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. 2007 Feb 20;56(10):1551–1563. doi: 10.1007/s00262-007-0298-z

The effect of dexamethasone on polyclonal T cell activation and redirected target cell lysis as induced by a CD19/CD3-bispecific single-chain antibody construct

Christian Brandl 1, Cornelia Haas 1, Sandrine d’Argouges 1, Tanja Fisch 1, Peter Kufer 1, Klaus Brischwein 1, Nadja Prang 1,2, Ralf Bargou 3, JoAnn Suzich 4, Patrick A Baeuerle 1,✉, Robert Hofmeister 1,5
PMCID: PMC11030660  PMID: 17310380

Abstract

BiTE molecules comprise a new class of bispecific single-chain antibodies redirecting previously unstimulated CD8+ and CD4+ T cells for the elimination of target cells. One example is MT103 (MEDI-538; bscCD19xCD3), a CD19-specific BiTE that can induce lysis of normal and malignant B cells at low picomolar concentrations, which is accompanied by T cell activation. Here, we explored in cell culture the impact of the glucocorticoid derivative dexamethasone on various activation parameters of human T cells in response to MT103. In case cytokine-related side effects should occur with BiTE molecules and other T cell-based approaches during cancer therapy it is important to understand whether glucocorticoids do interfere with the cytotoxic potential of T cells. We found that MT103 induced in the presence of target cells secretion by peripheral T cells of interleukin (IL)-2, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-6, IL-10 and IL-4 into the cell culture medium. Production of all studied cytokines was effectively reduced by dexamethasone at a concentration between 1 and 3 × 10−7 M. In contrast, upregulation of activation markers CD69, CD25, CD2 and LFA-1 on both CD4+ and CD8+ T cells, and T cell proliferation were barely affected by the steroid hormone analogue. Most importantly, dexamethasone did not detectably inhibit the cytotoxic activity of MT103-activated T cells against a human B lymphoma line as investigated with lymphocytes from 12 human donors. Glucocorticoids thus qualify as a potential co-medication for therapeutic BiTE molecules and other cytotoxic T cell therapies for treatment of cancer.

Keywords: CD3, CD19, BiTE, Bispecific antibody, Single-chain antibody, Dexamethasone, Tumor, T cell activation, T cell proliferation, Redirected lysis

Introduction

Numerous immune evasion mechanisms of tumor cells have been described that are directed against tumor-specific cytotoxic T cells. Such mechanisms include loss by tumor cells of MHC class I [6, 18, 28] and costimulatory molecules [33], impaired peptide antigen processing by loss of TAP transporters or alteration of proteasome subunits [13, 16, 30], expression of T cell-modulatory proteins such as TGF-β [11, 14], IL-10 [36] and indoleamine 2,3-dioxygenase (IDO) [12, 35], expression of T cell inhibitory or pro-apoptotic ligands such as FasL [1] and PD-L1/B7-H1 [4], and expression by tumor cells of proteins protecting from toxic T (and NK) cell granzymes, such as serpins [26] or membrane-bound cathepsin B [2]. The multitude of escape strategies provides evidence that a major threat to tumor cells are T cells, which seem to keep up a constant selection pressure leading to an accumulation of T cell-resistant tumor cell phenotypes during the course of the malignant disease.

The therapeutic potential of engaging T cells against tumor cells has long been recognized and precipitated in a number of promising approaches that either try to elicit a specific T cell response against tumor cells, or a polyclonal non-restricted response. The first class involves various vaccination approaches, where T cell survival and activity is frequently boosted by cytokines, most importantly IL-2, or other immune-stimulatory agents [22]. Tumor-associated peptide antigens for vaccination are delivered either in precursor form as inactivated tumor cells, proteins, cDNA, viruses, viral vectors or directly as peptides. In all cases, the goal is to prime, activate and expand tumor-specific T cells with the help of dendritic and other professional antigen-presenting cells. A different approach is the isolation, propagation and re-infusion of T cells isolated from a patient’s tumor, referred to as ‘adoptive T cell transfer’. Modifications to this approach have recently produced encouraging results in the treatment of late-stage melanoma [10]. The second class of T cell cancer therapies employ bi- or tri-specific antibodies targeting with one arm a ubiquitous T cell signal transducer, such as CD3, and with the other arm a tumor-associated cell surface antigen [39]. A number of such antibody constructs are currently in clinical testing [17, 25, 31].

One class of bispecific antibodies with unique properties are BiTE molecules [40]. These are polypeptides with a size of approximately 55 kDa comprising two flexibly linked single-chain antibodies. Their hallmarks are a very high potency of redirected tumor cell lysis at sub-ng/ml concentrations [5, 9, 24], support of serial killing by previously unstimulated polyclonal CD8+ and CD4+ T cells translating into activity at very low effector-to-target ratios [19], formation of proper cytolytic T cell synapses even on MHC class I negative tumor cells [27], and efficient production by eukaryotic cell fermentation.

One example is the CD3/CD19-bispecific BiTE MT103 (MEDI-538; bscCD19 × CD3) [9], which currently is in clinical testing for therapy-refractory non-Hodgkin lymphoma [3]. In healthy primates, MT103 has shown upon multiple dosing a transient and reversible T cell activation and cumulative loss of peripheral B cells [29]. Likewise, pro-inflammatory cytokines including TNF-α, IL-2 and IL-6 were transiently released into blood in response to each MT103 dose, which potentially can lead to cytokine related side effects. While a controlled local secretion of cytokines may be important for cell activation, differentiation, and proliferation, overshooting systemic levels of pro-inflammatory cytokines are known to cause fever, chills, vascular leak and/or a drop in blood pressure. A so called ‘cytokine release syndrome’ has been previously observed in response to protein therapeutics activating immune cells. For example, the administration of rituximab led to elevated serum levels of TNF-α and IL-6 shortly after start of infusion [38]. Likewise, the treatment of patients with a quadroma-derived CD3/CD19-bispecific antibody led to an increase in serum levels of a host of pro-inflammatory cytokines [8]. This prompted us to investigate the influence of the glucocorticoid derivative dexamethasone on T cell activation and function as induced by a BiTE molecule. Dexamethasone is widely used in cancer therapies to dampen side effects as can occur during first dosing with both cytostatics and biologicals.

We found that dexamethasone led to a significant reduction of cytokine release while the cytotoxic activity of T effector cells was not compromised. Co-medication with steroids may thus be suitable to control side effects related to systemic cytokine release of cytotoxic T cell therapies without significant impairment of their anti-tumor activity.

Material and methods

Reagents and antibodies

Dexamethasone (Sigma, Germany) was dissolved in PBS/10% FCS (Invitrogen, Germany) at a concentration of 1 × 10−2 M. For the use in assays, pre-dilutions were made in cell culture medium (RPMI 1640/10% FCS) (Invitrogen, Germany). MT103 was formulated at a concentration of 1 μg/ml in PBS/0.1% HSA (Baxter, Germany). Monomeric MT103 was used throughout this study and regularly controlled for the absence of aggregates. The stock solution of 100 μg/ml propidium iodide (Sigma, Germany) was prepared in PBS. The PKH-26 Red Fluorescent Cell Linker kit was from Sigma-Aldrich, Germany, the Vybrant CFDA-SE Cell Tracer Kit from Molecular Probes, Germany. All antibodies were purchased from BD Biosciences, Germany. The EpCAM-specific BiTE protein MT110 was used as control protein, and produced as described [5].

Cell lines

The CD19-positive cell lines NALM-6 and MEC-1 were used as target cells. The human gastric carcinoma line KATO III was used as a CD19-negative control cell line. All cells were purchased from the DSMZ, Germany.

Preparation of PBMC and T cells

PBMC were prepared from randomly selected blood filters, which were received from local blood banks. The cells were washed out from leukocyte filters with 3 × 50 ml PBS/20% Biocoll (Biochrom, Germany). PBMC were kept in an incubator at 37°C/5% CO2 until used in the cytotoxicity assay. CD3 positive T-cells were isolated using the Human T-Cell Enrichment Column Kit (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions.

Labeling of NALM-6 target cells with PKH-26

NALM-6 cells were labeled using the PKH-26 Red Fluorescent Cell Linker kit (Sigma, Germany). Briefly, 1 × 107 cells are washed twice with 10 ml PBS before the cells were re-suspended in 0.5 ml diluent C. In a second tube 0.5 ml Solution C were mixed with 5 μl of PHK-26 dye by vortexing. This solution was added to the cell suspension. Then, the cells were mixed and incubated for 2 min at room temperature. The staining reaction was quenched by addition of 1 ml FCS. Then, the tube was filled up with 15 ml complete cell culture medium. The cells were centrifuged for 5 min at 300g and once more washed with 15 ml complete medium. The number of viable cells was determined by Trypan Blue exclusion.

CFSE labeling

T cells were resuspended at a density of 2 × 106 cells/ml in 15 ml pre-warmed (37°C) PBS. 150 μl of 10 μM CFSE dye (Vybrant CFDA-SE Cell Tracer Kit, Molecular Probes) were added to a final concentration of 100 nM. After 15 min incubation at 37°C, labeling was quenched with complete RPMI 1640 cell culture medium. Cells were centrifuged and resuspended in fresh pre-warmed medium. Subsequently, cells were incubated for another 30 min at 37°C to ensure complete conversion of the dye by esterases. Then, they were washed once and resuspended in cell culture medium.

Cytotoxicity assay

To distinguish target from effector cells in a flow cytometer-based assay, NALM-6 target cells were labeled with the fluorescent dye PKH-26 as described under Material and methods. Unless otherwise noted effector and target cells were co-incubated in 96-well round-bottom plates at a ratio of 10:1. Per well, 5 × 105 PBMC effector cells were cultured with 5 × 104 PKH-26 labeled NALM-6 target cells in the absence or presence of the indicated MT103 concentrations. At indicated time points, the supernatants were harvested for cytokine quantification. The cells were washed twice with 200 μl PBS and re-suspended in FACS buffer. Before the cells were analyzed using a FACS Calibur flow cytometer (BD) propidium iodide at a final concentration of 1 μg/ml was added to the cells to distinguish live from dead cells. Cell lysis was calculated according to the following equation:

graphic file with name M1.gif

Dexamethasone treatment of cell cultures

Kinetics of cell lysis and cytokine release was investigated in the presence or absence of dexamethasone. Briefly, 5 × 105 PBMC were pre-incubated in round-bottom microtiter plates for 1 h either in medium supplemented with 3 × 10−7 M dexamethasone or medium alone in a volume of 80 μl. Then, 5 × 104 PKH-26 labeled NALM-6 target cells were added in 100 μl medium containing 3.6 × 10−7 M dexamethasone to adjust the dexamethasone concentration in the final 200 μl reaction volume. Subsequently, 20 μl culture medium with 10 ng/ml MT103 was added (final MT103 concentration of 1 ng/ml). For each time point, controls with PBMC and target cells, but without MT103 were prepared. Immediately after pipetting the cytotoxicity reactions and after 4, 8, 12, and 24 h the plates were centrifuged for 5 min at 300g. Supernatants were subjected to cytokine analysis; the cells were harvested and cell lysis determined as described under Material and methods.

Cytokine measurements

Cytokines were quantified in cell culture supernatants harvested in the above-mentioned experiments. Cytokine levels were determined using the human Th1/Th2 Cytokine Cytometric Bead Array (CBA) II kit from BD Pharmingen. Cytokine standards were prepared by limiting dilution according to the manufacturer’s protocol. The cytokines IFN-γ, TNF-α, IL-2, IL-6, IL-4 and IL-10 were measured applying a modified protocol. Duplicate samples were prepared in microtiter plates. For the preparation of the mixed capture beads, 3 μl aliquots of the individual capture beads were mixed in a vial. For the quantification of cytokines, 16 μl of sample supernatants were mixed with 16 μl of the mixed capture bead suspension and 16 μl of the PE-detection reagent. After the recommended incubation time of 3 h on a rocking platform (100 rpm), the plates were centrifuged at 200g for 5 min. The supernatants were carefully discarded and the pelleted beads were resuspended in 150 μl wash buffer. After a final centrifugation step the beads were resuspended in 200 μl wash buffer and transferred into FACS tubes. Cytometer setup, data acquisition and analysis were performed according to the manufacturer’s protocols.

The dexamethasone-induced cytokine reduction was calculated using the equation:

graphic file with name M2.gif

Data were analyzed using Wilcoxon matched pairs test.

Cell proliferation

CFSE-labeled T cells were preincubated overnight at 37°C with or without 3 × 10−7 M dexamethasone. Then, T cells were washed and co-cultured with MEC-1 target cells in the presence of 1 ng/ml MT103 (Micromet, Germany) or 10 ng/ml MT110 (Micromet, Germany) or medium as negative controls. For an E:T ratio of 5:1, 1.5 × 105 T cells were incubated with 3 × 104 MEC-1 cells in 96-well round-bottom plates for 5 days at 37°C.

The number of cell division cycles was determined after 5 days. To discriminate between CD4-positive and CD8-positive T cell proliferation, cells were labeled with an anti-CD3 PE antibody and a cocktail of anti-CD4 and anti-CD8 PE-Cy5 conjugated antibodies. Although anti-CD4 and anti-CD8 antibodies were labeled with the same fluorescent dye, CD4-positive T cells could be distinguished from CD8-positive T cells due to their weaker staining intensity, as shown in experiments with the single antibodies. Cells were analyzed using a FACS Calibur flow cytometer (Becton Dickinson). Acquisition time of events was 30 s. Data were evaluated by means of Cell Quest Software.

T Cell activation markers

PBMC were pre-incubated in absence or presence of 3 × 10−7 M dexamethasone for 24 h at 37°C and 5% CO2. After preincubation, PBMC were washed twice in PBS, resuspended in culture medium (RPMI 1640/10% FCS), and subsequently incubated with NALM-6 target cells in the presence of 2.5 ng/ml MT103. Effector cells (1 × 105) were incubated with 2 × 104 target cells (E:T ratio of 5:1). Total reaction volume was 200 μl. Expression of CD69, CD25 and CD11a/LFA-1, and CD2 was analyzed by staining with fluorescence dye-conjugated antibodies for 30 min at 4°C. The following antibody conjugates were used: anti-CD4-PE/Cy5, anti-CD8 PE/Cy5, anti-LFA-1 FITC, anti-CD2 FITC, anti-CD25 PE, and anti-CD69 PE.

Results

Dexamethasone inhibits cytokine release by MT103-activated T cells

Peripheral blood mononuclear cells (PBMC) and human pre-B lymphoma NALM-6 cells were incubated in the presence of 1 ng/ml MT103 at an effector-to-target (E:T) ratio of 10:1, a condition leading to efficient target cell lysis. PBMC rather than purified T cells were used for the experiments in order to pick up secondary cytokine effects and to more closely mimic the situation in peripheral blood of leukaemia patients. One ng/ml MT103 was previously shown to induce maximal redirected lysis of CD19-expressing normal B and B lymphoma cells [9].

The concentrations of IFN-γ, TNF-α, IL-2, IL-4, IL-6 and IL-10 in cell culture medium were recorded over 24 h. All six cytokines appeared in cell culture medium shortly after addition of MT103 (Fig. 1). While IFN-γ, IL-6 and IL-10 accumulated over the 24-h observation period, TNF-α, IL-2 and IL-4 levels peaked after 8 and 12 h, respectively, before they slightly dropped. TNF-α, IL-2 and IL-4 were induced fastest, IFN-γ and IL-6 more slowly, and IL-10 with the slowest kinetic. Basal cytokine release in the absence of MT103 was very low for TNF-α, IL-2, IL-4, IFN-γ and IL-10 consistent with an unstimulated phenotype of peripheral T cells. Only IL-6 showed a significant level of basal secretion. The kinetics of cytokine release by nine other PBMC donors was similar, but the cytokine levels of individual donors showed large variations (data not shown). When PBMC were pre-incubated with 3 × 10−7 M dexamethasone for 1 h before NALM-6 target cells and MT103 were added, both MT103-induced and, where detectable, basal production of all cytokines was substantially reduced (Fig. 1). Strongest inhibitory effects of dexamethasone were seen on TNF-α, IL-4 and IL-2 production.

Fig. 1.

Fig. 1

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Kinetics of cytokine release into the cell culture supernatant in response to T cell-mediated cell lysis. PMBC from a healthy donor and NALM-6 pre-B lymphoma cells were incubated at a ratio of 10:1 with 1 ng/ml MT103 in the presence or absence of 3 × 10−7 M dexamethasone. Controls were incubated without MT103 in the presence or absence of dexamethasone. At the indicated time points, cell culture supernatants were harvested and concentrations of the cytokines IFN-γ, TNF-α, IL-2, IL-4, IL-6, and IL-10 determined by means of Cytometric Bead Array kit. Mean values from duplicate determinations ± SEM are shown

The inhibitory effect of dexamethasone was studied for its variation with PBMC from ten healthy donors (data not shown). IL-2, IL-4 and TNF-α levels, as induced by MT103, were determined after 12 h, and IFN-γ, IL-6 and IL-10 levels after 24 h. Donors showed highly variable levels for the six cytokines after MT103 stimulation. In the presence of dexamethasone, the median level of IL-6 secretion dropped by a factor of 16 (P = 0.002). The IL-4 median dropped by a factor of 13 (P = 0.002). The median of TNF-α was reduced fivefold by dexamethasone treatment (P = 0.002), and the IL-2 median by a factor of 3.5 (P = 0.002). Dexamethasone inhibition was weakest for IL-10 with a median inhibition of 1.6-fold (P = 0.0039), and for IFN-γ with a factor of 1.9 (P = 0.084). The data suggest that dexamethasone is suitable to significantly inhibit BiTE-induced release of several cytokines in a larger population of human T cell donors.

Cytokine inhibition is maximal between 100 and 300 nM dexamethasone

Dose–response analyses were performed to identify a concentration of dexamethasone that would maximally inhibit release of all six cytokines tested in response to MT103 stimulation of T cells. PBMC effector cells from seven different donors were tested. Redirected lysis of NALM-6 target cells was assayed at an E:T ratio of 10:1 in the absence or presence of increasing dexamethasone concentrations covering a range from 1 × 10−9 up to 3 × 10−6 M. PBMC effector and NALM-6 target cells were pre-incubated for 1 h with dexamethasone before 1 ng/ml MT103 was added. The effect of dexamethasone on IL-2, IL-4 and TNF-α secretion into the cell culture supernatant was analyzed after 8 h, and the effect on IL-6, IFN-γ, and IL-10 levels after 24 h. With all seven PBMC donors and all six cytokines, a dose-dependent reduction of cytokine release was observed with the corticosteroid derivative (Fig. 2). Dexamethasone concentrations between 1 and 3 × 10−7 M were found to maximally reduce cytokine secretion.

Fig. 2.

Fig. 2

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Dose–effect curve of dexamethasone on cytokine release. PMBC from seven randomly picked healthy donors and NALM-6 pre-B lymphoma cells were incubated at a ratio of 10:1 with 1 ng/ml MT103 and six log3 serial dilutions of dexamethasone starting at 3 × 10−6 M. Controls were kept in complete culture medium alone. Peak levels of IL-2, TNFα, and IL-4 were measured after 8 h; peak levels of IFNγ, IL-6, and IL-10 were analyzed after 24 h. Mean values of duplicate determinations are shown

Marginal effect of dexamethasone on T cell activation markers

We have previously shown that T cells induce a number of activation markers and adhesion molecules upon stimulation by MT103, which is strictly dependent on the presence of CD19-positive target cells [23]. Here, we studied the effect of dexamethasone on expression of surface antigens CD69, CD25, CD2, and LFA-1 (CD11a) on CD4+ and CD8+ T cells in response to 2.5 ng/ml MT103 in the presence of NALM-6 target cells at an E:T ratio of 5:1.

Under control conditions, essentially all CD4+ and CD8+ T cells were negative for activation marker CD69, and only a small percentage of T cells expressed CD25 (Figs. 3a, b). Upon MT103 stimulation in the presence of target cells, approximately 60% of CD8+ and 80% of CD4+ T cells became CD69-positive between 24 and 48 h, followed by a decline of CD69 expression. After 6 days, 10% of CD8+ and 40% of CD4+ T cells still expressed CD69. The number of CD8+ T cells expressing CD25 reached close to 100% after 72 h, while CD4+ T cells reached after 24 h a plateau at 60% positivity. After 6 days, approximately 80 and 60% of CD4+ and CD8+ T cells, respectively, still expressed CD25. Expression of the adhesion molecules CD2 and LFA-1 increased by approximately sixfold to sevenfold upon stimulation of T cells with MT103 plus target cells (Figs. 3c, d). CD2 expression reached a maximum after 72 h, while LFA-1 expression continued to increase for 6 days.

Fig. 3.

Fig. 3

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Kinetics of expression of T cell activation markers CD69 and CD25 and adhesion molecules CD2 and LFA-1 in response to MT103 activation. PBMC isolated from the blood of a healthy donor were pre-incubated with 3 × 10−7 M dexamethasone for 24 h. After washing, PBMC were co-cultured with NALM-6 target cells at a ratio of 5:1 in the presence of 2.5 ng/ml MT103. At the indicated time points, the portion of CD69- (a) and CD25-positive cells (b) was analyzed by flow cytometry. The level of CD2 (c) and LFA-1 (d) surface expression on T cells is shown expressed as mean fluorescence intensity (MFI). Expression of activation markers and adhesion molecules was detected by three-color FACS staining using a cocktail of anti-CD4/CD8 PE/Cy5-conjugated antibodies, which was either combined with anti-LFA-1 FITC- and anti-CD25 PE-conjugated, or CD2 FITC- and CD69 PE-conjugated antibodies. Staining was controlled by isotype-matched antibodies. e Control experiments for CD69 expression were performed with medium control, BiTE molecules MT103 and EpCAM-specific MT110 (both at 2.5 ng/ml), CD19-positive NALM-6 cells, and the EpCAM-expressing human gastric carcinoma cell line Kato III in the absence and presence of dexamethasone. The E:T ratio was 5:1 and the assay duration 24 h

To study the effect of dexamethasone on upregulation of T cell surface markers in response to MT103, PBMC were pre-incubated with 3 × 10−7 M dexamethasone for 24 h before they were employed in cytotoxicity assays with NALM-6 cells at an E:T ratio of 5:1. As shown in Fig. 3, dexamethasone had only a small effect on the expression of all four surface markers.

As shown for the immediate-early T cell activation marker CD69 (Fig. 3e), significant T cell activation was only observed if the CD19-specific BiTE MT103 was present with CD19-positive NALM-6 cells, but not with CD19-negative Kato III cells. No T cell activation was seen in the presence of NALM-6 cells when MT103 was replaced by an EpCAM-specific BiTE molecule called MT110, that shares the same anti-CD3 single-chain antibody with MT103. MT110 however showed potent T cell activation when the EpCAM-positive cell line Kato III was present, but not with NALM-6. In the presence of Kato III cells, MT103 did no longer induce robust CD69 expression. These data support a highly conditional and specific T cell activation by BiTE molecules.

Small effect of dexamethasone on T cell proliferation

We next explored whether dexamethasone could influence the proliferation of T cells by using the fluorescent dye carboxyfluoresceine succinimidyl ester (CFDA) in flow cytometry. Once trapped inside cells, this compound forms adducts with cytoplasmic proteins, which upon cell division equally segregate between daughter cells. Sequential halving of fluorescence signals can be visualized as distinct peaks by FACS whereby the number of novel peaks correlates with the number of cell divisions. PBMC and the CD19-positive B lymphoma line MEC-1 were co-cultured either in the presence of MT103 or the EpCAM-specific control BiTE MT110. In the absence and presence of the control BiTE, neither CD8+ nor CD4+ T cells did proliferate as is evident by a single peak of CFSE-stained cells (Fig. 4, right panels). Upon addition of MT103, both CD8+ and CD4+ T cells were induced to strongly proliferate undergoing at least five cell division cycles within 5 days for CD8+ cells (Fig. 4, upper panels), and 2–3 cycles for CD4+ cells (Fig. 4, lower panels).

Fig. 4.

Fig. 4

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Effect of dexamethasone on T cell proliferation. PBMC isolated from the blood of a healthy donor were pre-exposed to 3 × 10−7 M dexamethasone for 24 h or kept untreated. After washing, CD3-positive cells were enriched using CD3+ T cell enrichment column kit. Isolated T cells were labeled with CFSE and co-cultured with CD19-positive MEC-1 B-CLL cells at a ratio of 5:1 in the presence of 1 ng/ml MT103. Controls were incubated with MT110, an Ep-CAM-specific BiTE, and cell culture medium alone, respectively. Cell proliferation was determined after 5 days. CD4- and CD8-positive T cell subsets were identified by a cocktail of anti-CD4 and anti-CD8 PE/Cy5-conjugated antibodies

PBMC were pre-exposed to 3 × 10−7 M dexamethasone for 24 h before addition of MT103 plus target cells. The corticosteroid did only marginally reduce proliferation of CD4+ and CD8+ T cell subsets. On average, one cell cycle less was observed with CD8+ and CD4+ T cell subsets in the presence of dexamethasone (Fig. 4, compare left panels).

No effect of dexamethasone on MT103-mediated cell lysis

Kinetics of redirected lysis of NALM-6 target cells were studied with a total of 12 healthy human PBMC donors by measuring uptake of propidium iodide in NALM-6 cell nuclei using flow cytometry. With a large majority of PBMC samples, more than 80% target cell lysis was achieved within 24 h in the presence of 1 ng/ml MT103 at an E:T ratio of 10:1 (Fig. 5a). PBMC donors fell into two groups. Redirected lysis with four donor PBMC was rapid and already reached a plateau after 12 h, while eight donors showed a 4–6 h delay before cell lysis started.

Fig. 5.

Fig. 5

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Kinetics of MT103-mediated lysis of NALM-6 cells. a PMBC from 12 healthy donors and NALM-6 pre-B lymphoma cells were incubated at a ratio of 10:1 with 1 ng/ml MT103. At the indicated time points, cell lysis was determined using a flow cytometry-based cytotoxicity assay. b Influence of dexamethasone on NALM-6 cell lysis. PMBC from healthy donors and NALM-6 pre-B lymphoma cells were incubated at a ratio of 10:1 with 1 ng/ml MT103 in the presence or absence of 3 × 10−7 M dexamethasone. At the indicated points in time, cell lysis was determined using a flow cytometry-based cytotoxicity assay. Kill kinetics of two fast and two slow responder PBMC are depicted. Mean values from triplicate determinations ± SEM are shown. c Control experiments for redirected lysis of CD19-positive NALM-6 cells were performed with medium control, BiTE molecules MT103 and EpCAM-specific MT110 (both at 1.0 ng/ml) in the absence and presence of dexamethasone. The E:T ratio was 10:1, and the assay duration 24 h

In the same experiment, redirected cell lysis was studied in the presence of 3 × 10−7 M dexamethasone added to PBMC 1 h prior to MT103 and target cells. In no single case did the corticosteroid inhibit the cytotoxic activity of MT103-stimulated T cells. Two representative examples from each two of the kinetic subpopulations are shown in Fig. 5b.

Control experiments (Fig. 5c) show that significant redirected lysis of NALM-6 is solely observed with MT103 while only background lysis was seen in the absence of BiTE or the presence of the EpCAM-specific control BiTE MT110. Dexamethasone alone or in combination with BiTE molecules did not detectably contribute to lysis of NALM-6 cells.

Discussion

This study explored the effect of dexamethasone, a glucocorticoid derivative frequently used with diverse cancer therapies, on the activation of T cells by the bispecific antibody MT103. In order to mimic the situation in blood from leukemia patients, human B lymphoma cells were added to PBMC containing normal B cells. Target cells for MT103 were thus a mix of normal and malignant B cells. Previous studies have shown that both are equally susceptible to redirected lysis by MT103 [9]. With 1–2.5 ng/ml meaningful concentrations of MT103 were tested. In preclinical experiments, these concentrations induced maximal lysis of normal B and B lymphoma cells [9]. In an ongoing phase I clinical study, a serum trough level of MT103 of 0.5–0.6 ng/ml induced partial and complete tumor responses in therapy refractory non-Hodgkin lymphoma patients [3].

MT103 was found to induce in the presence of these target cells the activation of CD69 and CD25 on a large percentage of peripheral T cells from healthy human donors, which is consistent with a polyclonal activation of CD8+ and CD4+ T cells via stimulation of the polymorphic T cell receptor signaling component CD3ε by MT103. Peripheral T cells from donors were previously unstimulated as is evident from low levels of basal cytokine production, low-level expression of activation markers and cell adhesion molecules, and lack of significant mitotic activity. In response to MT103 plus target cells but not to a mono-CD3-specific BiTE control in the presence of target cells, the CD19-specific BiTE induced cytokine production, upregulation of activation markers and cell adhesion molecules, and sustained cell cycling and division. This shows that bispecific MT103 presented to previously unstimulated T cells in the context of target cells can elicit a full-blown T cell activation in the absence of additional T cell stimulators, such as anti-CD28, lectins or IL-2. These agents are frequently required by other bispecific antibody formats for achievement of efficient redirected target cell lysis [7, 15, 20, 21].

How can BiTE molecules in the absence of any costimulatory agent trigger a sustained and comprehensive polyclonal activation of previously unstimulated T cells? We recently reported that BiTE molecules can induce with high frequency formation of regular cytolytic synapses between CD8+ T cells and antigen-positive target cells [27]. It is possible that within such synapses no additional signals and cell–cell contacts are required for T cell activation. The extreme proximity of two cell membranes in such synapses can likewise displace bulky negative regulators, such as the tyrosine phosphatase CD45 [32] from the synapse, an event, which in itself can provide a costimulatory signal. Because BiTEs are rather small (ca. 55 kDa) and their two arms closely spaced and freely moving, this bispecific construct may be optimal for synapse formation by juxtaposing two epitopes expressed on two different cell membranes. Moreover, formation of a matrix of otherwise monomeric CD3-binding arms on target cells may have an effect as seen by anti-CD3 antibodies, such as OKT-3, which can potently activate T cells when coated on a plastic surface or bound to Fcγ receptors on antigen-presenting cells.

For analysis of dexamethasone effects on BiTE-induced T cell activation, we studied the expression of six cytokines, four different cell surface markers and adhesion molecules, a larger number of human PBMC donors, and different steroid concentrations. PBMC rather than purified T cells were used for the experiments. This cell mix more closely mimicked the situation in peripheral blood and would capture secondary effects by co-stimulated other immune cells (NK and B cells, monocytes and macrophages). Dexamethasone was pre-incubated with effector cells for 1–24 h to increase the sensitivity of effects. Between 1 and 3 × 10−7 M dexamethasone were found to be the lowest concentrations for effective blunting of cytokine production. Serum levels of this magnitude can be reached with a single dose of 8 mg prednisolone, which is in the range this steroid is used for cytokine protection in the clinic. At this steroid concentration, T cell division and surface antigen expression were only marginally impaired, while redirected target cell lysis showed no impairment at all, as was studied with 12 human T cell donors. Similar results have been obtained with a trispecific T cell-recruiting antibody called BiUII [37]. Prednisolone significantly reduced TNF-α release in co-culture assays but did not impact the anti-tumor cell activity of BiUII. These findings provide a rationale to use dexamethasone or prednisolone in the clinic as a means to prevent cytokine release syndrome without affecting cytotoxic T cell function as is necessary for eradicating tumor cells.

We have observed in chimpanzees a ‘first dose effect’ whereby the first dose caused a robust cytokine release followed by a significant and long-lasting adaptation leading to low cytokine levels with subsequent doses [29]. Dexamethasone may thus only be necessary upon first dosing for a couple of days. The consequences of long-term treatment with steroids were therefore not tested in the present study.

How could dexamethasone affect various parameters of T cell activation in a selective fashion? A major mechanism by which glucocorticoid receptor is thought to inhibit cytokine expression is via its direct interaction with transcription factors NF-κB and AP-1, but also STST-3, STAT-5, CREB and NFAT [34]. NF-κB is controlling a multitude of pro-inflammatory genes in response to endogenous cytokine and exogenous pathogenic signals. Once activated by release from the cytoplasmic complex with its inhibitor IκB, NF-κB translocates into the nucleus to induce genes harboring its cognate motif in their promoters and enhancers. Glucocorticoid receptor can directly interact with the RelA subunit of NF-κB and downmodulate NF-κB activity in a hormone-dependent fashion. The impact of dexamethasone will thus be strongest at the transcriptional level and for genes that are inducibly regulated by NF-κB and other inducible transcription factors. T cell activation events that rely on pre-existing proteins and that involve transcription factors controlling cell proliferation may on the other hand be affected least. This notion is supported by our results. Target cell lysis, which relies on stimulus-coupled fusion of cytotoxic granules and discharge of pre-existing granzymes, cytolysin and perforin, was not detectably affected by the steroid hormone. Likewise, T cell proliferation, which may rely on ubiquitous cell cycle regulators, was barely affected. Dexamethasone had the strongest effect on genes known to be inducibly regulated by NF-κB and other inducible transcription factors.

The differential effect of dexamethasone on T cell activation parameters appears beneficial. For the therapeutic activity of BiTE molecules, target cell lysis and expansion of T cells is most important while systemic cytokine release is less desirable. Proliferation of T cells is increasing the effector-to-target ratio and counteracting apoptosis of T cells. Because dexamethasone does not completely inhibit cytokine production, local accumulation of cytokines in target tissue may still be possible as would be necessary for T cell function, and recruitment and activation of other kinds of immune cells.

An unexpected observation was that T cells in peripheral blood from human donors fell into classes with two different phenotypes of BiTE reactivity. In one group of donors, redirected target cell lysis had an immediate onset and reached a plateau within 4–8 h. In the other group, target cell lysis did not start before a 4-h lag phase. In both groups however high lysis rates were achieved after 24 h. The kinetic difference could be due to the “readiness” of effector memory T cells, which in vitro contribute strongest to redirected lysis among T cell subpopulations (unpublished observation). The lag phase in one group may be necessary to upregulate granzyme expression while, e.g., increased cytokine levels from an infection has in the other group prepared cytotoxic T cells for immediate cell lysis. Future experiments are required to decipher the basis for the kinetic difference in cytotoxicity.

The results of this study may be transferable to other T cell therapies being developed for treatment of cancer. The basic mechanism by which BiTE molecules activate T cells is a strictly target cell-dependent stimulation of CD3 leading to formation of immunological cytolytic synapses that are indistinguishable in size and subdomain structures from naturally induced synapses [27]. Other T cell therapies such as vaccination strategies or adoptive T cell therapy aim at priming, activating and expanding tumor-specific cytotoxic T cell populations. These specific T cells will ultimately also form cytolytic synapses on tumor cells. The effect of dexamethasone and other glucocorticoid derivatives on the activation and lytic activity of specific T cell clones may thus not be much different from that on BiTE-activated polyclonal T cells.

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