells before stimulation for 1, 2, and 4 h with PMA and ionomycin. Chromatin-bound and unbound proteins were isolated, and the levels of p65RelA, p75c-Rel, and p50 NF-{kappa}B subunits in each were detected by Western blotting. All subunits were chromatin-bound in T cells stimulated alone, but the amount of p65RelA and p75c-Rel bound to DNA is decreased in the presence of AML cells. B, Phospho-I{kappa}B (S32) (top) and total I{kappa}B (bottom) in unstimulated T cells, and in T cells cultured for 16 h with and without AML cells before being stimulated for 5, 15, 20, and 30 min, were detected by Western blotting. Maximal phosphorylation of I{kappa}B occurs by 15 min poststimulation with PMA and ionomycin, and it is then degraded. The presence of primary AML cells did not inhibit the phosphorylation of I{kappa}B or its degradation.

 
Phosphorylation and degradation of I{kappa}B is unaffected by the AML TSN

Activation and nuclear translocation of NF-{kappa}B is dependent on the phosphorylation of I{kappa}B, which is then rapidly degraded. Phosphorylation of I{kappa}B at S32 was detectable by 15 min after the addition of PMA and ionomycin, and had declined to background levels within 20 min (Fig. 5GoB, upper panel). Analysis of the levels of I{kappa}B protein (Fig. 5GoB, lower panel) showed a reduction after 20 min, consistent with its degradation. The presence of primary AML cells had no effect on the kinetics or extent of I{kappa}B phosphorylation or degradation (Fig. 5GoB). Therefore, inhibition of nuclear translocation of NF-{kappa}B cannot be explained by a failure to degrade I{kappa}B.

The presence of the AML cells does not affect activation of STAT5, ERK1/2, or p38, but JNK1/2 is delayed

To investigate whether the AML TSN inhibits T cell signaling in general, the kinetics of activation of the STAT5 transcription factor and the MAPK family of signal transduction proteins was analyzed. Activation of each protein is dependent on phosphorylation at specific sites, which was assayed by probing the blots with phosphorylation site-specific Abs. The presence of primary AML cells from three patients did not affect the kinetics of STAT5, ERK1/2, or p38 activation; however, there was a delay of ~5 min in the activation of JNK1/2 (Fig. 6Go). Therefore, a number of signaling pathways are activated normally in the presence of AML cells, which indicates that the AML TSN does not repress all T cell functions.



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  		FIGURE 6. Phosphorylation of STAT5, ERK1/2, and p38 are normal in the presence of primary AML cells, but phosphorylation of JNK1/2 is delayed. T cells were cultured overnight with or without AML cells and then stimulated for 0, 15, 20, and 30 min with PMA and ionomycin and phospho-STAT5 (Y694) (A), ERK1/2 phosphorylated at T202/Y204 (B), JNK1/2 at T185Y183 (C), and p38 at T180/Y182 (D) (upper panels) were detected by Western blotting. Blots of total STAT5A, ERK1/2, p38, or JNK1/2 protein present in each sample are shown in the lower panels. Each is representative of experiments using cells from three different patients. Phosphorylation of STAT5, ERK1/2, and p38 occurs within 5 min of stimulation with PMA and ionomycin, and the presence of primary AML cells did not affect the kinetics or magnitude of the response. Phosphorylation of JNK1/2 occurs by 15 min poststimulation, and this was delayed by 5 min in the presence of primary AML cells.

 
The presence of AML cells inhibits entry of T cells into S-phase, but not expression of the IL-2R, CD25

To determine whether the AML TSN affects T cell mitogenesis, we assessed the up-regulation of the IL-2R (CD25) and PCNA, a marker of cell entry into S-phase. When T cells were stimulated in the presence of primary AML cells from eight different patients, the levels of PCNA were reduced to varying degrees, but CD25 expression remained unchanged (Fig. 7GoA). Two examples of the range of responses seen in the presence of primary AML cells are shown: the first, in which ~50% inhibition was observed (AML1); and the second, in which there was essentially complete inhibition of PCNA production (AML2).



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  		FIGURE 7. Entrance into late G1 is delayed by the presence of primary AML cells, but expression of the IL-2R CD25 remains unaffected. CD25 and PCNA levels (A) and cyclin D3 and DNA content (PI staining) (B) in unstimulated T cells (a) and those stimulated with PMA and ionomycin in the absence (b) and presence (c and d) of AML cells were quantified by flow cytometry. The examples shown are representative of eight independent experiments. CD25 levels were unaffected in the presence of primary AML cells. Two patterns of PCNA and cyclin D3 expression were observed in the presence of AML cells: partial reduction (patient AML1) or almost total inhibition (patient AML2). The percentages of cells that express both CD25 and PCNA, and in G1 or S plus G2/M cell cycle phases are shown.

 
The AML-derived TSN blocks T cell proliferation by preventing phosphorylation of pRb and p130 and induction of p107 and c-Myc

To determine the point at which delay in cell cycle progression occurs, T cells were stained for cyclin D3, which is induced in mid/late G1. In comparison with those T cells activated alone, the presence of primary AML cells from eight different patients reduced the proportion of cells entering G1, but the extent of inhibition depended on the patient cells analyzed (Fig. 7GoB). Again, two examples are shown, partial (AML1) and complete (AML2) inhibition of entry into S and G2/M. However, these data also show a reduction in the proportion of cells with 2n DNA content in which there was detectable cyclin D3, indicating a delay or inhibition of entry into mid/late G1.

Phosphorylation of the pRb protein by cdk4/6-cyclin D occurs very early during entry into the cell cycle. We tested AML TSN from three patients that completely inhibited cell cycle entry. In each case, pRb phosphorylation was undetectable (Fig. 8GoA). p130, the second member of the pRb family responsible for maintaining cells in G0, was also not phosphorylated (Fig. 8GoB) and the third family member, p107, was not induced (Fig. 8GoC). These data are consistent with inhibition of the cell cycle very early during the transition from G0 to early G1.



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  		FIGURE 8. Phosphorylation of pRb and p130, and induction of p107 and c-Myc do not occur, or are delayed in T cells stimulated in the presence of primary AML cells. T cells stimulated for 24 h with PMA and ionomycin in the presence or absence of AML cells and phosphorylation of pRb on S807/811 (A), p130 (B) and induction of p107 (C) and c-Myc (D) was detected by Western blotting. Total pRb protein in each sample is shown in the lower panel. The hyperphosphorylated, inactive form of p130 is form 3, and the hypophosphorylated forms of the protein are forms 1 and 2. In the absence of primary AML cells phosphorylation of pRb and p130, and the induction of p107 occur 24 h poststimulation, but these events are all inhibited in the presence of AML cells. c-Myc induction was detectable by 5 h. Examples are shown of two responses in the presence of AML cells: delayed c-Myc induction by cells from patient AML1 and abolition by patient AML2. E, Analysis of FSC demonstrates that in the presence of AML TSN there is inhibition of the increase in T cell size upon stimulation.

 
Induction of the c-myc gene is an early event following mitogenic stimulation. The induction of c-Myc occurred 5 h poststimulation in the absence of primary AML cells, but was delayed or inhibited in the presence of leukemic cells from all four patients tested (Fig. 8GoD). In experiments using primary AML cells that completely prevented T cell cycling, e.g., AML2 (measured by PCNA expression as in Fig. 7GoA), there was no c-Myc induction, whereas in those cases where some T cells entered the cell cycle, e.g., AML1, c-Myc induction was delayed. T cells increase in size as they progress from G0 to G1, and this can be determined by measuring their forward scatter (FSC). The AML TSN inhibited the increase in FSC (Fig. 8GoE). Taken together, our data suggest that AML TSN prevent or delay cell cycle entry of T cells by maintaining them in G0.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In these studies we sought to determine whether the immunosuppressive TSN described in our previous work using the U937 leukemic cell line is also produced by primary tumor cells from patients with AML and, if so, its mechanism of action. In addition, we studied whether there is variation in inhibition using AML cells from different patients with the same disease. We have shown for the first time that primary AML cell supernatant has a profound inhibitory effect on the activation of normal T cells by PMA and ionomycin and the more physiological stimulus of CD3/CD28 cross-linking. In addition to the effect on T cell activation, we also demonstrated that AML TSN inhibits the recruitment of T cells into the cell cycle. Cells from 18 different AML patients all inhibited T cell activation and proliferation, although the degree of inhibition was variable. However, we could not correlate the degree of inhibition with the AML FAB type or with the percentage of blasts in each cell sample. Because inhibition of T cell activation and proliferation by AML cells has important clinical implications, we went on to further characterize its biological and functional properties.

First, we conducted a number of experiments to determine whether the TSN contained any of the previously characterized tumor-derived immunosuppressants. Since an inhibitory factor produced by renal cells has been shown to be a ganglioside (54), we used two methods to determine whether AML cells inhibit T cell activation in the same way. This is not likely to be the case for AML cells, because neither DL-PDMP nor neuraminidase had any effect on inhibition by AML cell supernatant. Similarly, addition of inhibitors of NO or PG production to AML cells had no effect. Furthermore, we showed previously that the U937 cell line (56) is similarly immunosuppressive and that this cell line does not produce PGE2, LTB4, LTC4, LTD4, or 5-HETE (57, 58, 59), or secrete arachidonic acid in its unprimed state (59, 60). Digestion of the AML cell supernatant with trypsin resulted in a marked reduction in the inhibition of IL-2 secretion, indicating that it contains secreted proteins that are responsible for the effect. This is further supported by the observation that brefeldin A and sodium monensin, inhibitors of Golgi function, also prevent production of inhibition. We excluded the presence of the three commonest immunosuppressive cytokines produced by tumor cells TGF-{beta}, IL-10, and VEGF (61), indicating that the AML TSN contains proteins which are distinct from previously described tumor-associated immunosuppressants. Identification of the proteins is beyond the scope of this study.

The effects of the AML TSN on T cells can be grouped into two categories: first, inhibition of Th1 cytokine production; and, secondly, inhibition of entry into the cell cycle. A number of cytokine genes, including IL-2 and IFN-{gamma}, are induced by costimulating T cells via CD3/CD28. Production of these cytokines, as well as others such as IL-3, IL-6, IL-8, and GM-CSF, is important in coordinating the activation, proliferation, and differentiation of a variety of hemopoietic cells during an immune response (62, 63). We demonstrate that production of both these cytokines is inhibited by the leukemia-derived TSN. It might be expected that failure to produce IL-2 is the reason that T cells do not proliferate. However, although we have shown that the high-affinity IL-2R is produced normally, addition of rIL-2 to the medium does not abrogate the block on cell proliferation caused by the TSN. Therefore, we can assume that the block occurs early during cell cycle entry from G0, before IL-2-dependent signals are required to stimulate entry from G1 into S-phase. Alternatively, signal transduction from the IL-2R may also be inhibited.

To characterize the effect of the AML-derived TSN more fully, we next examined its effects on T cell signal transduction pathways. T cell signal transduction in the cancer-bearing state has been heavily investigated since loss of the CD3-{zeta} chain in tumor-bearing mice was first reported by Mizoguchi et al. in 1992 (64). We have demonstrated loss of CD3-{zeta} in patients with myeloid malignancies (20); however, in our study this loss was generally seen in patients with advanced disease (20). This suggests that although this phenomenon would contribute to T cell immunosuppression, it is likely to be a late event and not the triggering mechanism. More recent work in our laboratory has shown that the CD3-{zeta} chain was down-regulated in normal T cells in the presence of AML cell supernatant, but only after 7 days in culture (A. Buggins, unpublished data). Thus, loss of CD3-{zeta} is a late event in T cell immunosuppression by AML cells.

Two of the earliest T cell signaling events affected was the nuclear translocation of NFATc and NF-{kappa}B. Because it has been proposed that nuclear accumulation of NFATc may be controlled by a balance between Ca2+/calcineurin and JNK/ERK/p38 signals (41), we investigated whether inhibition of these were the cause of the block in NFATc. We have demonstrated that the magnitude and duration of the Ca2+ signal triggered by PMA and ionomycin were not affected by the TSN generated by AML cells. Investigation of the kinetics of activation of JNK1/2, ERK1/2, and p38 pathways, as determined by assaying their phosphorylation, showed that neither ERK1/2 nor p38 was affected by the presence of AML cells, whereas JNK1/2 activation was delayed by ~5 min. It is unlikely that this delay would be sufficient to affect nuclear accumulation of NFATc, which should occur as a consequence of a more rapid activation of these kinases.

The kinetics of I{kappa}B phosphorylation and its subsequent degradation were not affected by the presence of AML cells, but nuclear accumulation of NF-{kappa}B was prevented. The AML TSN caused suppression of chromatin binding of the p65RelA and p75c-Rel rather than the p50 subunit. This is relevant, as the p65RelA-p50 and p75c-Rel-p50 heterodimers are active, whereas the p50-p50 homodimer is a poor activator of NF-{kappa}B-dependent transcription. The suppression of nuclear p65RelA and p75c-Rel could be due either to a failure to translocate to the nucleus even after I{kappa}B degradation or to degradation in the nucleus. In a study of suppression of T cell function by renal cell carcinoma, failure to activate NF-{kappa}B was caused in some cases by inhibition of phosphorylation and degradation of I{kappa}B (65) and in others by a lack of NF-{kappa}B nuclear accumulation (66). Degradation of I{kappa}B has been thought to be sufficient to cause the nuclear translocation of NF-{kappa}B, but it is not clear what mechanisms triggered by the AML TSN prevent nuclear accumulation and DNA binding of p65RelA or p75c-Rel.

We have shown previously that pRb is phosphorylated by cdk6 or cdk4-cyclin D2 early during the transition of human primary T cells from G0 to early G1 (G1A) (67). Phosphorylation of pRb at cdk6/4-cyclin D specific sites does not occur in T cells stimulated in the presence of AML cells, which is consistent with a block in cell cycle progression very early during the G0 to G1A transition. In agreement, induction of c-Myc that normally occurs in T cells within hours of PMA/ionomycin stimulation is also inhibited. Such early cell cycle inhibition would prevent events that occur after G1A and, consistent with this, we have demonstrated that the TSN also inhibits the induction of cyclin D3, which would normally occur in mid G1; p107, which is induced in mid/late G1; and PCNA, a cofactor of DNA polymerase {delta} that is rate-limiting for the G1 to S-phase transition (48, 49). The AML TSN also prevented the increase in cell size that normally accompanies the transition from G0 to G1, consistent with inhibition of entry into the growth cycle (68). In Drosophila, activation of cdk4 coordinates cell cycle entry with entry into the growth cycle (69, 70). Thus, if the same occurs in human cells, prevention of cdk4 activation by the AML TSN would be sufficient to account for inhibition of cell growth as well as preventing the quiescent T cells from entering the cell cycle. Cell cycle entry and proliferation accompany T cell activation, and it might be expected that inhibition of cell cycle entry would prevent the production of IL-2 and IFN-{gamma}. We show elsewhere that this is not the case and a number of effector functions are induced in quiescent T cells.3

Both NF-{kappa}B and NFATc are required for T cell proliferation and activation in vivo. Inactivation of NFATc in mice reduced thymic and peripheral T cell numbers and impaired the proliferation of peripheral lymphocytes (71, 72). Abrogation of NF-{kappa}B activity in the T cell lineage of mice caused a decrease in splenic T cells and suppressed proliferation of peripheral T cells in response to PMA/ionomycin or via CD3/CD28, as well as increasing their apoptosis (73, 74, 75). Further, production of IL-4, IL-10, and IFN-{gamma} were inhibited, but IL-2 and IL-2R were induced normally (73). NF-{kappa}B also induces pRb phosphorylation (76, 77) and directly activates the transcription of c-Myc (78, 79), p65RelA-p50 being the most potent transactivator (79). Therefore, inhibition of NF-{kappa}B by the protein produced by AML cells is sufficient to account for inhibition of IFN-{gamma} production, c-Myc induction, and entry into the cell cycle. Signal transduction pathways inhibited or unaffected by the AML TSN are shown in Fig. 9Go.



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  		FIGURE 9. A schema showing T cell signal transduction pathways affected by AML TSN. This figure shows the T cell signaling proteins analyzed in our study (boxed). Those affected are shaded in gray.

 
We have therefore demonstrated that primary AML blast cells from all 18 patients tested inhibit T cell activation to varying degrees. We have conducted experiments in which a highly inhibitory TSN was diluted and the effects on IL-2 production were evaluated. Once past an inhibitory threshold, the inhibition was dose-dependent (data not shown). Therefore, the difference in the extent of inhibition by TSN from different AML samples could be due to the production of different amounts of inhibitory factors. Nuclear translocation of NF-{kappa}B and NFATc, induction of c-Myc, and phosphorylation/induction of the retinoblastoma family of proteins are all essential T cell activation and proliferation events which are inhibited by AML TSN. This inhibition could be explained by the presence of a single secreted glycoprotein which inhibits NFATc and NF-{kappa}B nuclear translocation and thereby prevents cell cycle entry and the induction of T cell effector functions. Alternatively, the inhibition could be due to a more complex mechanism of action caused by the secretion of one or more factors that act at multiple levels on the signaling pathways. These data and the finding that the activity contains secreted glycoproteins open the way to identification by expression cloning methods and the development of therapeutic strategies to reverse the effect of AML TSN. This would have major implications for the success of immunotherapy protocols to eradicate minimal residual disease in patients with AML.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Andrea G. S. Buggins, Leukaemia Sciences, Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, U.K. E-mail address: andrea.buggins{at}kcl.ac.uk Back

2 Abbreviations used in this paper: AML, acute myeloid leukemia; TSN, tumor cell supernatant; DC, dendritic cell; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PCNA, proliferating cell nuclear Ag; PI, propidium iodide; FSC, forward scatter; VEGF, vascular endothelial growth factor; cdk, cyclin-dependent kinase; JNK, c-Jun N-terminal kinase; DL-PDMP, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. Back

3 N. C. Lea, S. Orr, K. Stoebert, G. J. Williams, M. A. Ibrahim, G. J. Mufti and N. S. B. Thomas. T-lymphocyte activation: effector functions are induced without engaging the cell cycle. Submitted for publication. Back

Received for publication July 3, 2001. Accepted for publication September 5, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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