Purpose: The dual BCR-ABL/SRC kinase inhibitor dasatinib entered the clinic for the treatment of chronic myeloid leukemia and Ph + acute lymphoblastic leukemia. Because SRC kinases are known to play an important role in physiologicT-cell activation, we analyzed the immunobiological effects of dasatinib on T-cell function. The effect of dasatinib on multiple T-cell effector functions was examined at clinically relevant doses (1-100 nmol/L); the promiscuous tyrosine kinase inhibitor staurosporine was used as a comparator. Experimental Design: Purified human CD3 + cells and virus-specific CD8 + Tcells from healthy blood donors were studied directly ex vivo ; antigen-specific effects were confirmed in defined T-cell clones. Functional outcomes included cytokine production (interleukin-2, IFNg, and tumor necrosis factor a), degranulation (CD107a/b mobilization), activation (CD69 up-regulation), proliferation (carboxyfluorescein diacetate succinimidyl ester dilution), apoptosis/necrosis induction, and signal transduction. Results: Both dasatinib and staurosporine inhibited T-cell activation, proliferation, cytokine production, and degranulation in a dose-dependent manner. Mechanistically, this was mediated by the blockade of early signal transduction events and was not due to loss of T-cell viability. Overall, CD4 + T cells seemed to be more sensitive to these effects than CD8 + Tcells, and naBve Tcells more sensitive than memoryT-cell subsets. The inhibitory effects of dasatinib were so profound that all T-cell effector functions were shut down at therapeutically relevant concentrations. Conclusion: These findings indicate that caution is warranted with use of this drug in the clinical setting and provide a rationale to explore the potential of dasatinib as an immunosuppressant in the fields of transplantation and T-cell^driven autoimmune diseases.
CD22 functions primarily as a negative regulator of B-cell receptor signaling. The Cd22a allele has been proposed as a candidate allele for murine systemic lupus erythematosus. In this study, we explored the possible expression of aberrant forms of CD22, which differ in the N-terminal sequences constituting the ligand-binding site due to synthesis of abnormally processed Cd22 mRNA, in several Cd22a mouse strains, including C57BL/6 Cd22 congenic mice. The staining pattern of splenic B cells obtained with CY34 anti-CD22 mAb, which was expected to bind poorly to the aberrant CD22, was more heterogeneous in Cd22(a) mice than in Cd22b mice. Moreover, CD22 detected on B cells of Cd22a mice was expressed more weakly and as a smaller-sized protein, compared with Cd22b mice. Significantly, analysis with a synthetic CD22 ligand demonstrated that Cd22a mice carried a larger proportion of CD22 that was not bound by cis ligands on the B-cell surface than Cd22b mice. Finally, the study of C57BL/6 Cd22 congenic mice revealed that Cd22a B cells displayed a phenotype reminiscent of constitutively activated B cells (reduced surface IgM expression and augmented MHC class II expression), as reported for B cells expressing a mutant CD22 lacking the ligand-binding domain. Our demonstration that Cd22a B cells express aberrant forms of CD22, which can potentially deregulate B-cell signaling because of their decreased ligand-binding capacity, provides further support for Cd22a as a potential candidate allele for murine systemic lupus erythematosus.
1123 Poster Board I-145 Introduction Immunosuppressive effects of the second generation tyrosine kinase inhibitor dasatinib (Sprycel®) on T cells and NK cells have been described in vitro. In contrast, in some CML or Ph+ ALL patients receiving dasatinib, the development of a chronic, oscillating lymphocytosis has been observed in vivo. We previously showed that dasatinib-induced lymphocytosis typically comprises of oligoclonal NK or CD8+ cytotoxic T cell large granular lymphocyte (LGL) expansions and was associated with HLA-A*0201, CMV reactivation and enhanced, long-lasting therapy responses in patients with advanced leukemia. Patients and Methods To further elucidate the mechanisms underlying LGL expansions during dasatinib, we analyzed T cell and NK cell effector functions directly ex vivo. Specifically, we evaluated activation, proliferation, cytotoxic activity, cytokine secretion, degranulation, KIR expression profile and apoptosis/necrosis susceptibility. Results We observed decreased NK cell cytotoxic activity against the cell line K562 only in patients with NK cell expansions, potentially due to exhaustion based on excessive in vivo proliferation. This reduced lytic ability was restored after pre-treatment with IL-2 for 18h. No correlation between functionality and KIR expression profile was found. Furthermore, patients with LGL expansions exhibited elevated IP10/RANTES levels in the plasma compared to non-LGL patients and healthy controls. Strikingly, within the CD8+ T cell population specific for the CMV epitope NLVPMVATV (pp65, residues 495-503) restricted by HLA-A*0201, we observed distinct CD8high and CD8low fractions in patients with LGL expansions (n=5) but not in patients without LGL expansions (n=4) or in healthy controls (n=3). The CD8high subpopulation was more resistant to inhibition by dasatinib compared to other cell fractions; in addition, dasatinib effectively abrogated down-regulation of both TCR and CD8 in the CD8high subpopulation in vitro. Conclusions Thus, we hypothesize that CMV reactivation is intimately linked to the observed LGL expansions in dasatinib-treated patients. The expanded cytotoxic cells may target both CMV-infected and leukemic cells by virtue of epitope sharing and thus explain the enhanced anti-leukemic control we observed. Further investigations based on the application of polychromatic flow cytometry and analysis of T cell clonality are underway to establish the nature of this association in more detail. Dasatinib and derivatives may prove to be useful in modulating anti-leukemic/anti-host immune responses in vivo. Disclosures Mustjoki: BMS: Honoraria. Ekblom:BMS: Honoraria. Porkka:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Seggewiss:BMS: Honoraria.
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