The tyrosine kinase inhibitor imatinib (imatinib, STI571, Glivec, and Gleevec) is increasingly used in patients undergoing allogeneic transplantation for leukemia. However, little is known regarding its potential immunoregulatory effects. Here, we investigate the effect of imatinib on T-cell receptor ( IntroductionImatinib mesylate (imatinib, STI571, Glivec, and Gleevec; Novartis, Basel, Switzerland) is a potent selective inhibitor of the tyrosine kinases (TKs) ABL, ARG, PDGFR ␣ and ␤, and c-KIT. It has proven clinical efficacy in the treatment of malignancies characterized by constitutive activation of these TKs: chronic myeloid leukemia (CML), Philadelphia chromosome-positive (Ph ϩ ) acute lymphocytic leukemia (ALL), myleoproliferative disorders due to chromosomal rearrangements in the PDGF-R locus and gastrointestinal stromal tumors (GIST) with mutations in c-KIT. [1][2][3][4][5] Imatinib can induce reversible dose-dependent hematologic side effects, predominantly neutropenia and thrombocytopenia. 3,6 In CML, this may in part be attributed to compromised normal hematopoiesis in addition to suppression of the BCR-ABLpositive clone that can dominate myelopoiesis. An additional mechanism may be inhibition of c-KIT in normal hematopoietic progenitor cells, which could account for the mild imatinib-induced myelosuppression observed in some patients with GIST. 2 However, these mechanisms are unlikely to account fully for the observed lymphopenia and hypogammaglobulinemia in patients with CML on long-term imatinib therapy. In one study, 25% of patients with CML on 400 mg imatinib daily developed mild lymphopenia and a gradual reduction in serum immunoglobulin levels over 3 to 12 months of therapy. 7 Another recent study found an inhibitory effect of imatinib on the development of progenitor cell-derived dendritic cells (DCs) and demonstrated that DCs exposed to imatinib were less potent at inducing primary cytotoxic T-cell reactions against tumor antigens and recall antigens in vitro. 8 The molecular target of imatinib in DCs was not defined but a role for c-KIT inhibition appeared unlikely. This raises the possibility that imatinib could affect normal hematopoiesis and immune function through inhibition of additional TKs.Effects of imatinib on T-cell activation and function have not been well defined. However, TKs play a prominent role in T-cell receptor (TCR) signal transduction and thus it is conceivable that imatinib may interfere with this process. Physiologic activation of T lymphocytes in response to antigen is controlled by the TCR. 9,10 The TCR is comprised of ␣ and ␤ chains, the signaling subunits CD3 ⑀, ␥, and ␦ chains, and TCR . TCR binding to cognate foreign peptide bound to major histocompatibility complex (MHC) molecules on the surface of antigenpresenting cells (APCs) triggers a signaling cascade that includes activation of the TKs LCK and FYN. 11 LCK phosphorylates the immunoreceptor tyrosine-based activation motifs (ITAMs) on the TCR subunits to which ZAP70 is recruited. LCK activates ZAP70, which...

Allogeneic hematopoietic stem cell transplantation (HSCT) has advanced to a common procedure for treating also older patients with malignancies and immunodeficiency disorders by redirecting the immune system. Unfortunately, cure is often hampered by relapse of the underlying disease, graft-versus-host disease, or severe opportunistic infections, which account for the majority of deaths after HSCT. Enhancing immune reconstitution is therefore an area of intensive research. An increasing variety of approaches has been explored preclinically and clinically: the application of cytokines, keratinocyte growth factor, growth hormone, cytotoxic lymphocytes, and mesenchymal stem cells or the blockade of sex hormones. New developments of allogeneic HSCT, for example, umbilical cord blood or haploidentical graft preparations leading to prolonged immunodeficiency, have further increased the need to improve immune reconstitution. Although a slow T-cell reconstitution is regarded as primarily responsible for deleterious infections with viruses and fungi, graft-versus-host disease, and relapse, the importance of innate immune cells for disease and infection control is currently being reevaluated. The groundwork has been prepared for the creation of individualized therapy partially based on genetic features of the underlying disease. We provide an update on selected issues of development in this fast evolving field; however, we do not claim completeness. (Blood. 2010; 115(19):3861-3868) IntroductionAllogeneic hematopoietic stem cell transplantation (HSCT) has become a common procedure for the therapy of hematologic malignancies and immune disorders. Donor bone marrow and mobilized peripheral blood stem cells are routinely used for the reconstitution of immune function in leukemia and lymphoma patients after radiation and/or chemotherapy. Recently, there have also been increasing attempts to treat solid tumors by immunotherapeutic approaches to induce a graft-versus-tumor effect, reviewed by Demirer et al. 1 Tempo of immune reconstitutionThe reconstitution of different immune cell subsets after an allogeneic HSCT occurs at different tempos. After the conditioning regimen there is the "aplastic phase" (neutropenia) until neutrophils recover at approximately 14 days after peripheral blood stem cell transplantation, approximately 21 days after bone marrow transplantation, and approximately 30 days after umbilical cord blood transplantation (UCBT). The infections encountered during the aplastic phase do not differ from those found in other neutropenic patients and consist primarily of bacterial infections. The first 100 days after HSCT are characterized by cellular immune deficiencies with a reduced number of cytotoxic lymphocytes, natural killer (NK) cells of the innate immune system, and T cells of the specific immune system (Figure 1). This renders the patient especially susceptible to viral and fungal infections. Rapid recovery of NK cells after HSCT is based on an expansion of the cytokine-producing CD56 bright NK-cell subset. ...

Flow cytometry with fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) tetramers has transformed the study of antigen-specific T-cells by enabling their visualization, enumeration, phenotypic characterization and isolation from ex vivo samples. Here, we demonstrate that the reversible protein kinase inhibitor (PKI) dasatinib improves the staining intensity of human (CD8+ and CD4+) and murine T-cells without concomitant increases in background staining. Dasatinib enhances the capture of cognate pMHC tetramers from solution and produces higher intensity staining at lower pMHC concentrations. Furthermore, dasatinib reduces pMHC tetramer-induced cell death and substantially lowers the T-cell receptor (TCR)/pMHC interaction affinity threshold required for cell staining. Accordingly, dasatinib permits the identification of T-cells with very low affinity TCR/pMHC interactions, such as those that typically predominate in tumour-specific responses and autoimmune conditions that are not amenable to detection by current technology.

In a proportion of patients with chronic myeloid leukemia (CML) being treated with dasatinib, we recently observed large granular lymphocyte (LGL) expansions carrying clonal T-cell receptor (TCR) ␥/␦ gene rearrangements. To assess the prevalence and role of clonal lymphocytes in CML, we collected samples from patients (n ‫؍‬ 34) at the time of diagnosis and during imatinib and dasatinib therapies and analyzed lymphocyte clonality with a sensitive polymerase chain reaction-based method of TCR ␥ and ␦ genes. Surprisingly, at CML diagnosis, 15 of 18 patients (83%) had a sizeable clonal, BCR-ABL1 negative lymphocyte population, which was uncommon in healthy persons (1 of 12; 8%). The same clone persisted at low levels in most imatinibtreated patients. In contrast, in a distinct population of dasatinib-treated patients, the diagnostic phase clone markedly expanded, resulting in absolute lymphocytosis in blood. Most patients with LGL expansions (90%) had TCR ␦ rearrangements, which were uncommon in patients without an LGL expansion (

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.