bluuebunny/crossref_metadata_2025_split huggingface dataset:
Correlation of IFNγ, CD107 and CD137 at the Single Cell Level Can Be Used To Monitor T Cell Responses in Patients after Immunotherapy.
Authors: Czerwinski Debra K. , Brody Joshua D. , Levy Ronald 
Published: April 2020

Abstract
As immunotherapies become increasingly important in the treatment of various cancers, monitoring the immune response to reflect the efficacy of the therapy also becomes increasingly important. Previously, tumor antigen-specific humoral responses in patients receiving vaccines for low-grade follicular lymphoma (FL) correlated with clinical outcomes, including tumor regression, molecular remission, progression free survival (PFS) and overall survival (OS). By contrast, T cell immune responses have been difficult to validate. T cell proliferation assays, mostly, measure CD4 T cell responses; whereas, CD8 T cells may be the important effectors generated by immunotherapies. However, assays designed to measure CD8 T cells, i.e. chromium release CTL assays, and IFN-γ ELISPOT and intracellular flow cytometry assays, are difficult to make reproducible. To address this issue, PBL were obtained from FL patients, cryopreserved, and thawed, then used to design a standardized method for detection of intracellular IFN-γ by flow cytometry. The combined stimulus of soluble anti-CD3 and anti-CD28 antibodies provides a robust stimulation, typically about 5% of normal PBL CD8+ T cells respond. By using a panel of irradiated B cell lymphoma cell lines as stimulators, we demonstrated that, on average, 1 – 2% of these T cells were capable of mounting a response in this assay. Surprisingly, CD8+ PBL T cells from several patients with FL were more responsive to combined anti-CD3 and anti-CD28 stimulation as well as to allo-stimulation, 15 – 22% and 2 – 6%, respectively. This response was accompanied by surface expression of CD107, a surrogate marker for CTL degranulation, in the same population of cells as demonstrated by multi-color flow cytometry. Both the IFN-γ and the CD107 responses were inhibited by an anti-class I antibody, W6/32, suggesting a class I restricted T cell receptor-mediated response. Furthermore, at later time points, these T cells also up-regulated CD137 on their surface. This activation molecule is upregulated on CD8 T cells in response to specific antigen recognition and provides an anti-apoptotic signal to the cells. In conclusion, immune competency of CD8 T cells isolated from FL patients can be assessed through allo-stimulation by a panel of B cell lymphoma cell lines. More importantly, correlation by flow cytometry of 3 independent indicators of response (IFN-γ, CD107 and CD137) within single populations of cells to both allo-stimulation and to the specific target, may lead to better understanding of the role of T cells in the immune response. Ultimately, these responses will need to be validated with patient outcomes in clinical trials of vaccines in lymphoma.
Photo-Reactive and Non-Natural Amino Acid Epitopes of Human WT1 Enhance Immunogenicity and Allow Kinetic Study of Antigen Processing.
Authors: Haro Kurtis J. , Gomez-Nunez Marta , Dao Tao , Chau Deming , Won Annie , Escobar-Alvarez Sindy , Zakhaleva Victoriya , Korontsvit Tatyana , Gin David , Scheinberg David A. 
Published: April 2020

Abstract
Wilms tumor protein (WT1) is a transcription factor selectively over expressed in several types of leukemia and solid tumors, making it a promising potential target antigen for immunotherapy. Several open clinical trials use native or altered peptide sequences derived from the WT1 protein in order to overcome the weak immunogenicity of the self-antigen. Here we report a new strategy to circumvent tolerance by designing peptides that incorporate non-natural amino acids into the native sequence of WT1 peptides. Starting from the nonamer sequences WT1 187–195 and WT1 235–243, eight peptides containing natural amino acids and nine peptides in which different chemical modifications (fluorination, photo-reactive azido groups or benzophenone groups) were introduced at major histocompatibility complex (MHC) and T cell receptor binding positions, were synthesized. The new non-natural peptides could stabilize MHC class I molecules better than the native sequences and were also able to elicit strong specific T-cell responses. Photo-reactive peptides were additionally modified with biotin handles to allow streptavidin-biotin pull down and western blot analysis of kinetics of binding and catabolism. Upon UV irradiation, these peptides covalently bound to MHC molecules on the live cells; clearance of the peptide-MHC covalent complex occurred over 24 hours, consistent with the T2 thermo-stabilization data for the same peptide. Further catabolic studies may elucidate the important or novel cellular proteins involved in antigenic peptide processing and cross presentation and should aid in vaccine development. We are investigating whether covalent interaction with the MHC may lead to alterations in immune responses as well. T cells stimulated with one of the synthetic peptides (WT1J-W4WF) cross-reacted with the native WT1J sequence and were able to kill WT1 positive HLA-A0201 matched acute lymphoblastic leukemia cell lines. In conclusion, this study shows that peptides with non-natural amino acids can be successfully incorporated into T cell epitopes to provide increased immunogenicity and novel biological information.
Differences in Survival Rates for Patients with Hodgkin Lymphoma, Who Were Treated Inside vs. Outside Therapy Optimisation Protocols in Germany.
Authors: Brillant Corinne , Terschueren Claudia , Gierer Sylke , Franklin Jeremy , Heidelbach Stephanie , Paulus Ursula , Diehl Volker , Hoffmann Wolfgang 
Published: April 2020

Abstract
Introduction: Despite the improvements and knowledge brought by clinical trials to cancer treatment, it remained unclear how beneficial participation in a clinical trial with Therapy Optimisation Protocol (TOP) is for patients (pts) with Hodgkin Lymphoma (HL) in relation to pts treated outside of trials. In the TOPiCS project, trial participants (TOP) were compared with non-trial pts (non-TOP).
Methods: In the population-based survey NLL, which aimed to register all incident HL-cases 356 pts were recorded in six counties of northern Germany with a first diagnosis of HL in 1988–1998. Data on staging, therapy, adverse events and survival were collected. The dataset was additionally screened for patients fulfilling inclusion and exclusion criteria for clinical trials of the German Hodgkin Study Group (GHSG). A total of 328 pts were documented of whom 198 pts (60%) met the inclusion criteria of the GHSG. Of these, 125 pts (63%) have not been recruited into GHSG trials (non-TOP pts). They were compared retrospectively with 4963 TOP pts randomised nation-wide between 1988–1998 in the GHSG trials HD4-HD9. Endpoints were Overall Survival (OS) and Progression Free Survival (PFS) which considered progression, relapse and death of any cause as events. Survival analysis was performed using Kaplan-Meier method and log-rank tests. Cox regression analysis was used for multivariable modelling of risk of death or progression and included 118 non-TOP pts and 4958 TOP pts.
Results: The demographic parameters were not well balanced between the two groups: TOP pts were younger, had more often advanced stage and diagnosis in the later study generation than the non-TOP pts. The median observation time for OS was 7 yrs for the TOP group and 10 yrs for the non-TOP group. The 5-yrs OS for TOP pts is 89% (95%–CI [88–90]) and for non-TOP 89% (95%-CI [82–94])(p=0.63). The 5-year PFS for TOP pts is 79% (95%–CI [78–80]) and for non-TOP pts 68% (95%–CI [59–76])(p&lt;0.001). According to Cox-regression analysis, 5 parameters were significantly (p&lt;0.01) associated with poor OS and PFS: male sex, older age, advanced stage (according to DHSG classification considering Ann Arbor stage, B-symptoms and risk factors) and earlier study generation. Participation in a TOP-trial did not contribute independently for OS (Hazard Ratio (HR)=1.12, 95%–CI [0.71–1.79]) but contributed independently and positively for PFS (HR=0.66, 95%–CI [0.49–0.89]). The difference is mainly due to a higher number of relapses.
Conclusion: For German patients fulfilling inclusion and exclusion criteria for TOP, a participation in therapy optimisation protocols results in a higher PFS, allowing for the influence of other factors, than for patients who were treated outside of TOP-trials. However, no difference was observed in OS.
Bifunctional siRNA Design Targeting Non-Hodgkin’s Lymphoma.
Authors: Höhn Britta , Soifer Harris S. , Forman Stephen J. , Rossi John J. 
Published: April 2020

Abstract
Non-Hodgkin’s lymphomas comprise a group of heterogeneous lymphoid malignancies that represent the fifth most common form of cancer in the United States. A hallmark of many types of B-cell lymphomas is the constitutive expression of oncogenes such as the transcription factors Bcl-6, STAT3 and c-Myc and the anti-apoptotic protein Bcl-2. Over expression of these genes causes uncontrolled proliferation and survival of malignant cells, making knockdown of these genes by RNA interference (RNAi) a rational strategy for therapeutic intervention. RNAi is a conserved endogenous mechanism in which small interfering RNAs (siRNAs) suppress target-specific gene expression by promoting mRNA degradation. We have designed potent Dicer-substrate siRNAs using different computer algorithms to predict accessible target sites in the mRNAs of B-cell lymphoma oncogene targets. The Dicer-substrate 27mers are designed asymmetrically, so that Dicer processing yields a predicted 21mer siRNA duplex for entry into RNA induced silencing complex (RISC). Dicer-substrate siRNAs show improved efficacy at lower concentrations compared with conventional 21mer siRNAs, suggesting the reduction of potential off-target effects. In addition, we have designed bifunctional siRNA duplexes that contain two fully target-complimentary antisense strands against two different target mRNAs, but that are only partially complementary to each other. In vitro cleavage assays indicate that our bifunctional siRNAs have sufficient complementarity to form stable duplexes and can be processed into smaller molecules by recombinant Dicer. When delivered to the Burkitt’s lymphoma cell line Raji by electroporation, the most effective siRNAs reduced target mRNA levels by ∼80% as determined by quantitative RT-PCR and immunoblot analysis. Silencing of transcription factors affected the expression of downstream target genes, indicating a relevant effect on growth on survival of lymphoma cells through oncogene down regulation by RNAi. One concern with RNAi-mediated therapy is the possible recognition of siRNA duplex by cell’s own response to double-stranded RNA (dsRNA) that could trigger an unwanted interferon response. To determine whether our Dicer-substrate siRNAs cause an interferon response, we monitored induction of the cellular dsRNA pathway by measuring gene expression of p56, OAS1 and interferon by quantitative RT-PCR after transfection of siRNAs in different cell lines. None of the analyzed siRNAs show a significant increase in the expression of interferon pathway related genes indicating that our selected siRNAs are powerful silencers of gene expression without inducing an interferon response. In future studies, these new identified siRNAs will be incorporated in nanoparticles or attached to antibodies / aptamers for cell-specific delivery to lymphoma cell lines to evaluate their potential alone or in combination with chemotherapeutic drugs in therapy for lymphoma.
Expression of a Glyco-Optimized Anti-CD20 Antibody in the Aquatic Plant Lemna with Enhanced ADCC Activity.
Authors: Gasdaska John R. , Sterling Jason D. , Regan Jeff T. , Cox Kevin M. , Sherwood Steven W. , Dickey Lynn F. ,   (Intr. by Klaus-Peter Radtke)
Published: April 2020

Abstract
Monoclonal antibodies (mAbs) are one of the fastest growing therapeutics in the drug sector. For many antibodies, the structure and extent of the N-glycans on the Fc region of the H-chain plays a significant role in their therapeutic function. A glyco-optimized anti-CD20 antibody (rituximab) was expressed in the clonal aquatic plant Lemna. The optimized glycosylation was accomplished by co-expressing an interfering RNA (RNAi) construct targeting the endogenous alpha-1,3-fucoslytransferase (FucT) and beta-1,2-xylosyltransferase (XylT) genes (Cox et al., 2006). The resulting glyco-optimized rituximab contained a single major G0 N-glycan without any detectable xylose or fucose. In cell-based functional assays, the glyco-optimized rituximab showed similar CD20-binding affinity and apoptotic effects as Rituxan® produced in mammalian cells but with significantly enhanced (up to 100-fold) antibody-dependent cellular cytotoxicity (ADCC). Using FACS based methods, comparable CD20-binding was demonstrated in the B-cell lines Raji, Daudi and Wil2S. Apoptotic measurements used Raji and Daudi cells. In the treatment of non-Hodgkin’s B-cell lymphoma (NHL), the patient response rate for Rituxan is only 50–60% and is significantly correlated with a FcgRIIIa receptor polymorphism (Cartron et al., 2002). However, the glyco-optimized rituximab showed enhanced ADCC with effector cells from all FcgRIIIa-158 genotypes. ADCC activity was determined by FACS-based methods using Raji as the target cells. PBMC genotyping was accomplished by PCR analysis (Koene et al., 1997). Up to a ten-fold decrease in complement dependent cytotoxicity (CDC) was also observed using Raji cells. This functional profile may offer the potential for an optimized anti-CD20 antibody therapeutic with improved efficacy and potency while simultaneously decreasing the side effects associated with CDC activity (Clark and Ledbetter, 2005).
Non-Clinical Pharmacology of Anti-CD22 Antibody Drug Conjugates.
Authors: Xie Dong , Baudys Jakub , Chan Pam , Deng Rong , Dere Randy , Ebens Allen , Fielder Paul , Fuh Franklin , Fuji Reina , Gray Alane , Huang Catherine , Jumbe Shasha , Makeever Kathleen , Saad Ola , Stephan Jean-Philippe , Tibbitts Jay , Williams Marna , Yu Shang-Fan , Prabhu Saileta 
Published: April 2020

Abstract
CD22 is a B-cell specific antigen expressed on both normal and malignant cells. It is internalized rapidly upon binding to its antibody, making it an ideal target for immunotoxin therapy. Anti-CD22 is a humanized monoclonal antibody that recognizes human and cynomolgus monkey CD22 but not the rodent CD22. When conjugated with mitotic inhibitors such as DM1 (a maytansinoid) or monomethylauristatin F (MMAF) as anti-CD22-MCC-DM1 or anti-CD22-MC-MMAF, the conjugates have been shown to regress tumors completely after a single IV bolus in a variety of xenograft mouse models. Pharmacokinetics (PK), pharmacodynamics (PD), efficacy and safety studies were conducted to explore the non-clinical pharmacology of the two anti-CD22 conjugates (ADC). Dose response of anti-tumor activity, characterized in the Bjab-Luc xenograft model, yielded an ED50 of ∼1 mg/kg for MC-MMAF. MCC-DM1 was about 2-fold less potent compared to MC-MMAF in this model. Anti-CD22 antibody demonstrated very limited antibody dependent cell mediated cytotoxicity and no complement dependent cytotoxicity in vitro. Antigen-independent pharmacokinetics were assessed in mouse (single IV bolus, 0.5 or 5 mg/kg) and rat (single IV bolus, 10 mg/kg), while the effect of target mediated disposition was studied in cynomolgus monkey (two IV bolus with three weeks apart, vehicle, 10, 20 or 30 mg/kg). Conjugation with DM1 or MMAF appeared to have minimal impact on the PK of anti-CD22 when the PK of naked anti-CD22 was compared with the total antibody PK profile in all three species. Pharmacokinetic parameters of both total antibody and ADCs were generally dose proportional at the dose range studied. Free DM1 in plasma increased with dose, while free MMAF was not detected. Safety and PD were also characterized in the cynomolgus monkey. Both ADCs were well tolerated up to 30 mg/kg through out the entire study period (42 days). For PD, B-cell depletion was analyzed via FACS. Substantial decreases of CD20+ B-cells were observed in peripheral blood at all dose levels. In tissues, reduction of CD20+ B-cells was observed in spleen, bone marrow and in the lymph nodes, although to a lesser extent. No anti-drug antibodies were detected in the cynomolgus monkey at the doses tested. Based on the preclinical data, human efficacious dose and therapeutic window were estimated to be ∼2–5 mg/kg and 5–10, respectively. In summary, both ADCs exhibit an encouraging nonclinical efficacy, pharmacokinetic, pharmacodynamic, and safety profile that supports clinical development for the potential treatment of NHL.
Tyrosine Kinase Inhibitors Dasatinib, Nilotinib and Imatinib Have an Impact on Both CD8+ T Lymphocytes and CD4+CD25+FoxP3+ Regulatory T Cells by Downregulation of the NF-κB Pathway.
Authors: Fei Fei , Yu Yingzhe , Schmitt Anita , Chen Jinfei , Chen Baoan , Rojewski Markus , Ringhoffer Mark , von Harsdorf Stefanie , Greiner Jochen , Goetz Marlies , Guillaume Philippe , Dohner Hartmut , Bunjes Donald , Schmitt Michael 
Published: April 2020

Abstract
Background: For CML/ALL patients with refractory disease or at relapse after allogeneic stem cell transplantation (allo-PBSCT), one might administer tyrosine kinase inhibitors (TKIs) like imatinib (Glivec), nilotinib (Tasigna) and dasatinib (Sprycel), or donor lymphocyte infusions (DLIs). TKIs will inhibit the proliferation of CML progenitor cells, but might also hamper the graft-versus-leukemia (GVL) effect considered to be crucial for the eradication of the disease. Moreover, CD8+ T cells specific for cytomegalovirus (CMVpp65) might be impaired.
Methods: Imatinib, nilotinib and dasatinib were added at concentrations of 0–20 μM, 0–4 μM and 0–50 nM respectively to proliferation assays of Tregs and CD8+ T cells. Mixed lymphocyte peptide cultures (MLPCs) were performed with peptides derived from
influenza matrix protein (IMP), CMVpp65 as a viral antigen and the receptor for hyaluronic acid mediated motility (RHAMM-R3) as a leukemia-associated antigen.
CD8+ T cells from these MLPCs from healthy donors and patients with CML after allo-PSCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as Tregs after 3 days of culture with anti-CD3 and anti-CD28. Activity of T cells from the peripheral blood of patients under dasatinib and after withdrawal of dasatinib. Western blots (WBs) for T cell receptor (TCR) related molecules and NFkB were performed.
Results: The release of interferon gamma and granzyme B by CD8+ HLA-A2/tetramer+effector T cells specific for IMP, CMVpp65 and RHAMM-R3 was inhibited by all TKIs in a dose-dependent fashion and correlating to the time of TKI exposure. The inhibition was reversible after removal of the drugs from the MLPC. The proliferation and function of Tregs were also significantly inhibited by TKIs in a dose-related fashion. In WBs, TKIs decreased the expression of ZAP70, Lck and Akt, as well as NFκB p65/p100/p105 and c-Rel. T cell activity was impaired when TKIs were clinically administered. The potency of T cell inhibition was imatinib: nilotinib: dasatinib = 1:2:40 under therapeutical serum respectively culture medium levels (2 μM:1 μM: 25 nM).
Conclusion: When administering TKIs to patients after allo-PBSCT, the inhibition of both CD8+ and Tregs must be taken into consideration with respect to GVL and anti-viral T cell response. Inhibition of CD8+ T lymphocytes was partially reversible after removal of TKIs from the cultures. Using western blotting analysis, we found that these effects are mediated at least in part by down-regulating the levels of phosphorylation of TCR and NF-κB family members.
Frequent CD7 Antigen Loss in Aggressive Natural Killer-Cell Leukemia Can Be a Useful Diagnostic Marker.
Authors: Kim Sun-Hee , Yoo Eun-Hyung , Kim Hee-Jin , Kim Won Seog 
Published: April 2020

Abstract
Aggressive natural killer-cell leukemia (ANKL) is a rare neoplasm characterized by systemic proliferation of NK cells with rapidly progressive clinical course and fatal outcome. Because of the aggressive clinical course, rapid and accurate diagnosis of ANKL is critical. However, the differential diagnosis of NK cell lymphoproliferative disorders including hemophagocytic lymphohistiocytosis is still challenging in the absence of a distinct diagnostic hallmark. Furthermore, cases with a low burden of malignant cell polpuation makes it more difficult. To find any diagnostic markers in ANKL, we analyzed clinical data and laboratory findings from bone marrow studies in Korean patients with bone marrow involvement of ANKL. From January 2000 to July 2007, a total of 20 cases were diagnosed with ANKL based on morphologic and immunophenotypic findings from bone marrow studies. The leukemic cells were surface CD3–CD16/56+ large granular lymphocytes with pale or lightly basophilic cytoplasm containing azurophilic granules. We retrospectively analyzed clinical features and laboratory findings including complete blood count (CBC), Epstein-Barr virus (EBV) status, serum lactate dehydrogenase (LDH) level, immunophenotype, and cytogenetic results from medical records. There were 6 (30%) women and 14 (70%) men with a median age of 44 years (range, 2–70 years). Hepatomegaly (70%), splenomegaly (60%), and lymphadenopathy (30%) were frequently observed. Peripheral blood counts were variable; anemia (hemoglobin &lt;10g/dL) was predominant in 14 patients and thrombocytopenia (platelet &lt;100×109/L) in 16. The proportion of leukemic NK cells ranged 3∼70%. EBV was detected in 15 of 18 cases (83%) by EBV in situ hybridization or EBV quantitative PCR. Cytogenetic studies were performed in 18 cases, and karyotypic abnormalities were observed in 50% (9/18). There were no recurrent cytogenetic abnormalities, except 6q abnormalities observed in 4 cases (4/18, 22%). The immunophenotype of the leukemic NK cells by flow cytometry was cytoplasmic CD3+, surface CD3−, CD16/56+, CD2+, and CD5−. Most cases were CD4− (13/16, 81%) and CD8− (11/14, 79%). Of note, loss of CD7 antigen was observed in 10 patients (10/20; 50%) (normal NK cells: CD2+, CD7+, and CD5−). There were no significant differences in clinical or laboratory parameters between the CD7+ and CD7− groups. All three cases with deletion of 6q revealed absent expression of CD7. When the CD7 loss was combined with cytogenetic abnormalities, clonal markers could be identified in 75% of ANKL cases. We observed frequent CD7 antigen loss in our series of Korean patients with ANKL. This characteristic immunophenotypic finding can provide a reliable and timely information as a diagnostic marker in ANKL along with cytogenetic findings. Therefore, immunophenotypic analysis of the expression of CD7 should be included in the diagnostic workup of NK cell neoplasms.