bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Human CD34(+) Subset That Constitutively Expresses the High Affinity Interleukin-2 Receptor Traffics to Lymph Nodes and Differentiates into CD56Bright Natural Killer Cells.
Authors: Freud Aharon G. , Becknell Brian , Roychowdhury Sameek , Mao Hsiaoyin C. , Ferketich Amy K. , Nuovo Gerard J. , Hughes Tiffany L. , Marburger Trent B. , Sung John , Baiocchi Robert A. , Guimond Martin , Caligiuri Michael A.
Published: October 2019

In adult humans, T cells differentiate in the thymus and B cells develop in the bone marrow, but the site(s) of natural killer (NK) cell differentiation are unclear. Here we describe, for the first time, a unique CD34(+) population found in human lymph nodes (LN) that differentiates into NK cells. CD56bright NK cells represent <10% of NK cells in peripheral blood (PB) yet predominate in LN where they can compete for endogenous T cell-derived IL-2 during immune activation due to their unique expression of functional high affinity (HA) interleukin (IL)-2 receptors (IL-2R). We hypothesized that a subset of CD34(+) hematopoietic precursor cells (HPC) might also express functional HA IL-2R and potentially differentiate into CD56bright NK cells via activation with low dose IL-2. We first identified a novel human CD34dimCD45RA(+) HPC in PB with constitutive expression of the HA IL-2R. When cultured in picomolar concentrations of IL-2 that selectively saturate the HA IL-2R, these cells give rise to CD56bright NK cells, and this effect is blocked when IL-2 cannot bind to its HA receptor. This unique CD34(+) population expresses IL-2Rα, CD2, CD7, c-kit, L-selectin, and NKR-P1A, all of which are also expressed by CD56bright NK cells. Unique among total PB CD34(+) cells, this novel population displays high integrin α4β7 expression. This attribute, in addition to its high L-selectin expression, suggested that these cells may traffic to LN where their progeny, CD56bright NK cells, represent the major NK subset. Indeed we found a distinct CD34dimCD45RA(+)α4β7bright population that resides in the T cell rich regions of human LN, and when stimulated in vitro with 10 pM IL-2, this cell gives rise to CD56bright NK cells. This novel population represents only ~6% of all PB CD34(+) HPC yet is the major if not exclusive CD34(+) subset in LN. While murine studies strongly support the notion that most if not all NK cells require IL-15 for their development, these new human data suggest a model for development of a minor human NK subset, the CD56bright NK cells, whereby CD34dimCD45RA(+)α4β7bright HPC constitutively expressing the HA IL-2R traffic to peripheral LN where endogenous T cell-derived IL-2 can drive CD56bright NK cell differentiation in vivo.
Cytomegalovirus pp65 - Recombinant Protein: A New Antigen for Efficient Restimulation of CD4+ and CD8+ pp65-Specific T Lymphocytes.
Authors: Richter Anne , Marschall Patricia , Mohn Marie , Odenthal Uwe , Gösling Silke , Skopnik Manuel , Nölle Volker , Assenmacher Mario
Published: October 2019

Short-term restimulation assays combined with the analysis of effector function, in particular the detection of cytokine production, are useful tools for the analysis and isolation of antigen-specific T cells. Until now, restimulations with soluble protein antigens failed to efficiently reactivate CD8+ T cells. We have developed a recombinant protein of the immunodominant cytomegalovirus (CMV) matrix protein pp65 for in vitro restimulation of pp65-specific CD4+ as well as CD8+ T cells. The efficiency of the CMV pp65 - Recombinant Protein to reactivate pp65-experienced CD4+ and CD8+ T cells and the specificity of the restimulated T cells were analysed.

PBMC from CMV seropositive donors were restimulated with CMV pp65 - Recombinant Protein or a complete pool of overlapping pp65 peptides. Afterwards T cells were analysed for intracellular IFN-γ production by flow cytometry. Interestingly, we observed that stimulation with CMV pp65 - Recombinant Protein results in IFN-γ production in CD4+ as well as CD8+ T cells with frequencies comparable to that using the peptide pool as antigen (n=17). In contrast, upon stimulation of PBMC from CMV seronegative donors with CMV pp65 - Recombinant Protein neither IFN-γ nor TNF-α were detectable in T cells (n=6).

Furthermore, we tested the specificity of CMV pp65 - Recombinant Protein-reactive CD4+ and CD8+ T cells. Therefore, IFN-γ-producing T cells were magnetically isolated after short-term stimulation with pp65 using the IFN-γ cytokine secretion assay and expanded for 7 days. Subsequently, the isolated and expanded CD4+ and CD8+ T cells were restimulated with pp65 peptide pool. More than 80 % of the CD4+ and CD8+ T cells produced IFN-γ and more than 80 % of the CD8+ T cells were positively stained with MHC class I/pp65 tetramers.

These results demonstrate that CMV pp65 - Recombinant Protein efficiently and specifically reactivates pp65-experienced CD4+ as well as CD8+ T cells. Therefore, CMV pp65 - Recombinant Protein is a useful antigen for the detection and isolation of pp65-experienced CD4+ and CD8+ effector/memory T cells.
Dependence of Vector Dose and Age of Mice in the Induction of Immune Response Against Factor VIII Following Liver-Directed Nonviral Gene Transfer.
Authors: Ye Peiqing , Miao Carol H.
Published: October 2019

Formation of inhibitory antibodies to transgene product may limit the success of gene therapy especially for the treatment of hemophilia A. The risk of forming inhibitory antibodies against factor VIII depends on multiple factors. Previously we have shown that following naked gene transfer of fifty micrograms of a liver-specific, high-expressing factor VIII plasmid, pBS-HCRHPI-FVIIIA into hemophilia A mice (at least 60 days old), a robust humoral response was induced two weeks post plasmid injection despite of initial high-level gene expression of factor VIII (Ye et al. (2004) Mol. Ther. 10, 117–126). This response completely inhibited the activity of circulating factor VIII although factor VIII was persistently expressed in the liver. In this study, the cytokine production was characterized in human factor VIII-activated T cells from plasmid-treated and untreated hemophilic A mice, consistent with a response predominantly mediated by Th2-induced signals. Injection of plasmid DNA into 4 groups of hemophilia A mice (n=5, 60 days old) with 4 different doses (0.4, 2, 10, & 50 microgram per animal) resulted in vector dose-dependent expression of factor VIII. In addition, the two groups of mice with lower doses of plasmid DNA (0.4 & 2 microgram per animal) did not elicit any antibody response against factor VIII, whereas the two groups of mice with higher doses of plasmid DNA (10 & 50 microgram per animal) induced inhibitory antibody formation. Nevertheless, when the two groups of animals (n=4) with lower doses were treated with second injection of fifty microgram of factor VIII plasmid 180 days post plasmid delivery, all mice developed inhibitors suggesting no immune tolerance was induced by first injection of plasmids. Furthermore, fifty micrograms of factor VIII plasmids were injected into 4 groups of hemophilia A mice (n=5) of 4 different age groups (36, 48, 60 & 72 days). It was found that none of the mice with age 36 days at the time of plasmid injection developed inhibitors, 1/5 mice with age 48 days developed inhibitors, whereas the two groups of mice with age 60 & 72 days all developed high-titer inhibitors. These results indicate that induction of anti-factor VIII antibody following gene therapy is strongly dependent upon the vector dose and age of the animals, which has important implication for developing immunomodulation or other strategies to avoid/eliminate antibody responses.
Isolation of Human CD34- Cells with High Aldehyde Dehydrogenase Activity Reveals a Novel Population with Hematopoietic Repopulating Potential.
Authors: Hess David A. , Herrbrich Phillip E. , Wirthlin Louisa , Craft Timothy P. , Nolta Jan A.
Published: October 2019

The successful development of stem cell-based therapies requires a thorough understanding of human hematopoietic stem cell (HSC) populations. Human CD34− cells engraft NOD/SCID mice with low efficiency by intravenous (IV) transplant. However, intra-femoral injection into immune deficient mice has identified potent human repopulating cells from CD34+ and CD34− subfractions. We recently described a novel strategy to purify reconstituting HSC from human umbilical cord blood (UCB) by lineage depletion (Lin−) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. Hematopoietic progenitor function and in vivo reconstituting ability were exclusively maintained within the ALDHhiLin− population, which demonstrated variable expression of CD34. Here, we compared the repopulating ability of purified CD34+ALDHhiLin− and CD34−ALDHhiLin− populations to traditionally isolated CD34+Lin− and CD34−Lin− cells. Sorting of Lin− cells from human UCB isolated CD34−ALDHhi and CD34+ALDHhi cells (>96% purity) at an overall frequency of 4.4±1.3% or 29.1±3.5%, respectively. In contrast to CD34−Lin− cells, ALDHhiCD34−Lin− cells demonstrated robust clonogenic progenitor function in vitro (1 CFU in 9 cells, n=3), and total colony production was further increased in ALDHhiCD34+Lin− cells (1 CFU in 4.5 cells, n=4) (p<0.05). Human hematopoietic repopulation was consistently observed in the bone marrow (17.2±4.2%), spleen (0.8±0.2%), and peripheral blood (0.7±0.3%) of NOD/SCID β2M null mice 6–8 weeks after IV transplant with 103–104 purified ALDHhiCD34+Lin− cells (n=14). Similarly, intra-femoral injection (IF) of ALDHhiCD34+Lin− cells resulted in robust human repopulation (n=5). IV injection of equivalent doses of either ALDHhiCD34+Lin− or CD34+Lin− cells showed similar levels and frequencies of human hematopoietic engraftment. Repopulating ALDHhiCD34+Lin− cells also differentiated into cells expressing markers for mature myeloid (CD33, CD14), B-lymphoid (CD19, CD20) cells and primitive repopulating cells (CD34+CD38−) at similar frequencies as CD34+Lin− cells (n=5). IV injection of 2x104–1x105 ALDHhiCD34−Lin− cells engrafted at 0.2–0.3% in the BM of 3 of 4 NOD/SCID β2M null mice, whereas IV injection of up to 4x105 CD34−Lin− cells produced no detectable human engraftment (n=6). IF-injected ALDHhiCD34−Lin− cells engrafted the injected bone in 2 of 3 NOD/SCID mice at low levels and did not efficiently migrate to the non-injected femur or tibiae. In summary, the human UCB ALDHhiLin− population includes both CD34+ and CD34− cells capable of bone marrow homing and hematopoietic reconstitution. Therefore, isolation of CD34− cells based on high ALDH activity may reveal a novel population of hematopoietic stem and progenitor cells.
The Role of CBFβ in T Cell Development.
Authors: Zhao Ling , Cannons Jennifer L. , Castilla Lucio H. , Schwartzberg Pamela L. , Liu Pu Paul
Published: October 2019

Core binding factor β (Cbfβ) is a transcription factor that heterodimerizes with Runx (Cbfα) family members, thereby stabilizing the interaction between the Runx proteins and DNA. Genetically manipulated mouse models of Runx and Cbfb genes have demonstrated their critical functions in hematopoiesis (Runx1, Runx3 and Cbfb), bone formation (Runx2, Cbfb), proliferation of gastrointestinal epithelia (Runx3) and differentiation of dorsal root ganglion cells (Runx3). Studies on T cell development showed that Runx1 and Runx3 repress CD4 expression at different stages of development. In addition, Runx 1 and Runx 3 are required for CD8 T cell development during thymopoiesis. No defects were found when Runx2 was inactivated, even though it is expressed throughout T cell development.

We have previously generated a knock-in mouse model expressing the Cbfb-MYH11 fusion gene (which is created by inv(16)(p13; q22) in human AML M4Eo). Heterozygous knock-in mice had a phenotype identical to that of the Cbfb and Runx1 null mice (embryonic lethality), suggesting that the fusion gene Cbfb-MYH11 functions in a dominant-negative manner. In order to study the function of Cbfb gene in T cell development, we used a mouse line with floxed exons 5 and 6 of Cbfb inserted 5′ to the Cbfb-MYH11 fusion cassette, which produced pseudo-normal mice (loxKI). By crossing the loxKI mice with mice expressing the Cre gene under the control of the T cell-specific Lck promoter (LckCre), we generated LckCre-loxKI double positive mice, in which the floxed exon 5 and 6 were deleted and Cbfb-MYH11 re-expressed only in the thymus when Lck started to express. The LckCre-loxKI mice were viable. However, their thymic development was severely impaired: The size of the thymuses in the mutant mice was about half the normal size, and the total number of thymocytes in the mutant mice was 10–20-fold reduced. FACS analysis of thymocytes from 4 to 12 week old mice showed a developmental blockade at the CD4/CD8-double negative (DN) stage, which was characterized by lower percentage of double positive cells and higher percentage of double negative cells. In addition, the CD4: CD8 ratio was altered. Furthermore, the mature T cell population size in the spleen of the mutant mice was lower than that of the control mice. Our preliminary data suggested that Cbfb plays an important role in T cell development. The mechanism through which Cbfb affects the T cell development is currently under investigation. It is likely that the phenotype reflects the combined effect of missing all three Runx genes, since the phenotype described here is more severe than either Runx1 or Runx3 null alone.
EBV Transformation of Tonsil Germinal Centre B Cells Carrying Functionally Inactivated IgV Gene.
Authors: Chaganti Sridhar , Pastor Noelia Begue , Drayson Mark T. , Bell Andy I. , Rickinson Alan B.
Published: October 2019

Somatic hypermutation of immunoglobulin (Ig) gene sequences in the germinal centres of lymphoid tissues is necessary for affinity maturation of B cell responses to antigen challenge. This process generates a few clones with improved affinity that are selected into B cell memory and many clones with other non favourable Ig mutations, including some cells with functionally inactivated Ig gene that normally die by apoptosis. It is postulated that infection with Epstein-Barr virus (EBV), a B lymphotropic agent linked to several types of B cell lymphoma, can rescue germinal centre cells with unfavourable mutations. This creates a pool of infected cells at greater risk of developing into lymphomas. In the present work, CD38+ germinal centre B cells were separated from tonsil by negative selection for IgD and CD39. Peripheral blood naïve and memory B cell subpopulations were FACS sorted as IgD+, CD27− and IgD−, CD27+ fractions respectively. These cells were infected with EBV (B95.8 strain) in vitro and seeded at limiting dilutions onto fibroblast feeders. EBV transformed lymphoblastoid cell lines (LCLs) from such cultures were analysed for surface Ig phenotype. Naïve B cell transformants were consistently IgM+, IgD+. Memory B cell transformants were IgM+ in some cases but more frequently IgG+ or IgA+. Germinal centre transformants showed the same spectrum of surface Ig phenotypes as memory cell transformants but in addition we identified six germinal centre derived LCLs which were consistently surface Ig negative. Sequencing from these lines confirmed that in at least three cases EBV had rescued cells with functionally inactivated Ig heavy chain gene.
Fc-Gamma Receptor IIIa and Interferon-Gamma SNPs Do Not Predict Responsiveness of Follicular Lymphoma to Galiximab (Anti-CD80 Antibody).
Authors: Czuczman Myron , Leigh Bryan R. , Witzig Thomas E. , Vose Julie M. , Younes Anas , Alkuzweny Baha , Larocca Anne , Cao Xianjun , Thall Aron
Published: October 2019

CD80 is an immune costimulatory molecule expressed on the cell surface of follicular and other lymphomas. Galiximab, a macaque-human chimeric anti-CD80 monoclonal antibody, has been shown to have antitumor activity in patients with relapsed or refractory, follicular lymphoma. Preclinical work suggested antibody-dependent cellular cytotoxicity (ADCC) as a mechanism for galiximab-induced cell lysis. Single-nucleotide polymorphisms (SNPs) for high-affinity Fc gamma receptor IIIa and high interferon-gamma production have been associated with increased ADCC activity. For this report, genomic DNA from peripheral blood of clinical study patients was analyzed for Fc gamma receptor IIIa and interferon-gamma gene SNPs after PCR amplification. In addition, the frequency of interferon-gamma secreting cells in peripheral blood samples was evaluated by ELISPOT. SNP and ELISPOT results were then evaluated for correlation with clinical response (CR or PR) to galiximab and reduction in tumor burden (SPD of indicator lesions). Of 35 follicular lymphoma patients evaluable for response to galiximab, 2 CRs and 1 PR were observed; time to best response was delayed (Months 3, 9, and 12). Of 34 patients with Fc gamma receptor IIIa SNP results, 6 were homozygous for the high-affinity receptor. Of 33 pts with interferon-gamma SNP results, 4 were homozygous for the high-producing allele. None of the 3 responders were homozygous for high-affinity Fc gamma receptor IIIa or high interferon-gamma production, and the degree of tumor burden reduction was smaller in these homozygotes than in the others. In addition, pretreatment interferon-gamma ELISPOT data did not predict clinical response to galiximab or tumor burden reduction. In summary, high-affinity Fc gamma receptor IIIa and high interferon-gamma production do not predict clinical responsiveness of follicular NHL to galiximab, suggesting that ADCC may not be a primary mechanism of action for this antibody. Ongoing studies are evaluating other potential mechanisms including the immunomodulatory effects of blocking CD80.
FDG PET Scan Alone Better Predicts Progression Free Survival at 2 Years Compared to CT Imaging in Aggressive Histology Non-Hodgkin’s Lymphoma Following Initial Anthracycline-Based Chemotherapy.
Authors: Wiseman Gregory A. , Juweid Malik E. , Rohren Eric M. , Wooldridge James E. , Graham Michael M.
Published: October 2019

Background: CT imaging has routinely been used for assessing therapy response in most malignancies including Non-Hodgkins Lymphoma (NHL). The presence of residual tumor is generally categorized using the International Workshop Criteria from CT imaging and bone marrow biopsies for assessing response after treatment. CT imaging has limitations in assessment of response to therapy in NHL with false positive results due to residual masses having viable tumor cells in less than 20% and the remainder being fibrosis or necrosis. In addition false negative CT results are seen due to viable tumor cells in nodes measuring less than 1.5 cm in size. F-18 FDG PET scans provide metabolic imaging of viable tumor cells due to uptake and retention of F-18 fluorodeoxyglucose preferentially in malignant cells.

Method: Forty-eight patients with aggressive NHL having completed anthracycline-based chemotherapy had the post therapy FDG PET scans and CT scans reviewed by experienced readers blinded from the comparison scan and from the clinical history. PET scans were read as positive or negative for abnormal FDG consistent with residual viable tumor and the CT was read as positive or negative for nodes greater than 1.5 cm in diameter. Records were reviewed for tumor histology and evidence of tumor relapse with a median follow-up of 35 months.

Results: The FDG PET and CT imaging prediction of PFS at 2 years had positive predictive values of 67% and 38%, negative predictive values of 88% and 78%, and accuracy of 81% and 50% respectively. The sensitivity and specificity of the FDG PET scan was 71% and 82% for predicting disease progression within 2 years from beginning treatment.

Conclusion: FDG PET imaging was compared with CT imaging done after completing initial chemotherapy for aggressive NHL and demonstrated superior prediction of tumor response status at 2 years. These results indicate that FDG PET imaging should be combined with bone marrow biopsy for restaging aggressive NHL after completion of chemotherapy. The use of FDG PET is more accurate and should replace response assessment by CT imaging in most pateints with aggressive NHL following treatment.
Outcome of Patients with Primary Mediastinal Large B-Cell Non-Hodgkin’s Lymphomas: A Single Institution Analysis from 1996-2004.
Authors: Hegenbart Ute , Rieger Michael , Witzens Matthias , Hensel Manfred , Ho Anthony D.
Published: October 2019

Primary mediastinal large B-cell lymphoma represents a distinct subset of large B cell lymphoma, and it has been shown to occur preferentially in young females with a bulky mediastinal mass. Between 1996 and 2004, a total of 43 patients (pts) with primary mediastinal lymphoma were diagnosed and treated at our institution, 34 of them are evaluable for response and long-term follow-up. Twenty females and 14 males have been treated, the median age at diagnosis was 33 years (range: 18–56), 10 pts had stage I, 19 pts stage II and 5 pts stage III disease, respectively. Fifteen pts presented with B symptoms, LDH was increased in 68% of pts. First-line chemotherapy included CHOP (2 pts), R-CHOP (3 pts), CHOEP (8 pts), R-CHOEP (13 pts) or Mega-CHOEP (8 pts). 16 pts received Rituximab in combination with chemotherapy as front-line therapy. Complete response after first-line therapy has been observed in 71% and PR in 21% of pts. Later on, two pts in CR and 1 pt with PR relapsed after conventional chemotherapy. Radiotherapy was administered in 22 pts (65%) presenting with initial bulky disease. 19 pts have been autografted, 15 of them as part of up-front therapy. Indications for up-front autologous HCT were treatment within the Mega-CHOEP protocol (n=8), PR after first-line chemotherapy (n=3), IPI Score of 2 (n=2) and individual decision in 2 pts. Allogeneic HCT was performed as relapse therapy in 4 patients. Of the 15 pts who had up-front autologous HCT, 13 remained in CR. Overall, thirty pts were alive at the date of last contact with a median follow-up since diagnosis of 25 months (range 3–99). Three of 4 pts who had undergone allogeneic HCT in a advanced phase of disease died of transplant-related complications. In conclusion, in this patient group addition of Rituximab and/or dose-intensified first-line chemotherapy lead to a high rate of complete remissions as compared to historic controls. Whether up-front high-dose chemotherapy with autologous HCT or the administration of radiotherapy to the initial bulk improve PFS and OS remains an open question for this special lymphoma entity. The achievement of complete remission with primary treatment strategies seems to be essential to achieve long-term cure for this disease.
Human Leukocyte Antigen (HLA) DR15 Is Associated with Reduced Incidence of Acute GvHD in HLA-Matched Allogeneic HSCT but Does Not Impact Survival.
Authors: Battiwalla Minoo , Hahn Theresa , Roy Hilary N. , Wahab Afrin , Duman Eileen A. , Becker Joanne , McCarthy Philip L.
Published: October 2019

HLA DR 15 is found more frequently in patients with bone marrow failure syndromes and is associated with response to immunosuppression. This suggests that HLA DR15 may preferentially present immunodominant myeloid antigens or that HLA DR15 positive cells may be more sensitive to immunosuppression. We investigated the role of HLA DR15 on graft-versus-leukemia effect and graft-versus-host disease (GVHD) in HLA-matched HSCTs performed for myeloid malignancies. We performed a retrospective review of 109 consecutive related and 42 consecutive unrelated allogeneic HSCT patients treated between 1991 and 2003 at Roswell Park Cancer Institute to investigate the influence of HLA DR15 on overall survival (OS), progression-free survival (PFS) and the incidence of grade 2–4 acute GVHD. HLA DR15 was determined by either molecular (n=92) or serologic (n=59) methods. The proportion of patients with one or two HLA DR15 alleles was 21% (32/151) which is similar to the general Caucasian population. Patient characteristics included: AML (n=74), CML (n=59), and MDS (n=18); median age 42 years (range 11–62); Males (n=93), Females (n=58); Caucasian (>95%); TBI-containing conditioning regimens (n=130); Busulfan + Cyclophosphamide (n=19), Busulfan + TBI (n=27), Cyclophosphamide + TBI (n=11), Etoposide + Cyclophosphamide + TBI (n=84), or other combinations (n=10). There was no difference in OS between the HLA DR15 positive versus negative groups in any disease or donor relation subgroups. There was no significant difference in PFS between the HLA DR15 positive and negative groups, however a trend toward late relapses after three years was observed in the HLA DR15 positive group. The HLA DR15 positive group experienced a significantly lower incidence of acute GVHD grade 2–4: 22% versus 41%, p=0.045. There was no difference between the HLA DR15 positive versus negative groups with respect to conditioning regimen, use of TBI or GVHD prophylaxis regimen. We are currently expanding this study to include a larger cohort from other centers. These results suggest that HLA DR15 may function as a surrogate for an altered response to the immunosuppression induced by GvHD prophylaxis. If these results are confirmed, this would be the first association between a specific HLA antigen and GvHD development, thus suggesting the need for studying GvHD prophylaxis modification based on specific HLA antigens.
Allogeneic Stem Cell Transplant (AlloSCT) for Hodgkin’s Lymphoma Which Has Relapsed Following Autologous SCT - Evidence for Potent Graft-Versus-Lymphoma Effect.
Authors: Klein Andreas K. , Geoffrey Chan , Sprague Kellie , Miller Kenneth B. , Foss Francine
Published: October 2019

With the advent of reduced intensity conditioning regimens, new treatment options are available to patients who suffer relapse of Hodgkin’s lymphoma (HL) following autoSCT, but the efficacy of this procedure is not proven. We retrospectively compared the cohort of adult patients who received alloSCT for relapse of HL following autoSCT to those who did not at a single institution. 64 patients underwent autoSCT for HL between 7/87 and 5/02: 41 for relapsed disease, 16 for primary refractory disease and 7 unclassified. Thirty-five patients (55%) relapsed at a median 12.5 months (range 0.9 to 140) after autoSCT. Eleven patients underwent alloSCT a median of 182 (35 – 645) days after relapse; 7/11 received reduced intensity conditioning, 5/11 received SCT from an unrelated donor. Of 24 patients who had relapsed after autoSCT (and did not receive an alloSCT), 16 survived at least 6 months and were considered in this analysis. Acute graft-versus-host disease (GVHD) > grade 2 occurred in only one patient, 5 /9 evaluable patients developed chronic GVHD (2 extensive). Five recipients of alloSCT have died: 2 with relapsed disease, 2 with disseminated fungal infections and 1 with interstitial pneumonitis. 6 patients remain alive, 5 remain free of disease. Median event free survival is12.5 months for recipients of alloSCT with median follow up of 3.1 years. Median overall survival is 4.1 years (64 days – 12.5+ years) and estimated overall survival is 71% at 3 years. This compares favorably with a median overall survival of 1.4 years in patients who survived at least 6 months, but did not receive alloSCT after relapse. Improved survival in the alloSCT cohort demonstrates that potent graft-versus-lymphoma activity can be achieved against HL without significant GVHD.
A Pilot Study Evaluating the Safety and Efficacy of AMD3100 for the Mobilization and Transplantation of HLA-Matched Sibling Donor Hematopoietic Stem Cells in Patients with Advanced Hematological Malignancies.
Authors: Devine Steven M. , Bonde Jesper , Hess David A. , Edwards Terri M. , Link Daniel C. , Sempek Diane , Calandra Gary , Badel Karin , Nolta Jan A. , DiPersio John F.
Published: October 2019

Cytokine mobilized peripheral blood (MPB) has become the preferred allograft source for patients with advanced hematological malignancies. Procurement of MPB currently requires from 5 to 6 days of granulocyte colony stimulating factor (G-CSF) administration and is associated with some donor morbidity and high cost. Recent studies suggest G-CSF induces mobilization by indirectly targeting the interaction between the chemokine stromal derived factor 1 (SDF-1) and its receptor CXCR-4. Here we report preliminary results using a direct antagonist of the SDF-1/CXCR-4 interaction, AMD3100, as a single agent to procure MPB from allogeneic donors. Two HLA-identical siblings, both males aged 55 and 58, received one or two doses of AMD3100 at 240mcg/kg followed 4 hours later by leukapheresis (LP). After successful collection and a one week washout period, the same donors were re-mobilized using G-CSF at 10mcg/kg/day followed by LP commencing day 5. The target CD34+ cell dose following each agent was >2.0x10e6/kg recipient weight. The results of the mobilizations and allograft composition are presented in the table.

Results of Mobilization

Agent AMD3100 G-CSF Donor 1 Donor 2 Donor 1 Donor 2 Days of Drug administration 2 2 6 5 # LP Procedures 2 1 2 1 Peak fold ↑ CD34 count in PB 7 6 17 25 CD34 dose (x10e6/kg) 2.6 2.1 2.9 5.7 CD3 dose (x10e8/kg) 4.9 1.5 1.3 1.3 CD4 dose (x10e8/kg) 3.1 1.0 0.9 0.9 CD8 dose (x10e8/kg) 1.7 0.4 1.0 0.4 CD56 dose (x10e7/kg) 3.4 2.1 2.2 1.8

Following AMD3100, donor 1 did not experience any drug related toxicity and donor 2 developed only grade 1 abdominal bloating. Both donors experienced grade 2 bone pain during G-CSF mobilization. The AMD3100 mobilized cells from donor 1 were transplanted into a 51 year old woman with AML following conditioning with cyclophosphamide (120mg/kg) and total body irradiation (single dose at 550cGy). Single agent cyclosporine was used for GVHD prophylaxis. She engrafted neutrophils >500/ul promptly on day +11 and platelets > 20,000/ul on day +14. A bone marrow biopsy performed on day +30 revealed an overall cellularity of 50% with normal trilineage hematopoiesis and full donor chimerism. By day 30, the peripheral blood CD3 count had reached 531/ul, CD4 count 305/ul, and CD8 count 211/ul. The CD4/8 ratio was 1.4. She is currently being followed as an outpatient and by day +39 is without evidence of GVHD. The allograft from the second donor was not given as the recipient’s disease progressed.

This is the first demonstration that a chemokine antagonist can induce the rapid mobilization of a functionally competent hematopoietic allograft without causing significant toxicity. Accrual is ongoing and additional donor/recipient pairs will be presented.
8-Amino-Adenosine Induces Loss of Phosphorylation of ERK1/2, p38 MAPK, and Akt Kinase: Role in Induction of Apoptosis in Multiple Myeloma.
Authors: Ghias Kulsoom , Ma Chunguang , Gandhi Varsha , Krett Nancy , Rosen Steven T.
Published: October 2019

Multiple Myeloma (MM) is a malignancy of monoclonal plasma cell proliferation in the bone marrow, which accounts for 10% of all hematologic cancers. This slow proliferating B-cell malignancy accumulates apoptosis-resistant and replication-quiescent cell populations, posing a challenge for current chemotherapeutics, which target rapidly replicating cells. MM remains an incurable disease in need of new therapeutic approaches. We have previously shown that the nucleoside analog, 8-chloro-adenosine (8-Cl-Ado), has significant activity for MM and that its sister compound, 8-amino-adenosine (8-NH2-Ado), appears to be even more potent, exhibiting greater activity in pre-clinical studies. 8-NH2-Ado is cytotoxic to MM cell lines both sensitive and resistant to conventional chemotherapies. This cytotoxic effect requires phosphorylation of 8-NH2-Ado to its tri-phosphate form, 8-NH2-ATP, and accumulation of 8-NH2-ATP results in a concomitant loss of endogenous ATP levels. 8-NH2-Ado induces apoptosis in MM cells as measured by an increase in Annexin V binding, a decrease in mitochondrial membrane potential, an increase in caspase activity, cleavage of caspase substrates, and an increase in cells with a subG1 DNA content. Here we demonstrate the novel effect of 8-NH2-Ado on the phosphorylation status of key cellular signaling molecules. MM cells treated with 8-NH2-Ado exhibit a dramatic loss of phosphorylation of several important signaling proteins, including ERK1/2, p38 MAPK and Akt kinase, within 30–120 minutes. Cells depleted of ATP by antimycin A or 2-deoxyglucose treatments to mimic 8-NH2-Ado-induced ATP depletion do not exhibit the same decrease in phosphorylation of vital cellular proteins. Therefore, the significant shifts in endogenous ATP pools caused by 8-NH2-Ado treatment cannot account for the changes in phosphorylation levels. Instead, 8-NH2-Ado may be affecting the activity of protein phosphatases involved in the negative regulation of these signaling molecules. Based on studies using the phosphatase inhibitor okadaic acid, it appears that PP2A may play a role in the 8-NH2-Ado-induced decrease in phosphorylation of p38. The distinctive effect of 8-NH2-Ado on the phosphorylation status of cellular proteins is a novel phenomenon for a nucleoside analog drug and appears to be unique to 8-NH2-Ado among this class of drugs. The kinetics of 8-NH2-Ado-mediated changes in phosphorylation levels of critical pro-survival and apoptosis-regulating proteins suggest that the modulation of these proteins by de-phosphorylation at early time-points may be an important mechanistic step in 8-NH2-Ado-induced programmed cell death.
Sensitization of Leukemic Cells for Glucocorticoids by Inhibition of NFκB Activity.
Authors: Cloos Jacqueline , Vink Josefien , van Miltenburg Marjolein , Oerlemans Ruud , van der Heijden Joost , Dijkmans Ben A.C. , Jansen Gerrit , Kaspers Gertjan J.L.
Published: October 2019

Based on their immunosuppressive and cytotoxic effects, glucocorticoids (GCs) find widespread use as immune-modulatory agents and in (childhood) leukemia, lymphomas and myelomas. Unfortunately, resistance to GCs is a major adverse prognostic factor and occurs in about 20% in newly diagnosed childhood acute lymphoblastic leukemia (ALL) and in more than 50% in relapsed ALL, while acute myeloid leukemia (AML) is largely unresponsive to GCs. In addition, adult AML is more often GC resistant compared to childhood AML. Recently, we observed (van der Heijden et al, Ann Rheum Dis 63: 131-137,2004) that prolonged exposure of human CEM-C7 T cell leukemia cells to the anti-rheumatic drug sulfasalazine (SSZ) sensitized these primary sensitive cells even further for GCs. SSZ is an inhibitor of activation of NFκB that is often therapeutically used in combination with methotrexate and prednisone. Following these observations, two human myeloid leukemia cell lines (THP-1 and U937) with inherent resistance to GCs (IC50 for prednisolone > 250 μM, IC50 dexamethasone > 25 μM) were exposed to SSZ to establish whether this would provoke GC sensitization. Indeed, prolonged SSZ exposure sensitized both myeloid leukemia cells for prednisolone (IC50 < 1 μM) and dexamethasone (IC50 < 0.05 μM). In order to elicit the mechanistic basis for the GC sensitizing effect of SSZ expression levels of Glucocorticoid Receptor α (GRα), NFκB p65 and Inhibitor of NFκB (IκBα) were analyzed. In contrast to GC-sensitive CEM-C7 cells, parental THP-1 and U937 cells had no detectable and constitutive expression of GRα and NFκB p65 protein, even though mRNA levels for both parameters were readily detectable. In SSZ-exposed THP-1 and U937 cells protein expression of NFκB p65 was markedly increased although it does not represent the active form of NFκB since protein expression of IκB increased concomitantly. Consistent with the sensitization for GCs in SSZ-exposed THP-1 and U937 cells was a marked increase in GRα protein expression, suggesting that GRα is post-transcriptionally prevented from degradation. In line with this concept is that co-incubation with a 26S proteasome inhibitor Bortezomib further enhanced (5-fold) GC sensitivity in SSZ-exposed THP-1 and U937 cells. Collectively, this study reveals that SSZ-exposure impairs activation of NFκB and facilitates stabilization of GRα protein, conditions which confer GC-sensitization in leukemic cells with primary resistance to GCs. In conclusion, modulation of GC resistance in acute leukemias by NFκB inhibitors is a novel, clinically attractive approach that warrants further preclinical and subsequent clinical investigations.
Rituximab-Mediated Molecular Cell Signaling Via CD20 in B-NHL Cell Lines: Autonomy from Cross-Linking with Tumor FcRs.
Authors: Vega Mario I. , Huerta-Yepez Sara , Chinn Paul , Hariharan Hari , Hanna Nabil , Bonavida Benjamin
Published: October 2019

Rituximab (chimeric anti-CD20 mAb) is currently being used in the therapy of Non-Hodgkin’s B-cell Lymphoma (NHL). It has also been reported that the combination of rituximab and chemotherapy is effective in patients with NHL. Several proposed mechanisms have been suggested for rituximab-mediated effects in vivo and include ADCC, CDC and apoptosis. In addition, we have reported that rituximab can trigger the tumor cells and modifies several intracellular survival signaling pathways resulting in the sensitization of tumor cells to various chemotherapeutic drugs (Alas et al., Clin. Cancer Res.8:836, 2001; Jazirehi et al., Mol. Cancer Therapy2:1183, 2003; Vega et al., Oncogene23:3530, 2004). Rituximab-mediated signaling might have resulted from directly triggering CD20 alone or in conjunction with triggering FcRs on the tumor cells via the Fc fragment of rituximab. This study was designed to distinguish between these two possibilities. Since the CH2 domain in Fc is responsible for its interaction with the Fc receptor, we have genetically engineered a rituximab mAb in which the CH2 domain was deleted. The CHO cells secreted one monomeric form with properly formed disulfides rituximab (CH2−) and a dimeric form rituximab (CH2−) Dimer. The functions of the two CH2-deleted domain antibodies were examined in several systems and compared with those of the native rituximab. We used Ramos and 2F7 B-NHL cell lines as model systems. As expected, we demonstrate that the CH2-deleted antibodies are defective in their ability to mediate ADCC and CDC. However, their biological functions and their ability to signal the cells were not altered and were similar to those observed with rituximab. Hence, the CH2-deleted antibodies inhibited cell proliferation, induced cell aggregation in culture, and induced apoptosis when cross-linked with a secondary anti-human IgG. In addition, the CH2-deleted antibodies triggered the cells similar to rituximab and inhibited the Src kinase pLyn, the downstream p38 MAP kinase, the transcription factors NF-κB, YY1, SP1, and STAT3, and Bcl-2/Bcl-xL expression. These modifications resulted in the sensitization of B-NHL cell lines to both chemotherapeutic drugs and Fas-induced apoptosis. Altogether, these findings demonstrate that rituximab-mediated signaling and modification of intracellular signaling pathways can take place in the absence of any contribution of the Fc fragment cross-linked to the cell Fc receptors. These studies suggest that chemo and immuno-sensitization by rituximab can take place in vivo in the absence of Fc-FcR contributions and thus are of potential clinical relevance.
Cellular and Molecular Characterization of Rituximab-Resistant CD20+ NHL Ramos (Ramos RR1) and Daudi (Daudi RR1) Clones: Development of Cross-Resistance to Cytotoxic Stimuli.
Authors: Jazirehi Ali R. , Bonavida Benjamin
Published: October 2019

Numerous studies have documented the efficacy of rituximab (either alone and/or in combination with drugs) in eradicating tumor cells in vitro and in vivo. However, a subset of patients does not respond or relapse after initial rituximab treatment. In an attempt to recapitulate the non-responders and/or relapsed situations, rituximab-refractory B-NHL Ramos and Daudi clones were generated by growing the cells in the presence of step-wise increasing concentrations of rituximab followed by multiple rounds of limiting dilution assay. Single cells were then propagated as clones and assayed for cellular and molecular alterations when compared to the parental cell lines. This study investigates two rituximab-resistant clones, namely, Ramos RR1 and Daudi RR1. A partial decrease in surface CD20 expression, namely, decreased MFI compared to parental cells was observed in the clones. Both clones exhibited resistance to complement-dependent cytotoxicity (CDC). Rituximab was incapable of either inhibiting cellular growth or inducing apoptosis (both in monomeric and cross-linked forms). The clones developed significant cross-resistance to chemotherapeutic drugs and importantly, rituximab had lost its chemo-sensitizing effect on these clones. Detailed analysis revealed a hyper-activated status of the ERK1/2 and NF-κB signaling pathways and rituximab was inefficient in inhibiting these pathways in the clones. Strikingly, selective over-expression of Bcl-xL and the exhibition of higher resistance to a wide array of anti-neoplastic agents were observed in the clones concordant with the protective role of Bcl-xL. However, pharmacological inhibition of the NF-κB and the ERK 1/2 pathways as well as the proteasome inhibitor bortezomib (Velcade) efficiently sensitized the clones to structurally and functionally distinct drugs including topoisomerase II inhibitor, DNA damaging agents, and microtubule poisons, albeit to varying degrees. The above inhibitors also reduced Bcl-xL levels further confirming the pivotal role played by Bcl-xL is clone resistance. The generation of rituximab-resistant clones provides novel means to further investigate the underlying mechanisms that may govern rituximab resistance in patients. Further, these results provide a rational molecular basis for the utilization of pharmacological inhibitors of constitutive signaling pathways in combination with cytotoxic stimuli in the treatment of rituximab-refractory NHL cells.
A Monoclonal Antibody to High Molecular Weight Kininogen Reduces Inflammation in the HLA-B27 Transgenic Rat Model of Inflammatory Bowel Disease and Arthritis.
Authors: Pixley Robin A. , Sainz Irma M. , Keith James C. , Isordia-Salas Irma , Leatherby Yelena , Albert Leo M. , Colman Robert W.
Published: October 2019

The HLA-B27 rat is a well-characterized model of human bowel disease, rheumatoid arthritis and psoriasis. Previous studies of chronic inflammation in other rat models of inflammatory disease have demonstrated activation of the kallilrein-kinin system (KKS) as well as modulation by a kallikrein inhibitor and HK deficiency. The effects of C11C1, a monoclonal antibody acting to inhibit cellular binding of high molecular weight kininogen (HK), were studied in the HLA-B27 transgenic rat. Thrice weekly intraperitoneal injections of C11C1 (1.9 mg/kg) or IgG1 (6mg/kg) were given to male HLA-B27 transgenic rats for 3 weeks, beginning when the rats were 23 weeks old. Stool character was scored daily as a measure of intestinal inflammation, and the rear limbs were scored daily for clinical signs of arthritis, tarsal joint swelling and erythema. At the end of the experiment the animals were euthanized, and the colon and tarsal joint histologic lesions were examined. The histology sections were assigned a numerical score for colonic inflammation, synovitis, and cartilage damage. Activation of KKS was assessed by assays of plasma prekallikrein, HK, factor XI, and factor XII. Administration of the monoclonal antibody directed against HK rapidly decreased the clinical scores of inflammatory bowel disease from 3.0 ± 0 to 1.2 ± 0.2 (p<0.005) and arthritis from 12.0 ± 0 to 0.8 ± 0.8 (p<0.001). Histologic analyses confirmed significant reductions in colonic lesions from 7.7 ± 0.8 to 2.8 ± 0.9 (p=0.004) and synovitis from 9.5 ± 0.8 to 5.0 ± 1.0 (p=0.009). Decreased prekallikrein and HK levels were reversed by monoclonal antibody C11C1, providing evidence of KKS activation. A monoclonal antibody to kininogen appears to have therapeutic potential in human inflammatory bowel disease and arthropathies.
Gene Transfer of CD40-Ligand to Human Dendritic Cells Induces Growth Arrest and Apoptosis of Tumor Cells Mediated by Up-Regulated TRAIL and its Receptors.
Authors: Tomihara Kei , Kato Kazunori , Masuta Yukari , Noguchi Makoto , Hiratsuka Hiroyoshi , Hamada Hirofumi
Published: October 2019

Interaction of CD40 and its ligand CD40L is essential for the activation of dendritic cells (DC) followed by the induction of antigen-specific T-cell responses. Recent studies have also indicated that ligation of CD40 on tumor cells directly modulate tumor cell growth. In this study we examined whether gene transfer of CD40L to DC could exhibit direct anti-tumor effect on CD40-expressing tumors. First, we constructed a modified adenoviral vector driving human CD40L gene with CA promoter/enhancer and incorporating the integrin-binding motif, RGD, in the H1 loop of the fiber knob (AxCAhCD40L-F/RGD). At the same multiplicity of infection, cell surface expression of human CD40L on DC after infection with AxCAhCD40L-F/RGD (CD40L-DC) was induced to express superior higher levels of T-cell costimulatory molecules (CD80, CD86, and CD54), DC maturation markers (CD25 and CD83), MHC molecules and the production of IL-12 than soluble CD40L or TNF-a treated DC. In addition to the phenotypic alternation of CD40L-DC, we found that cultivation of tumor cells (including lymphoma, leukemia, glioma, head and neck carcinoma) with CD40L-DC significantly inhibited the expression of phospho-AKT and tumor cell growth in vitro. Importantly, these tumor cells in transwell cocultured with CD40L-DC, but not with TNF-a-treated DC or immature DC, were significantly induced to express apoptosis-inducing death receptors such as TRAIL-R1, TRAIL-R2 and Fas. The ability of CD40L-DC to inhibit tumor-cell growth and TRAIL receptor induction could not be abrogated by neutralizing antibodies specific for CD40L, IFN-b, IFN-g, and IL-12, indicating that these factors were not involved. Additionally, natural killer cells cocultured with CD40L-DC displayed marked up-regulation of TRAIL on their cell surface. To confirm the biological function of TRAIL and its receptors, we assessed the cytotoxic activity of NK cells to various tumors that were stimulated with CD40L-DC. Tumor cells stimulated with CD40L-DC were susceptible to apoptosis by recombinant TRAIL protein and natural killer cells expressing TRAIL. Overall, these results reveal that CD40L-transfected DC could induce direct tumor cell growth arrest mediated by AKT pathway and susceptibility of apoptosis by TRAIL, indicating that CD40L-DC have potential therapeutic implications which we were currently exploring.