bluuebunny/crossref_metadata_2025 huggingface dataset:
ICSBP Haplo-Insufficiency Synergizes with SHP2-Activation for Apoptosis Resistance and Cytokine Hypersensitivity in Myeloid Progenitors.
Authors: Konieczna Iwona , Horvath Elizabeth , Wang Hao , Platanias Leonidas , Eklund Elizabeth
Published: April 2020

The interferon consensus sequence binding protein (ICSBP or IRF8) is an interferon regulatory transcription factor which functions as a myeloid leukemia tumor-suppressor. In mice, ICSBP-deficiency was found to induce a myeloproliferative disorder (MPD) which progressed to acute myeloid leukemia (AML) over time. This result suggested that ICSBP-deficiency was sufficient for myeloproliferation, but additional genetic lesions were required for differentiation block and therefore AML. ICSBP-deficiency has been described in human myeloid malignancies including myelodysplastic syndrome (MDS), AML and uncontrolled chronic myeloid leukemia (CML). Other previous studies determined that cytokine-induced ICSBP-tyrosine-phosphorylation was required for differentiation-stage-specific target-gene transcription during myelopoiesis. Based on these results, we hypothesized that impaired ICSBP-tyrosine-phosphorylation might exacerbate the impact of ICSBP-deficiency and induce differentiation block and progression from MPD to AML. However, leukemia-associated mutations which impaired tyrosine-phosphorylation had not been described in the gene encoding ICSBP. Therefore, we hypothesized that mutations influencing the molecular pathways involved in ICSBP-post-translational-modification might synergize with ICSBP-deficiency to accelerate progression to AML. SHP2 protein tyrosine phosphatase was previously found to dephosphorylate ICSBP in immature myeloid cells. Additional studies determined that a leukemia-associated, constitutively-active form of SHP2 de-phosphorylated ICSBP through out myelopoiesis. In the current studies, we investigated the ability of constitutive SHP2-activation to synergize with ICSBP haplo-insufficiency to induce cytokine-hypersensitivity and apoptosis-resistance. We also investigated the impact of constitutive SHP2-activation on the tumor suppressor effect of ICSBP. Both ICSBP-deficiency and constitutively active mutants of SHP2 have been described in subjects with therapy related MDS/AML. Also, activity of the bcr/abl kinase induces both SHP2 activation and ICSBP-deficiency. Therefore, our studies identified a molecular mechanism which might be involved in disease progression in these human myeloid diseases.
G-CSF Stimulates Expression of Non-Phosphorylated Stathmin (Oncoprotein, Op18) in Hematopoietic Stem Cells.
Authors: Lannert Heinrich , Able Thomas , Franz Thomas , Leicht Stefan , Wuchter Patrick , Lenze Angela , Eckstein Volker , Saffrich Rainer , Ho Anthony D.
Published: April 2020

Stathmin/Op18 is a cytosolic phosphoprotein which regulates the dynamics of microtubules. This regulation is important in mitosis during cell division and in the migration of cells in modification of the cytoskeleton. In this study we investigated native hematopoietic CD34+ stem cells (HSCs) from BM in comparison to mobilized peripheral blood stem cells (mPBSCs) from G-CSF stimulated healthy donors. All the cell fractions were highly enriched (>99%). In comparative proteome analysis mPBSCs showed high levels of Stathmin compared to native HSCs from BM. We monitored Stathmin by fluorescent-microscopy of bone marrow smears and cytospins. We performed microarray-based gene expression profiles of the aforementioned cells focused on kinases regulating Stathmin’s activity. Western blotting and ion trap mass spectrometer (Bruker) were used to determine the differential phosphorylation. Mononuclear cells were isolated by a standard Ficoll separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions.

Sample preparation: Total RNA was isolated from sorted 1 × 10e6 cells by standard methods using RNA isolation kit (Qiagen). For gene expression analysis topic-defined PIQOR™ stem cell microarrays (936 genes) were performed. Proteomics started with the determination of protein concentrations, 2D-gel-electrophoresis were described in Proteome Works System (BioRad). Sypro ruby and/or coomasie stained gels were used for protein identification after spot cutting by Q-TOF and ion trap mass spectrometer analyses.

The subcellular localization of the identified Stathmin and tubulin were performed by fluorescence and confocal microscopy of all cell fractions. The microarray gene expression correlation shows different regulated genes e.g. kinases: PAK1, MAPK, ATM1, CDKN2A, cdk, PKA, PKCB, PKCD, PKCZ. Stathmin (oncoprotein Op18) is expressed at very high levels in mPBSCs (Q-TOF), and remains non-phosphorylated (western blotting, ion trap mass spectrometry).

Our findings show that G-CSF stimulates Stathmin expression in CD34+ cells from BM and plays a key role in migration into peripheral blood. In microarray analyses we show that Stathmin phosphorylation will be regulated by different kinases in mPBSCs. But Stathmin is highly expressed in mPBSCs and is non-phosphorylated in its active form.
Identification of a Hierarchy of Multipotent Hematopoietic Progenitors in Human Cord Blood.
Authors: Majeti Ravindra , Park Christopher Y. , Weissman Irving L.
Published: April 2020

Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC have been identified, as have downstream lineage-committed progenitors, but not multipotent progenitors. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow, and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. While, the function of the CD90- subpopulations is unknown, the CD90+CD45RA- subpopulation presumably contains HSC. We report here in vitro and in vivo functional studies of these three subpopulations from normal human cord blood. In vitro, CD90+CD45RA- cells formed all types of myeloid colonies in methylcellulose and were able to replate with 70% efficiency. CD90-CD45RA- cells also formed all types of myeloid colonies, but replated with only 33% efficiency. CD90-CD45RA+ cells failed to form myeloid colonies in methylcellulose. In liquid culture, CD90+CD45RA- cells gave rise to all three subpopulations; CD90-CD45RA- cells gave rise to both CD90- subpopulations, but not CD90+ cells; CD90-CD45RA+ cells gave rise to themselves only. These data establish an in vitro differentiation hierarchy from CD90+CD45RA- to CD90-CD45RA- to CD90-CD45RA+ cells among Lin-CD34+CD38- cord blood. In vivo, xenotransplantation of CD90+CD45RA- cells into NOD/SCID/IL-2R?-null newborn mice resulted in long-term multilineage engraftment with transplantation of as few as 10 purified cells. Secondary transplants from primary engrafted mice also resulted in long-term multilineage engraftment, indicating the presence of self-renewing HSC. Transplantation of CD90-CD45RA- cells also resulted in long-term multilineage engraftment; however, secondary transplants did not reliably result in long-term engraftment, indicating a reduced capacity for self-renewal. Transplantation of CD90-CD45RA+ cells did not result in any detectable human hematopoietic cells, indicating that the function of these cells is undetermined. Finally, transplantation of limiting numbers of CD90-CD45RA- cells (less than 100) resulted in multilineage human engraftment at 4 weeks, that was no longer detectable by 12 weeks. Thus, the CD90-CD45RA- subpopulation is capable of multilineage differentiation while exhibiting limited self-renewal ability. We believe this study represents the first prospective identification of a population of human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.
Comprehensive Biomarker and Genomic Analysis Identifies p53 Status as the Major Determinant of Response to MDM2 Inhibitors in Chronic Lymphocytic Leukemia.
Authors: Malek Sami N. , Ouillette Peter , Kujawski Lisa , Shangary Sanjeev , Talpaz Moshe , Kaminski Mark , Erba Harry , Shedden Kerby , Wang Shaomeng , Saddler Chris
Published: April 2020

To understand determinants of sensitivity or resistance to MDM2 inhibitor therapy in chronic lymphocytic leukemia (CLL), we comprehensively analyzed highly pure CD19+ cells derived from 106 CLL patients for response to the MDM2 inhibitors MI-63 and Nutlin3 ex vivo and correlated these responses with clinically important biomarkers, including hierarchical FISH, IgVH-mutations and ZAP70 expression. P53 exons 5–9 were sequenced for all cases, and p53 induction pre- and post-inhibitor treatment was determined by immunoblotting for all 106 samples. Furthermore, we employed high-density 50kSNP-arrays to analyze changes of copy number and allele status, including that of p53 on chromosome 17p, in 171 CLL patients and correlated this finding with p53 mutations and p53 expression. Patient-derived samples were incubated for 36 hours with both inhibitors in parallel and apoptotic (annexin-V) and necrotic cell death (PI) was assayed for all incubations using flow cytometry. IC50 values were calculated using XL-fit. Nineteen CLL cases demonstrated either p53 mutations or aberrant expression and all were relatively resistant to MDM2 inhibitor treatment; eighty-seven cases had functional p53 and all but 3 cases demonstrated sensitivity to MDM2 inhibitor treatment. Very few cases with functional p53 demonstrated IC50 values for both inhibitors that were within the range of p53 mutant cases. Del17p patient samples were resistant to MDM2 inhibitor-mediated cell kill, while all other CLL biomarker-based subgroups appeared equally sensitive. Univariate time to first therapy (TTFT) estimates demonstrated a statistically significant (p=0.02 using the log-rank test) shorter TTFT for the group of 19 CLL cases with aberrant p53 function as opposed to the group of 87 cases with functional p53. Importantly, TTFT analysis for the 87 patients with functional p53 found that patients with low IC50 values (high sensitivity to MDM2 inhibitors) had more aggressive CLL (shorter TTFT), identifying for the first time a susceptible target group for these inhibitors that demonstrates aggressive disease and wt p53. In conclusion, the results of these studies conclusively demonstrate that p53 status is the major determinant of response to MDM2 inhibitors in CLL. Additional defects in the p53 regulatory cascade do not appear operational in this leukemia. This comprehensive dataset provides supportive rationale for the design of early phase human trials using these compounds and supports ex vivo measurements of p53 induction pre- and post-MDM2 inhibitor treatment as part of future trial schemas.
Measurement of Disease Severity in Patients with Sickle Cell Disease: A Systematic Review.
Authors: van den Tweel Xandra W. , van der Lee Johanna H. , Howard Jo , Fijnvandraat Karin ,   (Intr. by Marinus H.J. Van Oers )
Published: April 2020

Background: The clinical picture in sickle cell disease (SCD) is highly heterogeneous. Knowledge of the determinants of a severe disease course will help us to unravel the pathophysiological mechanisms underlying the disease process and provide novel targets for therapeutic interventions. To generate this knowledge well designed etiological studies are needed, using a valid outcome measure for disease severity.

Objective: The aim of this systematic review is to identify all indices used to discriminate SCD patients by their current disease severity and to evaluate the methodological foundations of the indices and utility for clinical research.

Method: We performed a systematic search in MEDLINE (Pubmed) (1966- February 2006) using the search terms: anaemia, sickle cell/ [MeSH]; health status indicators/ [MeSH]; severity; severe and clinical spectrum. Reference lists of relevant studies and reviews were screened to identify additional articles. Information was extracted in duplicate by two independent reviewers (XT, KF).

Results: The Medline search yielded 1346 abstracts from which we selected 91 articles. Reference tracking resulted in 20 additional articles. After evaluation of the full text of these 111 articles, 28 articles (27 in English) were included in the review. These 28 indices contained 50 different items, including age at diagnosis, symptoms, findings at physical examination, laboratory values, organ damage, treatments and socio-economic consequences. The five most frequently used items were painful vaso-occlusive crises (86% of the indices), central nervous system abnormalities (59%), aseptic/avascular necrosis of the bone (52% of the indices), acute chest syndrome (48% of the indices) and leg ulcers (48% of the indices). Many indices (37%) were calculated by adding up scores on individual items without making a difference in the weight of the constituent items. In indices that did differentiate between items by allocating scores differentially, the rationale for weighing of the items was not explained. There were no scores that clearly distinguished devastating complications from complications that leave no sequelae. Most severity indices (89%) were not validated.

Conclusion: In order to increase our understanding of the pathofysiology of SCD and identify potential targets for new therapeutic interventions, we need to identify the determinants of a severe phenotype. For this purpose, consensus on the concept of severity in SCD and an instrument or index to measure it are necessary. This review reveals that there is no consensus on a definition of SCD severity and an index to measure it. Given the current state, efforts should be made to reach international consensus on a definition of SCD severity and a core set of items to measure this concept should be developed.
Low Socioeconomic Status, Not Silent Cerebral Infarcts, Is Associated with Grade Failure in Children with Sickle Cell Anemia.
Authors: King Allison A. ,   the Silent Infarct Transfusion Trial Investigators
Published: April 2020

Background: Among students with sickle cell anemia (SCA), cerebral infarcts are an established risk factor for poor cognition and academic achievement. We tested the hypothesis that in children with SCA, lower socioeconomic status (SES) is associated with grade failure.

Methods: We evaluated the association between the SES as measured by annual income per person in household per year, age of student, gender, medical history and grade failure in children with SCA in 24 sites participating in the Silent Cerebral Infarct Multi-Center Clinical Trial.

Results: A total of 404 students were evaluable. The mean age was 9.2 years (range 6–13), 37% had a silent infarct, 15% failed a grade, and 18% had an Individualized Education Plan. SES was divided in tertiles based on annual income/person in the household/year < $5000, $5000–$9999, > $10,000. After adjustment for covariates, the risk of grade failure was 1.4 (95% CI 1.2, 1.6) for each increasing year of age, and 1.9 (95% CI 1.03, 3.5) for male gender. Children from households of the lowest tertile were 4 times more likely to fail a grade than children from the top tertile. A silent cerebral infarct was not associated with grade failure 1.4 (95% CI 0.8, 2.6). No student with a history of grade failure was hospitalized > 3 times per year over the past three years.

Conclusion: Among students with SCA, poverty is the greatest risk factor for grade failure; whereas the presence of silent cerebral infarct is not. Targeted strategies to improve educational attainment in students with SCA are needed.
Heat Shock Protein Inhibition in Sickle Cell Disease: A Novel Approach to Attenuating the Inflammatory Response?.
Authors: Natarajan Kavita , Catravas John , Cannon Joseph , Li Tao , Kutlar Ferdane , Sloan Gloria , Fields Sabine , Clair Betsy , Kutlar Abdullah
Published: April 2020

Sickle cell disease (SCD) is a genetic disorder resulting from a single amino acid substitution on the β globin chain. It is well established that inflammation is a key component of the protean manifestations and complications of SCD. The heat shock proteins are a group of evolutionarily conserved proteins that are up-regulated in response to cellular stress. However, it has been recognized that an exaggerated response may be detrimental rather than beneficial. In our exploratory study, we look at effects of Hsp90 inhibition in mononuclear cells from controls and patients with SCD after stimulation with lipopolysaccharide (LPS). Whole blood was drawn from 2 African-American controls and 6 patients with SCD in steady state (3 on hydroxyurea (HU) and 3 off HU). Mononuclear cells were separated using Ficoll Hypaque density gradient centrifugation and were washed and suspended in RPMI containing penicillin, streptomycin, and heat inactivated autologous serum. Dilution of the final cell suspension was adjusted to achieve a mononuclear cell count of 400,000/0.5 ml. Each subject’s cell suspension was divided into 4 aliquots: control (V), cells stimulated with LPS (L), and cells treated with heat shock protein inhibitors 17-AAG (A) and Radiciol (R) followed by stimulation with LPS. Subsequently cell suspensions were incubated for 24 hours in a CO2 incubator at 37°C. Supernatants were then extracted and frozen at −70°C. Multiplex cytokine bead assay was performed. No significant change in response to LPS challenge or Hsp90 inhibition was observed for IFNγ, IL-2, IL-4, and IL-5. TNFα and IL-10 showed significant increases with LPS challenge, which could be almost completely inhibited by preincubation with 17-AAG and Radiciol. The magnitude of the TNFα and IL-10 responses to LPS challenge in SCD patients was significantly greater than controls. This held true for patients on and off HU. While there seemed to be a trend towards a more blunted response of TNFα to LPS challenge in HU treated patients, the numbers in this exploratory study are too small to allow statistical comparisons. These data confirm the previous observations that mononuclear cells in SCD are in an activated state and respond to LPS challenge in a more pronounced manner. The increase in IL-10 is more likely the result of a counteractive response to TNFα elevations. Significant observation of this preliminary study is that the inhibition of Hsp90 blunts the TNFα response to LPS challenge and may potentially offer a mechanism of modulation of inflammation in SCD. Determinations of other inflammatory cytokines are currently underway.

Figure Figure
Novel Findings Support a Patophysiological Role of the Arachidonic Cascade in Hodgkin Lymphoma.
Authors: Sjoberg Jan , Schain Frida , Tryselius Ylva , Backman Linda , Porwit Anna , Liu Cheng , Xu Dawei , Wei Wenbin , Baumforth Karl , Murray Paul , Bjorkholm Magnus , Claesson Hans-Erik
Published: April 2020

Lipoxygenases (LO) are a family of structurally related enzymes which catalyze the conversion of the fatty acid arachidonic acid to biologically active metabolites. The key enzyme in leukotriene synthesis, 5- LO, catalyzes the first step in the metabolism of arachidonic acid to leukotriene (LT) C4. This compound and its metabolites LTD4 and LTE4, collectively called cysteinyl leukotrienes (cysLT), can bind to two high-affinity receptors, named cysLT1 and cysLT2. A sister enzyme to 5-LO is 15-LO-1 which also can catalyze the formation of bioactive metabolites. Arachidonic acid metabolites have traditionally been linked to inflammation and asthma but several studies also indicate a role of these metabolites in carcinogenesis In our studies of arachidonic acid metabolism in human lymphomas, we have identified the expression of 15-LO-1 and receptors for cysLT in primary as well as cultured Hodgkin-Reed Sternberg (H-RS) cells. In tumor tissue, H-RS cells positive for 15-LO-1 and the cysLT1 receptor were detected immunohistochemically in 13/15 and 12/16 cases, respectively. The presence of mRNA for 15-LO-1 and the cysLT1 and 2 receptors was confirmed by microarray analysis of laser dissected H-RS cells. Studies of 15-LO-1 gene transcription revealed that STAT6 activation and chromatin remodeling by DNA demethylation and histone acetylation are crucial for transcriptional activation of this gene in cultured H-RS cells. Incubation of the Hodgkin lymphoma-derived cell lines KMH2 and L1236 with LTD4 led to a robust calcium response that was completely abolished in presence of the cysLT1 receptor inhibitor zafirlukast. In L1236 cells, LTD4 induced increased DNA synthesis, interleukin-13 transcription and release of TNF-alpha, interleukin-6 and interleukin-8 in a dose-dependent manner, all of which were inhibited by zafirlukast. Thus, as the H-RS cells lack the expression of 5-LO, we hypothesize that that tumor-associated cysLT-producing inflammatory cells, such as eosinophils and mast cells, contribute to the pathogenesis of Hodgkin lymphoma through induction of tumor cell proliferation and cytokine release. Furthermore, immunohistochemical studies of 20 non-Hodgkin lymphomas revealed the expression of cysLT1 receptors also in primary mediastinal B cell lymphomas (PMBCL), and the functionality of these receptors was confirmed in the PMBCL-derived cell line MedB1. Taken together, these novel findings support a role for arachidonic acid metabolites in the pathogenesis of lymphoma.
Photopheresis in HIV-1 Infected Patients Utilizing (Benzoporphyrin Derivative Verteporfin/BPD-MA) and Light.
Authors: Bernstein Zale P. , Dougherty Thomas , Gollnick Sandra , Schwartz Stanley , Mahajan Supriya , Kepner James , Sumlin Adam , Stewart Carleton , Wallace Paul , Walder Harold , Poiesz Bernard
Published: April 2020

An ex vivo trial utilizing photopheresis with Benzoporphyrin Derivative as the photoactive compound, identified the minimum energy levels of light and concentrations of BPD that eradicated both cell-free and cell-associated HIV-1 infectivity without destroying the virus particles or infected leukocytes. Leukocytes remained viable with altered chemokine/cytokine expression. Apoptosis was induced in a minority of CD4 but not CD8 positive cells with a statistically significant increase in cytolytic T-cell activity. In the 24 week clinical trial in seven HIV-1 infected patients. Three who had rapidly rising viral loads prior to initiating therapy stabilized. Two had a (sustained) greater than .5 log decrement and 5 had stable plasma viral loads (less than a .5 log increment or decrement) with varied effects on absolute CD4 and CD8 positive lymphocytes counts. One achieved a greater than 1 log decrement in HIV-1 plasma viral load and undetectable in vivo cell-free and cell-associated HIV-1 infectivity with an increased in vitro lymphocyte mitogen stimulation index. Under amended protocol 5 additional 12 month courses were administered to three additional patients and two of the previous enrollees. Area under the curve for viral load showed a significant decrease from pre to post therapy (p 0.007). No associated toxicities observed.
Long-Distance Tunneling Nanotubules Shuttle Viral Immunoglobulin Class Switch-Suppressing Factors from HIV-Infected Macrophages to B Cells.
Authors: Xu Weifeng , Santini Paul A. , Shan Meimei , Chen Kang , Ball Susan C. , Chiu April , Kingsley Lawrence A. , Rinaldo Charles R. , Chadburn Amy , Knowles Daniel M. , He Bing , Cerutti Andrea
Published: April 2020

Class switching from immunoglobulin M (IgM) to IgG, IgA or IgE is central to immunity against viruses and requires activation of B cells by CD4+ T cells through CD40 ligand (CD40L). Human immunodeficiency virus (HIV) evades protective T cell-dependent IgG and IgA responses against viral proteins and vaccines through mechanisms that remain unknown. Recent findings from our lab suggested the involvement of negative factor (Nef), an HIV accessory protein that suppresses CD40 signaling in B cells. The present work addressed the mechanism by which Nef invades B cells. Fluorescence microscopy-based studies showed that accumulation of Nef in infected lymphoid follicles was associated with severe loss of activation-induced cytidine deaminase (AID), a Nef-targeted DNA-editing enzyme essential for class switching. Loss of follicular AID correlated with in vivo inactivation of CD40 signaling and formation of long-distance membrane tethers connecting Nef-positive B cells with HIV-infected macrophages. By combining confocal microscopy, videomicroscopy and electron microscopy with standard molecular biology techniques, we demonstrated that macrophages harboring HIV or expressing a chimeric Nef-green-fluorescent protein were capable of transferring Nef-containing cargos to B cells via long-distance, actin-propelled and energy-dependent membrane channels, including tunneling nanotubules. Transfer of Nef to B cells involved a clathrin-dependent pathway that required the guanine nucleotide exchange factor Vav as well as downstream Rho, Rac and Ccd42 small GTPases. This pathway led to Nef-mediated inhibition of CD40-induced AID expression and IgG and IgA class switching. In summary, our findings indicate that HIV evades T cell-dependent IgG and IgA responses by shuttling virus-encoded Ig class switch-suppressing factors from infected macrophages to non-infected B cells via long-range membrane tethers, including tunneling nanotubules.
The Histone Acetyltransferase CBP Is Essential for Conventional T Cell Development.
Authors: Fukuyama Tomofusa , Boussouar Fayçal , Kasper Lawryn H. , van Deursen Jan M. , Brindle Paul K.
Published: April 2020

Defining the epigenetic mechanisms (e.g. chromatin modifications) that underlie T cell fate decisions is a major challenge. The transcriptional coactivators CREB binding protein (CBP) and the closely related p300 comprise a two-member family of histone/protein acetyltransferases that interact with over 50 T lymphocyte-essential transcriptional regulators. Rather than having distinct regulatory roles, CBP and p300 are often thought to confer utilitarian transactivation and histone modifying functions to transcription factors that mediate T cell fate. In contrast to this view, we show here that CBP acts uniquely in conventional T cell development. Inactivation of CBP, but not p300, starting at the double negative stage of T cell development yielded thymocytes with partial activation of an effector/memory- or innate-T cell program. CD8SP thymocytes from CBP mutant mice expressed genes that define professional CD8 cells such as Il-2/Il-15 receptor β chain, granzyme A, interferon γ (Ifnγ), Fas ligand, perforin, and the chemokine receptors Ccr5, and Cxcr3. CD4SP thymocytes from CBP mutant mice also expressed effector genes such as Ifnγ, Il-4, and Ccr5. In addition, CD8SP and CD4SP thymocytes from CBP mutant mice produced Ifnγ protein when the cells were stimulated with phorbol ester and ionomycin. Mechanistically, loss of CBP acted cell non-autonomously to induce the expression of the CD8 T cell master regulatory transcription factor eomesodermin (Eomes). This suggests that CBP in thymocytes or T cells controls an extracellular factor that helps demarcate conventional naïve T cell development in the thymus from effector/memory T cell differentiation in the periphery.
Novel Human CD4+ T Lymphocyte Subpopulations Defined by CD300a/c Molecule Expression.
Authors: Hart Derek N.J. , Rao Min , Ju Xinsheng , Clark Georgina J.
Published: April 2020

CD300a and CD300c are leukocyte membrane glycoproteins encoded by genes in the CD300 complex on human chromosome 17. These molecules function as immunoregulatory molecules that initiate triggering and inhibitory responses following ligation with ligands. The CMRF-35 monoclonal antibody binds to an epitope common to both molecules, expressed on most human leukocyte populations, apart from B lymphocytes and a subpopulation of CD4+ and CD8+ T lymphocytes. We used it to study the distribution of the CD300a and CD300c molecules on peripheral blood CD4+ T lymphocytes and defined novel CD300a/c++ , CD300a/c+ and CD300a/c− CD4+ T lymphocyte subpopulations. Resting peripheral CD4+ T lymphocytes express CD300a mRNA and very low amounts of CD300c. Activation results in an initial decrease in CD300a gene expression before an increase in both CD300a and CD300c gene expression. The upregulated expression of these genes was associated with increased CMRF-35 binding to activated T lymphocytes. The CD300a/c− fraction of CD4+ T lymphocytes proliferated to a greater extent than the CD300a/c++ fraction, in response to mitogens or allogeneic antigen. The poor proliferation of the CD300a/c++CD4+ in response to mitogens was explained by increased apoptosis within this subpopulation. The recall antigen, tetanus toxoid, stimulated the CD300a/c++CD4+CD45RO+ but not the CD300a/c−CD4+CD45RO+ subpopulation. Resting CD300a/c++CD4+ lymphocytes express low levels of IFNγ mRNA. Within 18 hours following in vitro activation, CD300a/c++CD4+ lymphocytes express more IFNγ mRNA and protein compared to the CD300a/c−CD4+ lymphocytes, however, after 24 hours both the CD300a/c+ and CD300a/c− CD4+ T lymphocytes were able to produce IFNγ. Th1 and Th17 effector populations are found within the CD300a/c++CD4+ T lymphocyte population, whilst the Treg population are restricted to the CD300a/c−CD4+ T lymphocyte population. Thus, CD300a/c subdivides CD4+ subpopulations into functional populations with the potential to immunoregulate immune and allogeneic responses. We are currently monitoring these populations in allogeneic haemopoietic stem cell recipients.
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

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

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

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<0.001). According to Cox-regression analysis, 5 parameters were significantly (p<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

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

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

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.