Mechanistic Insights into the Inhibition of Regulatory T Cells by Dasatinib in Chronic Myeloid Leukaemia Patients with Clonal Lymphocytosis - European Medical Journal
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Mechanistic Insights into the Inhibition of Regulatory T Cells by Dasatinib in Chronic Myeloid Leukaemia Patients with Clonal Lymphocytosis

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Authors:
Patrick Harrington,1,2 Richard Dillon,1,2 Deepti Radia,1 Donal McLornan,1 Philippe Rousselot,3 Katy Rezvani,4 Shahram Kordasti,1,2 Claire Harrison,1,2 *Hugues de Lavallade2,5
Disclosure:

The current research was supported by a grant from Bristol Myers Squibb; scientific design of the research is independent from Bristol Myers Squibb and the data generated is not affected or controlled by Bristol Myers Squibb. Dr Harrington reports grants from Bristol Myers Squibb, during the conduct of the study; and personal fees from Incyte, outside the submitted work. Dr de Lavallade reports grants and personal fees from Bristol Myers Squibb and Incyte; and personal fees from Novartis and Pfizer, during the conduct of the study. Dr Dillon reports grants, personal fees, and nonfinancial support from AbbVie; personal fees from Novartis and Menarini; grants and personal fees from Pfizer; personal fees from Astellas; grants and nonfinancial support from Amgen; and personal fees and nonfinancial support from Jazz Pharmaceuticals, outside the submitted work. Dr Kordasti reports grants from Celgene and Novartis; and personal fees from Alexion, outside the submitted work. The other authors have declared no conflicts of interest.

Citation
EMJ Hematol. ;8[1]:63-65. Abstract Review No: AR5.

Each article is made available under the terms of the Creative Commons Attribution-Non Commercial 4.0 License.

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BACKGROUND AND AIMS

Dasatinib is a potent inhibitor of lymphocyte-specific protein tyrosine kinase (Lck), which plays a pivotal role in T-cell receptor (TCR) signalling, with immediate downstream targets including ZAP70 and linker of activated T cells (LAT), which are also implicated in regulatory T cell (Treg) development. Signal transducer and activator of transcription 5 (STAT5), the downstream target of IL-2, also plays a critical role in Treg differentiation and maintenance of FOXP3 expression through its binding of the promoter region of the FOXP3 gene. A subset of patients on dasatinib develop clonal large granular lymphocytosis (LGL) which is associated with immune-mediated toxicity and improved outcome.1,2 The authors hypothesised that a reduction in Treg frequency and function would correlate with the expansion of clonal LGL populations in patients receiving dasatinib.

METHODS

Phosphoflow cytometry was performed in Tregs and T effectors to assess the effect of dasatinib on TCR signalling.3 Cells were activated with the phosphatase inhibitor H2O2 and were analysed for phosphorylation of ZAP70, LAT, and STAT5. A gating strategy of CD4+/CD25+/FOXP3+/CD127lo cells was used for identification of Tregs, with FOXP3hi/CD45RA-ve cells denoting effector Tregs. Intracellular flow cytometry was also performed on T effectors after stimulation with OKT3, assessing the impact of dasatinib on cytokine expression, including TNFα, interferon-γ, IL-2, -4, and -10.4

RESULTS

Fifteen patients with chronic myeloid leukaemia (dasatinib [n=11], imatinib/nilotinib [n=4]) and five healthy controls were recruited. Patients on dasatinib had lower Tregs compared with the non-dasatinib group (mean CD3+ cells: 1.3% versus 2.0%; mean CD4+ cells: 2.6% versus 4.3%; p=0.01 and p=0.007, respectively). Dasatinib-treated patients also had a lower percentage of effector Tregs: 11.5% versus 22.1% (p=0.0097) (Figure 1A).

Patients on dasatinib had significantly reduced phosphorylation of ZAP70, LAT, and STAT5 compared with the non-dasatinib group, in CD4+ cells, CD8+ cells, and Tregs, following stimulation (phosphorylated STAT5 mean increase in median fluorescence intensity [MFI] of 4.1 versus 21.7 in CD4+ cells; 6.1 versus 28.2 in CD8+ cells; and 4.4 versus 22.6 in Treg [p=0.0001, p=0.0001, and p=0.001, respectively]) (Figure 1B).

Mean absolute increase in IL-2 expression was also lower in patients on dasatinib (0.9 versus 7.0 in CD4+ cells; 0.3 versus 3.0 in CD8+ cells; p=0.001 and p=0.014, respectively). Five patients on dasatinib had reversal of CD4:CD8 ratio and lymphocytosis, in line with clonal LGL populations, with TCR clonality confirmed by PCR. These patients had lower Tregs compared with other patients on dasatinib, with a mean CD3+ cells percentage of 0.9% versus 1.8% (p=0.035). A lower increase in MFI within isolated Tregs following stimulation was seen in this group, when compared with patients on dasatinib with normal CD4:CD8 (phosphorylated ZAP70: 1.8 versus 0.8, p=0.024; phosphorylated LAT: 4.4 versus 1.4, p=0.05; phosphorylated STAT5: 7.4 versus 2.0, p=0.15) (Figure 1C).

Figure 1: Effects of dasatinib on regulatory T cells.

A) Proportion of effector regulatory T cells (Tregs) in the dasatinib group compared with the non-dasatinib group. B) RFI of pSTAT5 in Tregs in the dasatinib group, other TKI group, and healthy controls. C) RFI of ZAP70 in Tregs in dasatinib patients with CD8+ lymphocytosis compared with the dasatinib group with normal CD4:CD8.

pSTAT5: phosphorylated signal transducer and activator of transcription 5; RFI: relative fluorescence intensity; TKI: tyrosine kinase inhibitor; Treg: regulatory T cell.

CONCLUSION

Dasatinib potently inhibits signalling from the TCR in T effectors but also in Tregs, as indicated by inhibition of phosphorylation of ZAP70 and LAT, as well as STAT5, which is essential for transcription of FOXP3. Dasatinib-treated patients have a reduction in proinflammatory cytokine expression within T effectors, with the most significant inhibitory effect seen against IL-2. Tregs have abundant expression of the IL-2 receptor on the cell surface and binding leads to STAT5 signalling.

A subset of patients on dasatinib with clonal LGL populations have further reduction in frequency and function of Tregs as assessed by signalling from the TCR and IL-2 receptor. These findings may explain the mechanism of lymphocytosis in this group and could be used to predict improved outcomes with dasatinib.

References
Mustjoki S et al. Clonal expansion of T/NK-cells during tyrosine kinase inhibitor dasatinib therapy. Leukaemia. 2009;23(8):1398-405. Kreutzman A et al. Mono/oligoclonal T and NK cells are common in chronic myeloid leukemia patients at diagnosis and expand during dasatinib therapy. Blood. 2010;116(5):772-82. de Lavallade H et al. Tyrosine kinase inhibitors impair B-cell immune responses in CML through off-target inhibition of kinases important for cell signaling. Blood. 2013;122(2):227-38. de Lavallade H et al. Repeated vaccination is required to optimize seroprotection against H1N1 in the immunocompromised host. Haematologica. 2011;96(2):307-14.