A Combination of the Immunotherapeutic Drug Anti-Programmed Death 1 with Lenalidomide Enhances Specific T Cell Immune Responses against Acute Myeloid Leukemia Cells.

Immune checkpoint inhibitors can block inhibitory molecules on the surface of T cells, switching them from an exhausted to an active state. One of these inhibitory immune checkpoints, programmed cell death protein 1 (PD-1) is expressed on T cell subpopulations in acute myeloid leukemia (AML). PD-1 expression has been shown to increase with AML progression following allo-haematopoeitic stem cell transplantation, and therapy with hypomethylating agents. We have previously shown that anti-PD-1 can enhance the response of leukemia-associated antigen (LAA)-specific T cells against AML cells as well as leukemic stem and leukemic progenitor cells (LSC/LPCs) ex vivo. In concurrence, blocking of PD-1 with antibodies such as nivolumab has been shown to enhance response rates post-chemotherapy and stem cell transplant. The immune modulating drug lenalidomide has been shown to promote anti-tumour immunity including anti-inflammatory, anti-proliferative, pro-apoptotic and anti-angiogenicity. The effects of lenalidomide are distinct from chemotherapy, hypomethylating agents or kinase inhibitors, making lenalidomide an attractive agent for use in AML and in combination with existing active agents. To determine whether anti-PD-1 (nivolumab) and lenalidomide alone or in combination could enhance LAA-specific T cell immune responses, we used colony-forming immune and ELISpot assays. Combinations of immunotherapeutic approaches are believed to increase antigen-specific immune responses against leukemic cells including LPC/LSCs. In this study we used a combination of LAA-peptides with the immune checkpoint inhibitor anti-PD-1 and lenalidomide to enhance the killing of LSC/LPCs ex vivo. Our data offer a novel insight into how we could improve AML patient responses to treatment in future clinical studies.

Increasing Role of Targeted Immunotherapies in the Treatment of AML.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The standard of care in medically and physically fit patients is intensive induction therapy. The majority of these intensively treated patients achieve a complete remission. However, a high number of these patients will experience relapse. In patients older than 60 years, the results are even worse. Therefore, new therapeutic approaches are desperately needed. One promising approach in high-risk leukemia to prevent relapse is the induction of the immune system simultaneously or after reduction of the initial tumor burden. Different immunotherapeutic approaches such as allogenic stem cell transplantation or donor lymphocyte infusions are already standard therapies, but other options for AML treatment are in the pipeline. Moreover, the therapeutic landscape in AML is rapidly changing, and in the last years, a number of immunogenic targets structures eligible for specific therapy, risk assessment or evaluation of disease course were determined. For example, leukemia-associated antigens (LAA) showed to be critical as biomarkers of disease state and survival, as well as markers of minimal residual disease (MRD). Yet many mechanisms and properties are still insufficiently understood, which also represents a great potential for this form of therapy. Therefore, targeted therapy as immunotherapy could turn into an efficient tool to clear residual disease, improve the outcome of AML patients and reduce the relapse risk. In this review, established but also emerging immunotherapeutic approaches for AML patients will be discussed.

Characteristics and mechanisms to control a COVID-19 outbreak on a leukemia and stem cell transplantation unit.

Immunosuppressed patients like patients with leukemia or lymphoma, but also patients after autologous or allogeneic stem cell transplantation are at particular risk for an infection with COVID-19. We describe a COVID-19 outbreak on our leukemia and stem cell transplantation unit (LSCT-Unit) originating from a patient with newly diagnosed acute myeloid leukemia. The patient was treated with intensive induction chemotherapy and we characterize the subsequent outbreak of COVID-19 on a LSCT-Unit. We describe the characteristics of the 36 contacts among the medical team, the results of their PCR and antibody tests and clinical aspects and features of infected employees. Of these 36 close contacts, 9 employees of the LSCT-Unit were infected and were tested positive by PCR and/or antibody-testing. 8/9 of them were symptomatic, 3/9 with severe, 5/9 with mild symptoms, and one person without symptoms. Due to stringent hygiene measures, the outbreak did not lead to infections of other patients despite ongoing clinical work. Moreover, we demonstrate that incubation period and clinical course of a COVID-19 infection in an immunosuppressed patient could be unusual compared to that of immunocompetent patients. Consistent PCR and antibody testing are helpful to understand, control, and prevent outbreaks. For the safety of health-care workers and patients alike, all employees wore FFP2 masks and were trained to adhere to several further safety guidelines. The implementation of rigorous hygiene measures is the key to controlling an outbreak and preventing infections of other patients.

Immune Checkpoint Expression on Immune Cells of HNSCC Patients and Modulation by Chemo- and Immunotherapy.

Endogenous control mechanisms, including immune checkpoints and immunosuppressive cells, are exploited in the process of tumorigenesis to weaken the anti-tumor immune response. Cancer treatment by chemotherapy or immune checkpoint inhibition can lead to changes of checkpoint expression, which influences therapy success. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were isolated from head and neck squamous cell carcinoma (HNSCC) patients (n = 23) and compared to healthy donors (n = 23). Immune checkpoint expression (programmed cell death ligand 1 (PD-1), tumor necrosis factor receptor (TNFR)-related (GITR), CD137, tumor necrosis factor receptor superfamily member 4 (TNFRSF4) (OX40), t-cell immunoglobulin and mucin-domain containing-3 (TIM3), B- and T-lymphocyte attenuator (BTLA), lymphocyte-activation gene 3 (LAG3)) was determined on immune cells by flow cytometry. PD-L1 expression was detected on tumor tissue by immunohistochemistry. Immune cells were treated with immuno- and chemotherapeutics to investigate treatment-specific change in immune checkpoint expression, in vitro. Specific changes of immune checkpoint expression were identified on PBL and TIL of HNSCC patients compared to healthy donors. Various chemotherapeutics acted differently on the expression of immune checkpoints. Changes of checkpoint expression were significantly less pronounced on regulatory T cells compared to other lymphocyte populations. Nivolumab treatment significantly reduced the receptor PD-1 on all analyzed T cell populations, in vitro. The specific immune checkpoint expression patterns in HNSCC patients and the investigated effects of immunomodulatory agents may improve the development and efficacy of targeted immunotherapy.

Specific T-cell immune responses against colony-forming cells including leukemic progenitor cells of AML patients were increased by immune checkpoint inhibition.

The efficacy of immunotherapies in cancer treatment becomes more and more apparent not only in different solid tumors but also in hematological malignancies. However, in acute myeloid leukemia (AML), mechanisms to increase the efficacy of immunotherapeutic approaches have to be further elucidated. Targeting leukemic progenitor and stem cells (LPC/LSC) by specific CTL, for instance, in an adjuvant setting or in minimal residual disease, might be an option to prevent relapse of AML or to treat MRD. Therefore, we investigated the influence of immune checkpoint inhibitors on LAA-specific immune responses by CTL against leukemic myeloid blasts and colony-forming cells including leukemic progenitor cells (CFC/LPC). In functional immunoassays like CFU/CFI (colony-forming units/immunoassays) and ELISpot analysis, we detected specific LAA-directed immune responses against CFC/LPC that are postulated to be the source population of relapse of the disease. The addition of nivolumab (anti-PD-1) significantly increases LAA-directed immune responses against CFC/LPC, no effect is seen when ipilimumab (anti-CTLA-4) is added. The combination of ipilimumab and nivolumab does not improve the effect compared to nivolumab alone. The anti-PD1-directed immune response correlates to PD-L1 expression on progenitor cells. Our data suggest that immunotherapeutic approaches have the potential to target malignant CFC/LPC and anti-PD-1 antibodies could be an immunotherapeutic approach in AML. Moreover, combination with LAA-directed vaccination strategies might also open interesting application possibilities.

Immunological and Clinical Impact of Manipulated and Unmanipulated DLI after Allogeneic Stem Cell Transplantation of AML Patients.

Allogeneic stem cell transplantation (allo-SCT) is the preferred curative treatment for several hematological malignancies. The efficacy of allo-SCT depends on the graft-versus-leukemia (GvL) effect. However, the prognosis of patients with relapsed acute myeloid leukemia (AML) following allo-SCT is poor. Donor lymphocyte infusion (DLI) is utilized after allo-SCT in this setting to prevent relapse, to prolong progression free survival, to establish full donor chimerism and to restore the GvL effect in patients with hematological malignancies. Thus, there are different options for the administration of DLI in AML patients. DLI is currently used prophylactically and in the setting of an overt relapse. In addition, in the minimal residual disease (MRD) setting, DLI may be a possibility to improve overall survival. However, DLI might increase the risk of severe life-threatening complications such as graft-versus-host disease (GvHD) as well as severe infections. The transfusion of lymphocytes has been tested not only for the treatment of hematological malignancies but also chronic infections. In this context, manipulated DLI in a prophylactic or therapeutic approach are an option, e.g., virus-specific DLI using different selection methods or antigen-specific DLI such as peptide-specific CD8+ cytotoxic T lymphocytes (CTLs). In addition, T cells are also genetically engineered, using both chimeric antigen receptor (CAR) genetically modified T cells and T cell receptor (TCR) genetically modified T cells. T cell therapies in general have the potential to enhance antitumor immunity, augment vaccine efficacy, and limit graft-versus-host disease after allo-SCT. The focus of this review is to discuss the different strategies to use donor lymphocytes after allo-SCT. Our objective is to give an insight into the functional effects of DLI on immunogenic antigen recognition for a better understanding of the mechanisms of DLI. To ultimately increase the GvL potency without raising the risk of GvHD at the same time.

Donor lymphocyte infusion leads to diversity of specific T cell responses and reduces regulatory T cell frequency in clinical responders.

T cell responses against malignant cells play a major role in maintaining remission and prolonging overall survival in patients after allogeneic stem cell transplantation and donor lymphocyte infusion (DLI) due to graft-versus-leukemia effect. For better characterization of the T cell responses, we assessed frequency and diversity of leukemia-associated antigen (LAA)-specific cytotoxic T cells using ELISpot and pMHC multimer assays and analyzed the frequency of regulatory T cells (Treg) as well as cytokine profiles before/after DLI. The data were correlated to the clinical course of patients. Significantly more LAA-derived T cell epitopes (p = 0.02) were recognized in clinical responders (R) when compared to nonresponders (NR). In addition, pMHC multimer-based flow cytometry showed a significantly higher frequency of LAA-specific T cells in R versus NR. The frequency of Treg in R decreased significantly (p = 0.008) while keeping stable in NR. No differences in T cell subset analysis before/after DLI were revealed. Clinical responders were correlated to specific immune responses and all clinical responders showed an increase of specific immune responses after DLI. Cytokine assays using enzyme-linked immunosorbent assay showed a significant increase of IL-4 after DLI. Taken together, an increase of specific CTL responses against several LAA after DLI was detected. Moreover, this study suggests that enhanced LAA diversity in T cell responses as well as decreasing numbers of Treg contribute to clinical outcome of patients treated with DLI.

Peptide vaccination in the presence of adjuvants in patients after hematopoietic stem cell transplantation with CD4+ T cell reconstitution elicits consistent CD8+ T cell responses.

Rationale: Patients receiving an allogeneic stem cell graft from cytomegalovirus (CMV) seronegative donors are particularly prone to CMV reactivation with a high risk of disease and mortality. Therefore we developed and manufactured a novel vaccine and initiated a clinical phase I trial with a CMV phosphoprotein 65 (CMVpp65)-derived peptide. Methods: Ten patients after allogeneic stem cell transplantation received four vaccinations at a biweekly interval. All patients were monitored for CMVpp65 antigenemia. Flow cytometry for CMV-specific CD8+ and γδ T cells as well as neutralizing anti-CMV antibodies were correlated to clinical parameters. Results: The vaccination was well tolerated. Seven of nine patients cleared CMVpp65 antigenemia after four vaccinations and are still free from antigenemia to this day. Two patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An increase of up to six-fold in frequency of both CMV-specific CD8+ T cells and/or Vδ2negative γδ T cells was detected. Titers of neutralizing antibodies increased up to the tenfold. Humoral and cellular immune responses correlated with clearance of CMV. Conclusion: In summary, CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing.

Leukemic progenitor cells are susceptible to targeting by stimulated cytotoxic T cells against immunogenic leukemia-associated antigens.

Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. We investigated the role of cytotoxic T-lymphocytes (CTL) counteracting and recognizing LSC. Leukemia-associated antigens (LAA) represent immunogenic structures to target LSC. We enriched the LSC-containing fraction of 20 AML patients and hematopoietic stem cells (HSC) of healthy volunteers. Using microarray analysis and qRT-PCR we detected high expression of several LAA in AML cells but also in LSC. PRAME (p = 0.0085), RHAMM (p = 0.03), WT1 (p = 0.04) and Proteinase 3 (p = 0.04) showed significant differential expression in LSC compared with HSC. PRAME, RHAMM and WT1 are furthermore also lower expressed on leukemic bulk. In contrast, Proteinase 3 indicates a higher expression on leukemic bulk than on LSC. In colony forming unit (CFU) immunoassays, T cells stimulated against various LAA indicated a significant inhibition of CFUs in AML patient samples. The LAA PRAME, RHAMM and WT1 showed highest immunogenic responses with a range up to 58-83%. In a proof of principle xenotransplant mouse model, PRAME-stimulated CTL targeted AML stem cells, reflected by a delayed engraftment of leukemia (p = 0.0159). Taken together, we demonstrated the expression of several LAA in LSC. LAA-specific T cells are able to hamper LSC in immunoassays and in a mouse model, which suggests that immunotherapeutic approaches have the potential to target malignant stem cells.

Frequent T cell responses against immunogenic targets in lung cancer patients for targeted immunotherapy.

To date, lung cancer is one of the leading causes of cancer mortality with short overall survival despite adequate therapy. New immunotherapeutic strategies using peptides derived from tumor-associated antigens (TAAs) can induce a specific cytotoxic T cell (CTL) response leading to a targeted tumor cell death. In the present study, we addressed whether there are further significant immunogenic candidate targets that may induce strong immune reactions with a high frequency in lung cancer patients eligible for cellular immunotherapeutic approaches, such as in a polyvalent vaccination approach. In this study, we investigated specific CTL responses of 14 HLA-A*0201-positive patients (of 33 screened patients) with non-small cell lung cancer (NSCLC; n=12) or small cell lung cancer (SCLC; n=2) against several known and novel TAA-derived peptides from lung cancer and/or other tumor entities, by measuring granzyme B (GrB) and/or interferon γ (IFNγ) secretion using enzyme-linked immunospot (ELISpot) analysis. Specific T cell responses could be detected for hTERT (4/13), two MAGE-A3-derived peptides (4/13 and 3/13, respectively), RHAMM (4/14), PRAME (8/14), G250 (7/12), survivin (3/13), HER2 (5/10) and WT1 (2/14), but also novel epitopes derived from Aurora kinase A (4/13) and B (5/13). Additionally, simultaneous CTL responses against the different peptides were examined and specific T cell responses against at least one of these TAAs could be detected in 13/14 (93%) patients. It could be shown that all patients with immune reactions against RHAMM and hTERT showed also immune responses against PRAME. Furthermore, patients with CTL responses against the Aurora kinase A peptide (Aura A1) also demonstrated a response against the Aurora kinase B peptide (Aura B1). Taken together, we showed that these TAA-derived peptides induce frequent specific T cell responses in patients with metastatic lung cancer and are, therefore, novel candidates for targeted immunotherapies and polyvalent approaches.

PRAME-induced inhibition of retinoic acid receptor signaling-mediated differentiation--a possible target for ATRA response in AML without t(15;17).

PURPOSE: In acute myeloid leukemia (AML) without retinoic acid receptor (RAR) rearrangement, the effect of all-trans-retinoic acid (ATRA) is still poorly understood despite an association of NPM1 mutation and ATRA response. Recently, preferentially expressed antigen in melanoma (PRAME) has been shown to be a dominant repressor of RAR signaling. EXPERIMENTAL DESIGN: Thus, we further investigated ATRA response mechanisms, especially the impact of PRAME expression on ATRA responsiveness. We profiled gene expression in diagnostic samples derived from our AML HD98B trial, in which ATRA was administered in addition to intensive chemotherapy. RESULTS: Our data revealed a PRAME expression-associated gene pattern to be significantly enriched for genes involved in the retinoic acid metabolic process. In leukemia cell line models, we could show that retinoic acid-regulated cell proliferation and differentiation are impacted by PRAME expression. In patients with primary AML, repressor activity of high-PRAME levels might be overcome by the addition of ATRA as indicated by better outcome in 2 independent studies (P = 0.029). CONCLUSIONS: PRAME seems to impair differentiation and to increase proliferation likely via blocking RAR signaling, which might be reversed by ATRA. PRAME therefore represents a promising target for both ATRA treatment and possibly future immunotherapeutic approaches in AML.

Specific immune responses against epitopes derived from Aurora kinase A and B in acute myeloid leukemia.

Aurora kinases are serine/threonine kinases which play an important role in the process of mitosis and cell cycle regulation. Aurora kinase inhibitors are described to sensitize malignant cells to cytosine arabinoside and specific antibodies by mediating apoptosis. Aurora kinases are overexpressed in most acute leukemias but also in solid tumors. In this study we investigated whether epitopes derived from Aurora kinase A and B are able to elicit cellular immune responses in patients with acute myeloid leukemia (AML) to investigate their role as potential targets for specific immunotherapy. Samples of eight patients with AML were analyzed in enzyme-linked immunosorbent spot (ELISpot) assays and compared with immune responses of nine healthy volunteers (HVs). Specific CD8 + T cell responses were detected against the epitopes Aura A1, A2, B1, B2, B3, B4 and B5. Immune responses for epitopes derived from Aura B were induced more frequently compared to Aura A. The antigens with the most frequent cytotoxic T-lymphocyte (CTL) responses were Aura B3, B4 and B5, although the number of patients tested for these antigens was low. Aura B5 did not elicit specific CTL responses in HVs. For epitope Aura B6 no immune response was detected in HVs or patients. Taken together, with the combination of Aurora kinase inhibitors and an immunotherapeutic approach, an effective blast and minimal residual disease elimination might be achieved.

Immunogenic targets for specific immunotherapy in multiple myeloma.

Multiple myeloma remains an incurable disease although the prognosis has been improved by novel therapeutics and agents recently. Relapse occurs in the majority of patients and becomes fatal finally. Immunotherapy might be a powerful intervention to maintain a long-lasting control of minimal residual disease or to even eradicate disseminated tumor cells. Several tumor-associated antigens have been identified in patients with multiple myeloma. These antigens are expressed in a tumor-specific or tumor-restricted pattern, are able to elicit immune response, and thus could serve as targets for immunotherapy. This review discusses immunogenic antigens with therapeutic potential for multiple myeloma.

Mutated regions of nucleophosmin 1 elicit both CD4(+) and CD8(+) T-cell responses in patients with acute myeloid leukemia.

Mutations in the nucleophosmin gene (NPM1(mut)) are one of the most frequent molecular alterations in acute myeloid leukemia (AML), and immune responses may contribute to the favorable prognosis of AML patients with NPM1(mut). In the present study, we were able to demonstrate both CD4(+) and CD8(+) T-cell responses against NPM1(mut). Ten peptides derived from wild-type NPM1 and NPM1(mut) were subjected to ELISPOT analysis in 33 healthy volunteers and 27 AML patients. Tetramer assays against the most interesting epitopes were performed and Cr(51)-release assays were used to show the cytotoxicity of peptide-specific T cells. Moreover, HLA-DR-binding epitopes were used to test the role of CD4(+) T cells in NPM1 immunogenicity. Two epitopes (epitopes #1 and #3) derived from NPM1(mut) induced CD8(+) T-cell responses. A total of 33% of the NPM1(mut) AML patients showed immune responses against epitope #1 and 44% against epitope #3. Specific lysis of leukemic blasts was detected. To obtain robust immune responses against tumor cells, the activation of CD4(+) T cells is crucial. Therefore, overlapping (OL) peptides were analyzed in ELISPOT assays and OL8 was able to activate both CD8(+) and CD4(+) T cells. The results of the present study show that NPM1(mut) induces specific T-cell responses of CD4(+) and CD8(+) T cells and therefore is a promising target for specific immunotherapies in AML.

Effects of nilotinib on regulatory T cells: the dose matters.

BACKGROUND: Nilotinib is a tyrosine kinase inhibitor with high target specificity. Here, we characterized the effects of nilotinib for the first time on CD4+CD25+ regulatory T cells (Tregs) which regulate anti-tumor/leukemia immune responses. DESIGN AND METHODS: Carboxyfluorescein diacetate succinimidyl ester (CFSE) and 5-bromo-2-deoxy -uridine (BrdU) were used to assess the proliferation and cell cycle distribution of Tregs. The expression of the transcription factor forkhead box P3 (FoxP3) and the glucocorticoid-induced tumor necrosis factor receptor (GITR) were measured by flow cytometry. Western blotting analysis was used to detect the effects of nilotinib on the signal transduction cascade of T-cell receptor (TCR) in Tregs. RESULTS: Nilotinib inhibited the proliferation and suppressive capacity of Tregs in a dose-dependent manner. However, the production of cytokines secreted by Tregs and CD4+CD25- T cells was only inhibited at high concentrations of nilotinib exceeding the mean therapeutic serum concentrations of the drug in patients. Only high doses of nilotinib arrested both Tregs and CD4+CD25- T cells in the G0/G1 phase and down-regulated the expression of FoxP3 and GITR. In western blotting analysis, nilotinib did not show significant inhibitory effects on TCR signaling events in Tregs and CD4+CD25- T cells. CONCLUSIONS: These findings indicate that nilotinib does not hamper the function of Tregs at clinical relevant doses, while long-term administration of nilotinib still needs to be investigated.

High-dose RHAMM-R3 peptide vaccination for patients with acute myeloid leukemia, myelodysplastic syndrome and multiple myeloma.

BACKGROUND: Recently, we demonstrated immunological and clinical responses to a RHAMM-R3 peptide vaccine in patients with acute myeloid leukemia, myelodysplastic syndrome and multiple myeloma. To improve the outcome of the vaccine, a second cohort was vaccinated with a higher dose of 1,000 microg peptide. DESIGN AND METHODS: Nine patients received four vaccinations subcutaneously at a biweekly interval. Immunomonitoring of cytotoxic CD8(+) as well as regulatory CD4(+) T cells was performed by flow cytometry as well as by enzyme-linked immunospot (ELISpot) assays. Parameters of clinical response were assessed. RESULTS: In 4 of 9 patients (44%) we detected positive immunological responses. These patients showed an increase of CD8(+)RHAMM-R3_tetramer(+)/CD45RA(+)/CCR7(-)/CD27(-)/CD28(-) effector T cells and an increase of R3-specific CD8+ T cells. Two of these patients showed a significant decrease of regulatory T cells (Tregs). In one patient without response Tregs frequency increased from 5 to 16%. Three patients showed clinical effects: one patient with myelodysplastic syndrome RAEB-1 showed a reduction of leukemic blasts in the bone marrow, another myelodysplastic syndrome patient an improvement of peripheral blood counts and one patient with multiple myeloma a reduction of free light chains. Clinical and immunological reactions were lower in this cohort than in the 300 microg cohort. CONCLUSIONS: High-dose RHAMM-R3 peptide vaccination induced immunological responses and positive clinical effects. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematologic malignancies. However, higher doses of peptide did not improve the frequency and intensity of immune responses in this trial.

Dasatinib inhibits the proliferation and function of CD4+CD25+ regulatory T cells.

CD4+CD25+ regulatory T cells (Tregs) can influence various immune responses. Little is known about the effects of the Abl/Src kinase inhibitor dasatinib on Tregs which regulate anti-tumor/leukaemia immune responses. The present study demonstrated that dasatinib inhibited proliferation of Tregs and CD4+CD25- T cells in a dose-dependent manner, which was associated with the decreased production of corresponding cytokines. Treatment of Tregs with dasatinib inhibited the suppressive capacity of Tregs. The mechanisms of this inhibition included arrest of cells in the G0/G1 phase of cell cycle, down-regulation of the transcription factor forkhead box P3, glucocorticoid-induced tumour necrosis factor receptor and the cytotoxic T lymphocyte associated protein 4 as well as inhibition of signaling events through Src and nuclear factor kappaB. Dasatinib showed an inhibitory effect on the proliferation and function of both Tregs and CD4+CD25- T cells at therapeutically relevant concentrations of the drug. Clinical administration of dasatinib might influence not only the graft-versus-leukaemia effect but also the graft-versus-host-disease in patients receiving dasatinib after allogeneic stem cell transplantation and/or donor lymphocytes infusion as the function of both Tregs and effector T cells are hampered in a similar way by dasatinib.