julilili42/papers huggingface dataset:
Heatshockprotein-60/90 Are Overexpressed in Leukemia Stem Cells and Stabilize Oncogene Signaling.
2012-11-16

Abstract Abstract 2472 Introduction: Using a global phosphoproteome screen, we have recently identified heat shock proteins (HSPs) to be crucial effectors to stabilize KIT oncoprotein function in the presence of tyrosine kinase inhibitors in mutant-KIT leukemia models (Kampa-Schittenhelm et al., ASH annual meeting 2010). We now show that HSP60 and (p)HSP90 are frequently expressed in acute leukemia, arguing for an integrative function of HSPs to stabilize leukemia-driving oncoproteins. Importantly, HSPs are highly expressed in leukemia stem cells (LSCs), which provides a rationale for therapy insensitivity. Targeting HSPs may therefore be an attractive strategy to target the LSC clone. Methods: Intracellular protein levels of (p)HSPs in leukemic blasts were studied by flow cytometry. Three dimensional detection of the putative CD34+/CD38-/HSP+ leukemia stem/progenitor cell fraction was performed using -FITC, -PE and APC-coupled secondary antibodies using standard techniques. Cellular proliferation and induction of apoptosis in leukemia cells treated with the HSP90 inhibitor IPI-504 was determined by XTT- and annexin V-based assays. Immunoblot experiments to assess IPI-504 interaction on the chaperone level were set up using standard protocols including a LICOR imaging system. Results: Of 16 evaluated patients with AML, 50% and 56% demonstrated significant HSP90 and HSP60 expression levels (with a threshold level set as &gt; 3× of the relative mean fluorescence compared to IgG controls), respectively. Interestingly, HSP expression was predominantly seen in patients with core–binding factor leukemia (associated with KIT mutations). Moreover, five evaluable freshly harvested patient samples were used to assess the putative CD34+/CD38-/HSP90 and HSP60 population by flow cytometry. Tantalizingly, all tested CD34+/CD38- samples revealed high co-expression of HSPs. Consequently, an HSP90 inhibitor (IPI-504) potently inhibited cellular proliferation in in vitro leukemia models as well as freshly harvested leukemia cells at highly variable doses starting in low nanomolar ranges up to lower micromolar concentrations with regard to IC50s. Several cell lines with defined leukemia-driving oncogenes (MOLM14 – FLT3 ITD, HMC1.1 – KIT V560G, Kasumi1 – KIT N822H) were treated with IPI-504, and degradation of FLT3 and KIT was confirmed by immunoblotting. Consequently, oncogene-degradation led to induction of apoptosis in the nanomolar range which was confirmed in ex vivo blasts. Noteworthy, treating isolated freshly harvested CD34+/CD38- progenitor cells lead to potent reduction of the viable cell fraction. Conclusion: HSPs are frequently expressed in acute leukemias, including LSCs, arguing for a function as mechanism to protect and uphold LSC function in the presence of cell stress such as antileukemic treatment. Targeting the HSP-expressing stem cell pool may be an attractive novel therapeutic strategy in acute leukemia. Disclosures: No relevant conflicts of interest to declare.
Human Peripheral CD34+ γδ T Cells Can Transdifferentiate into αβ T Cells.
2012-11-16

Abstract Abstract 2312 Human peripheral CD34+ γδ T cells can transdifferentiate into αβ T cells Haploidentical transplantation of peripheral mobilized T-cell depleted stem cells is associated with a delayed immune recovery resulting in severe and often lethal infectious complications after transplantation. For T-cell depletion, either positive selection of CD34+ stem cells as well as negative depletion of CD3+ T lymphocytes is used. However, delayed reconstitution of the adaptive T-cell immune system is seen with both approaches. Since adaptive immune cells are the progeny of haematopoietic precursors, one reason for the delayed immune reconstitution might be the depletion of a progenitor for the adaptive immune system with CD34+ positive selection and CD3 negative depletion. Therefore, we hypothesized the existence of a peripheral CD34+ CD3+ lymphoid progenitor cell. And indeed, we could identify a subset of peripheral circulating cells with a weaker expression of the CD34 antigen compared to CD34+ myeloid progenitor cells which coexpressed CD3, γδ TCR(Vδ1), and CD4lo and additionally express the hematopoietic progenitor markers CD105, CD117, CD135 and CXCR4. In an inflammatory environment, this CD34+ subset can transdifferentiate in vitro into αβ T cells. Upon its extrathymic route of differentiation, that resembles thymic development, CD34+ Vd1+ CD4+ cells increase CD4+ coreceptor expression, develop Vδ1+ CD4+CD8+ double positive cells, show heterodimeric CD8αβ, transcribe RAG and preTα and express a particular Vβ chain on their surface. Simultaneously inflammation confers controlled initiation of rearrangement in the TCRα locus. Transdifferentiation of Vδ1+ T cells at the clonal and bulk-culture level into functional CD4+ or CD8+ αβ T cells upon inflammatory stimuli, is in line with the findings that HSCs participate directly in the primary response to both acute and chronic infections. The identification of CD34+ Vδ1+ T-cells as precursor for adaptive immune cells under inflammatory conditions is of utmost clinical importance, since (i) this subset is selectively lost with the clinically used CliniMACS method of CD34 positive selection, which preferentially enriches for stem cells with a stronger expression of the CD34 antigen due to the binding of more magnetic particles and better retention of the CD34++ cells in the magnetic field and (ii) this subset is also lost using CD3 negative depletion due the coexpression of the CD3 antigen on the CD34+ Vd1+ cells. Our results might be an explanation why a more rapid T-cell recovery is seen after the transplantation of peripheral stem cells depleted of TcRαβ T-cells, which retains the CD34+ Vδ1+ cells in the graft. Furthermore, the assignment of this fundamental role for γδ T cells as a reservoir of an as-yet unappreciated lineage-committed extrathymic αβ T-lymphoid progenitor opens a new vista in immunology and necessitates reevaluation of adaptive immune responses in infection, autoimmunity and cancer. Disclosures: No relevant conflicts of interest to declare.
Detection of Th1 Driven T-Cell Responses in Peripheral Could Guide Individualized Immunosuppression and Risk of Viral Reactivation Under GvHD Prophylaxis Following Allogeneic Stem Cell Transplantation.
2012-11-16

Abstract Abstract 3047 Allogeneic stem cell transplantation (SCT) can expose patients to a transient but marked immunosuppression, during which viral infections are an important cause of morbidity and mortality. The control of these infections will ultimately depend on the restoration of adequate T-cell immunity. Most viral infections after SCT are caused by endogenous reactivation of persistent pathogens such as cytomegalovirus (CMV), adenovirus (ADV) and Epstein-Barr-virus (EBV). Risk of viral complications is even higher under GvHD treatment or prophylaxis like calcineurin inhibitors and steroids. Post transplant often the immunosuppression needs to be reduced to improve viral complications with the risk of GvHD. The virus-specific T-cell responses in peripheral blood have been shown to be a good marker of immunological protection, but has not been used for clinical decision making and the guidance of drug plasma levels. Therefore, we performed a prospective clinical trial in 33 adult and pediatric patients after allogeneic stem cell transplantation receiving pharmacologic immunosuppression with steroids, Cyclosporin A, Tacrolimus, Everolimus or Mycophenolate. Median Age was 16 years. T-cell responses were analyzed ex vivo against Cytomegalovirus (pp65), Adenovirus (hexon antigen) and Epstein-Barr Virus (EBNA, LMP) using intracellular cytokine staining. In addition in vitro analysis of the proliferation responses using CFSE were performed. Responses were compared to healthy donors. The T-cell responses in vitro under low, high and supraphysiologic plasma concentrations of the respective drugs were investigated. Under the direct influence of steroids, activated, virus-specific T-cells underwent apoptosis. Among the Calcineurin inhibitors, Tacrolimus had the strongest inhibition on virus-specific T-cell immunity, followed by Cyclosporin A. But, under low therapeutic levels, Virus speciffic T-cell responses have been able to develop in PBMCs. Mycophenolate had only in high concentrations a strong effect on the T-cell response against viral pathogens. Relevant differences in the frequency of virus-specific T-cells secreting IFN-g could be detected within the CD4 compartment in correlation to the level of immunosuppression. In conclusion we could show that detection of virus-specific T-cells could be used to guide the level of immunosuppression in case of viral complications after allogeneic stem cell transplantation, since emergence of in vivo T-cell responses was closely associated with a clearance or reduction of the viral load. Disclosures: No relevant conflicts of interest to declare.
The Role of Her2/Neu in ALL: Long Term Clinical Follow-up of Surface-Positive Patients and Induction of NK Cell ADCC by Trastuzumab in Vitro
2012-11-16

Abstract Abstract 1421 In about one third of patients, acute lymphoblastic leukemia (ALL) cells express Her2/neu (p185Her-2). In epithelial malignancies Her2/neu contributes to oncogenic transformation and metastatic potential, and overexpression often is associated with poor prognosis. Previous reports also suggested an inferior prognosis in patients with Her2/neu-positive ALL, but as of now no study has performed survival analyses. In breast cancer patients, targeting of Her2/neu with the monoclonal antibody Trastuzumab results in improved disease outcome, and induction of antibody dependent cellular cytotoxicity (ADCC) and cytokine release of NK cells contributes to the same. Most recently, application of Trastuzumab in patients with relapsed or refractory ALL was shown to yield promising results, but the mechanisms by which Trastuzumab mediates its efficacy have not been studied so far. Here, we studied patient records and flow cytometry data on Her2/neu expression of patients treated for ALL at our hospital between 2000 and 2011 and determined the effect of Trastuzumab and/or Rituximab on NK cell reactivity against ALL blasts in vitro. In 57 patients (37 men, 20 women, median age at diagnosis 42 years, range 17–83 years) data on Her2/neu expression were available. Her2/neu expression was detected in 15/57 patients (26%). Clinical follow up was assessed for all 57 patients. Median follow-up was 44 months (range 0.5–356 months) until death or last follow-up visit. Overall, 60% of the patients had died at the end of follow-up (25 of 42 Her2/neu-negative and 9 of 15 Her2/neu-positive patients, respectively). Median survival times were 43 (Her2/neu-negative, range 0.5–356 months) and 41 months (Her2/neu-positive, range 3.5–138 months; p = 0.81. Kaplan Meyer regression analysis). Also, median time to relapse was comparable in both groups (22.5 months vs. 15.5 months; p = 0.77). In functional analyses with NK cells, incubation of Her2/neu-positive CD20-positive ALL blasts with Trastuzumab or Rituximab comparably induced ADCC and cytokine release of NK cells. Only minor additional effects were observed upon combined application of both antibodies. When blasts from Her2/neu-negative ALL patients were employed (phentotype CD20-positive Her2/neu-negative or CD20-negative Her2/neu-negative), no effect of Trastuzumab was observed, and Rituximab exerted effects solely with blasts from CD20-positive ALL patients. Taken together, in our patient cohort Her2/neu expression on ALL blasts was not associated with time to relapse or overall survival and thus did not appear to be an additional risk factor. Rather, Her2/neu is a promising target for antibody therapy, especially in patients with Her2/neu-positive CD20-negative ALL, as treatment with Trastuzumab is suitable to induce ADCC and cytokine production of NK cells also against Her2/neu-positive CD20-negative ALL blasts. Disclosures: No relevant conflicts of interest to declare.
Induction of NK Cell Reactivity Against AML Cells by Fc-Engineered RANK-Ig Fusion Proteins.
2012-11-16

Abstract Abstract 2625 NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. A clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of anti-tumor antibodies. In acute myeloid leukemia (AML) NK cells largely contribute to the therapeutic efficacy allogenic stem cell transplantation (SCT). Recently we demonstrated that AML cells functionally express the TNF family member RANK ligand (RANKL) which impairs NK cell anti-leukemia reactivity (Schmiedel et al., ASH annual meeting 2011). Here we developed a strategy to combine blocking of the NK inhibitory effects of RANKL with targeting of AML cells for NK cell ADCC. To this end we generated fusion proteins consisting of the extracellular domain of RANK and a human IgG1 Fc part that was modified by amino acid exchange. Compared to wild type RANK-Fc fusion protein (RANK-Fc-WT), our mutant RANK-Fc-ADCC (S239D/I332E) displayed highly enhanced affinity to FcγRIIIa (CD16) on NK cells. Primary AML cells expressed substantial levels of RANKL in 53 of 78 (68%) investigated patient cases, and our RANK-Ig fusion proteins bound to AML cells in a target antigen-specific manner. Treatment with both RANK-Fc-WT and RANK-Fc-ADCC clearly reduced the release of RANKL-induced immunomodulatory factors like TNF, IL-6, IL-8 and IL-10 by AML cells. When the effects of the fusion proteins on NK cell ADCC were studied we found that treatment with RANK-Fc-WT only slightly enhanced NK cell reactivity against RANKL-positive patient AML cells. However, RANK-Fc-ADCC potently induced NK cell ADCC and cytokine production in response to AML targets in a target antigen-dependent manner due to the functional properties of its engineered Fc moiety. Taken together, our Fc-engineered RANK-Fc-ADCC fusion protein may serve to modulate the cytokine milieu involved in AML pathophysiology and target RANKL-expressing leukemia cells for NK anti-tumor reactivity. Thus, RANK-Fc-ADCC constitutes an attractive immunotherapeutic means for the treatment of AML, e.g. for elimination of minimal residual disease after conventional therapy including SCT. Disclosures: No relevant conflicts of interest to declare.
Sequential Chemotherapy Followed by Reduced Intensity Conditioning and Allogeneic Stem Cell Transplantation (allo-SCT) in Adult Patients with Relapsed or Refractory Acute Myeloid Leukemia (AML): A Survey From the Acute Leukemia Working Party of EBMT
2012-11-16

Abstract Abstract 2025 Patients with refractory or relapsed AML have a very dismal outcome. In the light of poor results with conventional therapy, allo-SCT is the recommended treatment for refractory AML. However, results are limited by a high relapse incidence (RI) and high non-relapse mortality (NRM). To improve the dismal outcome of patients with refractory or relapsed AML, the sequential application of cytoreductive chemotherapy, followed by reduced-intensity conditioning (RIC) allo-SCT, may represent an attractive treatment option. The aim of this multicentre analysis was to assess the outcome of 239 AML patients who received such a so-called sequential chemotherapy and allo-SCT and were reported to the EBMT registry. Sequential chemotherapy included fludarabine (30 mg/m2), cytarabine (2g/m2) and amsacrine (100mg/m2) for 4 days followed by RIC with busulfan (Bu) in 73 patients, or cyclophosphamide (80–120mg/kg) and 4Gy. TBI (Cy-TBI) in 166 patients. Median age was 62 and 51 years (range, 19–73) and median year of transplant was 2009 and 2008 in the two groups respectively (p=0.0001). Patients with primary induction failure (PIF), 1st or 2nd relapse AML did not differ significantly between the Bu and Cy-TBI patients. There were more unrelated donor transplants in the Bu group (p=0.05). CR rate post transplant, engraftment, acute and chronic GVHD were similar between the two groups. One year NRM, RI, OS and leukemia free survival (LFS) were 24+/−5% vs 16+/−3%, 44+/−6% vs 51+/−4%, 46+/−7% vs 47+/−3% and 32+/−6% vs 33+/−4% in the Bu and Cy-TBI groups respectively (p=ns). Recipients of unrelated grafts had a lower probability of RI (Hazards ratio (HR)=0.64, p=0.02) and better LFS (HR=0.67; p=0.02) compared to recipients of HLA identical sibling allo-SCT. One year probability of relapse and LFS were 43+/−4% and 38+/−4% using unrelated donors as opposed to 60+/−6% and 24+/−5% when the donors were HLA identical siblings. In the multivariate analysis, conditioning by Bu or Cy-TBI, age, disease stage or year of transplant had no significant impact on NRM, RI, OS or LFS. In conclusion, the current data suggest that a sequential strategy of intensive chemotherapy, followed by RIC allo- SCT, might represent a step forward in the treatment of refractory AML. Results from the current survey suggest that this strategy might be considered early in the course of a patient with AML not responding to conventional chemotherapy. Controlled prospective studies are warranted and currently being performed (e.g. ClinicalTrials.gov Identifier: NCT01188174). Disclosures: Hallek: Roche: Consultancy, Honoraria, Research Funding.
Human Multiple Myeloma and Breast Cancer Cells Evade Immune Rejection Through Expression of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6.
2012-11-16

Abstract Abstract 2942 Tumor-specific cytotoxic T cells are common in tumor patients, but ineffectively react against autologous tumor cells. Here, we demonstrate in multiple myeloma and breast cancer that human tumor cells escape recognition by tumor-specific CD8+ T cells through carcinoembryonic antigen-related cell adhesion molecule-6 (CEACAM-6) expression. We demonstrate for the first time CEACAM-6 expression in primary and established myeloma and examined the effects of altered CEACAM expression on cytotoxic T cell activity and cytokine secretion against myeloma and breast cancer cells in vitro —, and in vivo, using a xenotransplant mouse model. Cytotoxic T cells from multiple myeloma patients reacted against myeloma antigens presented by dendritic cells, but not against autologous myeloma cells, which expressed CEACAM6. Gene knockdown or blocking of CEACAM6 on myeloma cells restored CD8+ T-cell reactivity against malignant plasma cells. SiRNA-mediated CEACAM6 knockdown or inhibition by specific mAbs also restored cytokine secretion, cytotoxic activity, and antigen-specific lysis of CEACAM6-positive breast cancer cells. Moreover, CEACAM-6 inhibition was a prerequisite for efficient treatment of xenotransplanted breast tumors by adoptive T cell transfer. CEACAM6 thus plays an important role in inhibiting CD8+ T-cell responses against hematological and epithelial human tumors. Therapeutic targeting of CEACAM6 may be a promising strategy for improving cancer immunotherapy. Disclosures: No relevant conflicts of interest to declare.
Immunosuppressive Drugs up-Regulate the Immunoinhibitory Receptor Osteoactivin On Human Monocyte-Derived Dendritic Cells
2012-11-16

Abstract Abstract 3284 Introduction: Dendritic cells (DC) abundantly express the type I transmembrane glycoprotein Osteoactivin (OA) - also known as transmembrane glycoprotein NMB and DC-HIL - compared to low expression levels on monocytes. Antigen-presenting cells interact via OA with the type I transmembrane proteoglycan syndecan-4 (SD-4) on T cells which inhibits T cell activation. We previously reported on increased expression of OA upon exposure of monocyte-derived DC (moDC) to immunosuppressive drugs (e.g., Gutknecht et al ASH annual meeting 2011). Here we extended these analyses and comparatively analyzed the impact of various immunsuppressive drugs (ID) on moDC phenotype and function. Methods: The moDC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Clinically relevant concentrations of ID were added to the culture medium every second day starting with the first day of culture (cyclosporine A: 1μg/ml, prednisolone: 3.5μg/ml, tacrolimus: 10ng/ml, mycophenolat-mofetil 0.1μM, methotrexat 230ng/ml). Cells were harvested for immunophenotyping by flow cytometry, western-blotting and real-time PCR. Cytokine release by moDC was determined on day 7 by ELISA. Functional properties were determined by mixed lymphocyte reactions (MLR) on day 7 of culture. Results: Exposure of moDC to therapeutic concentrations of prednisolone resulted in significantly reduced expression of the costimulatory molecules CD83 and CD86 and increased levels of the monocyte marker CD14, indicative of impaired differentiation. Tacrolimus significantly increased CD14 expression and reduced CD83 expression, while the other ID did not cause significant alterations. All ID altered the release of the immunomodulatory cytokines IL-10, IL-6 and TGF-ß. Notably, all ID except cyclosporine A caused a substantial upregulation of the immunoinhibitory receptor OA in moDC. The extent of OA expression increased over time of exposure to ID during differentiation and resulted in reduced capacity of the moDC to stimulate allogenic T cells which could be restored by disruption of OA/SD-4 interaction using a blocking OA antibody. Conclusion: Increased expression of OA on moDC upon exposure to ID contributes to inhibition of T-cell activation. The mechanisms underlying the differential effect of cyclosporine A are presently under study. Our results indicate that targeting OA/SD-4 interaction may hold promise for modulation of T cell responses in various pathophysiological conditions and immunotherapeutic strategies. Disclosures: No relevant conflicts of interest to declare.
Involvement of NFAT Signaling in Function and Anti-Tumor Reactivity of NK Cells
2012-11-16

Abstract Abstract 1887 NK cells play an important role in anti-tumor immunity. They significantly contribute to the clinical success of allogeneic stem cell transplantation (SCT). Their reactivity as a consequence of an integrative response mediated by various activating and inhibitory surface receptors results in the induction of yet only partially defined signal transduction pathways. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells). While its role in T cell development and function is meanwhile well defined, surprisingly little is known on its function in NK cells. NFAT seems to be dispensable for NK cell development, but several lines of evidence clearly point to its involvement in NK reactivity and function. Cyclosporin A (CsA) and tacrolimus are immunosuppressive drugs that are widely used in transplant medicine. They mediate their immunosuppressive effects through inhibition of the serine/threonine phosphatase calci-neurin, which dephosphorylates and thereby activates NFAT. Here we studied the role of NFAT in NK cells and found that all five NFAT family members are expressed in NK cells with their levels being dependent on NK cell activation state. CsA and tacrolimus, but not mycophenolic acid which mediates its immunosuppressive effects by inhibiting inosine monophosphate dehydrogenase, reduced activation and degranulation of NK cells, resulting in impaired cytotoxicity and IFN-γ production in response to leukemia targets. NK reactivity was also suppressed by the specific NFAT inhibitors VIVIT and INCA-6, indicating that the calcineurin inhibitors CsA and tacrolimus in fact modulate NK reactivity by inhibition of NFAT proteins and not by potential “off target”-effects. These results provide evidence for the critical involvement of the transcription factor NFAT in NK cell reactivity and also indicate that potential effects on NK cell immunosurveillance should be considered upon choice and dosing of immunosuppressive treatment regimens after SCT. Disclosures: No relevant conflicts of interest to declare.