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

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-alpha (TNF-alpha), interferon-gamma (IFN-gamma), 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 3x10(-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.

Fig. 1

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

Fig. 2

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

Fig. 3

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

Fig. 4

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

Fig. 5

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