Dasatinib promotes Th1-type responses in granzyme B expressing T-cells

Abstract

Tyrosine kinase inhibitors (TKIs) have dramatically improved the outcome of chronic myeloid leukemia (CML). Besides inhibiting target kinases in leukemic cells, 2nd generation TKI dasatinib also inhibits off-targets in immune effector cells resulting in atypical immune responses in some patients. Dasatinib has been described to increase the proportion of late effector memory T-cells, however, to date no follow-up studies have been performed in first-line patients. In this study, we explored the functional properties of T-cells using primary samples from CML patients (n = 28) on TKI therapy. Granzyme B (GrB) was used as a marker for late phase antigen experienced CD4+ and CD8+ T-cells. Dasatinib treatment increased the numbers of both GrB expressing memory CD4+ and CD8+ T-cells when compared with healthy controls. Functionally, the GrB+CD4+ T-cells were highly active and differentiated into Th1-type T-cells capable of producing IFN-γ, which is important for tumor control. Similar kind of increase was not observed during imatinib or nilotinib therapy. These data support the dual mode of action of dasatinib: potent BCR-ABL1 inhibition in leukemic cells is accompanied by the enhancement of cellular immunity, which may have implications in the long-term control of leukemia.

Keywords: CML; Th1-immune response; dasatinib; granzyme B; tyrosine kinase inhibitors.

Figure 1. The proportion of granzyme B positive (GrB+) T-cells is increased in CML patients at diagnosis and further expands during dasatinib therapy. Fresh or frozen PBMNCs were first stained for surface markers (α-CD45, α-CD3, α-CD4 and α-CD8), and after fixation and permeabilization intracellular GrB was stained, and cells were analyzed with flow cytometry. (A) The relative proportions of GrB+CD4+ T-cells and (B) GrB+CD8+ T-cells in samples obtained from healthy controls (n = 5) and CML patients at diagnosis (dg) (n = 18). (C) The proportion of GrB+CD4+ T-cells and (D) GrB+CD8+ T-cells 6 mo after start on dasatinib (DA, n = 7), imatinib (IM, n = 6), or nilotinib (NI, n = 6) therapy. The absolute number of GrB+CD4+ (E) and GrB+CD8+ T-cells (F) was measured in CML patients 6 mo after the start of DA (n = 8), IM (n = 6), or NI (n = 5) therapy. Panels A and B were analyzed by nonparametric Mann Whitney t test and panels C, D and E by 1way ANOVA.

Figure 2. Patients treated with dasatinib have increased proportions of memory CD4+ T-cells. The memory cell subsets of CD4+ and CD8+ T-cells were examined by staining the fresh or frozen PBMNCs with α-CD45, α-CD3, α-CD4-, α-CD45RA and α-CCR7-PE antibodies after which they were analyzed by flow cytometry. The proportion of (A) naïve (CCR7+CD45RA+), (B) CD45RA+ effector memory (T_EMRA, CCR7negCD45RA+) (C) central memory (T_CM, CCR7+CD45RAneg) and (D) effector memory T_EM (CCR7negCD45RAneg) CD4+ T-cells from the whole CD4+ T-cell population was measured from healthy (n = 7), untreated CML patients (n = 9) and patients treated with dasatinib (DA, n = 6), imatinib (IM, n = 5) or nilotinib (NI, n = 5) at least 12 mo after start of treatment. Same analysis was performed on CD8+ T-cells (E–H). Statistical analyses in all panels were done by 1way ANOVA.

Figure 3. Granzyme B (GrB+) positive T-cells in dasatinib-treated CML patients are sensitized to produce Th1-type cytokines upon stimulation. Fresh PBMNCs were stimulated with OKT3 and co-stimulatory molecules (α-CD28 and α-CD49d) for 6 h in the presence of Golgi STOP. After the stimulation, Th1-type cytokine (TNF-α and IFN-γ) production in T-cells was measured by flow cytometry. Panel (A) presents representative cases showing one healthy volunteer, one dasatinib-treated patient (patient nr 7 in Table 1), one imatinib-treated patient (patient nr 15), and one nilotinib-treated patient (patient nr 17). Each plot shows 20 000 events. (B) Cytokine production by GrB+CD4+ and GrB+CD8+ T-cells in a representative dasatinib-treated patient (patient nr 7 in Table 1). (C) The percentage of cytokine-producing GrB+ T-cells in healthy volunteers (n = 5) and patients treated with dasatinib (DA, n = 9), imatinib (IM, n = 4) or nilotinib (NI, n = 6) were compared by 1way ANOVA.

Figure 4. Granzyme B positive T-cells in dasatinib-treated CML patients produce mainly IFN-γ. Fresh PBMNCs were stimulated with OKT3 and co-stimulatory molecules (α-CD28 and α-CD49d) for 6 h in the presence of Golgi STOP. After the stimulation, Th1-type cytokine (TNF-α and IFN-γ) production in T-cells was measured separately by flow cytometry. The figure presents a representative dasatinib- (A), nilotinib- (B), and imatinib- (C) treated patient (patient nr 6, 23, and 14 in Table 1).

Figure 5. Dasatinib intake decreases the absolute number of Granzyme B positive (GrB+) CD4+ and CD8+ T-cells and decreases cytokine production. Fresh PBMNCs were stimulated with OKT3 and co-stimulatory molecules (α-CD28 and α-CD49d) for 6 h in the presence of Golgi STOP. After the stimulation, Th1-type cytokine (TNF-α and IFN-γ) production in T-cells was measured by flow cytometry. The absolute counts of GrB+CD4+ and GrB+CD8+ T-cells in CML patients before (0h) and after (1 or 2 h) the patients’ daily dose of dasatinib (DA, panel A), imatinib (IM, panel B), and nilotinib (NI, panel C). Panel (D) shows the percentage of cytokine-producing GrB+ T-cells in these patients before and after the drug intake. Cytokine production before and after drug intake was analyzed with paired t test.