bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of AMG 531 Efficacy in Splenectomized Patients with Chronic Immune Thrombocytopenic Purpura (ITP) in a Randomized Placebo-Controlled Phase 3 Study.
Authors: Gernsheimer Terry B. , Pullarkat Vinod , Senecal Francis M. , Aledort Louis M. , Kessler Craig M. , Sanz Miguel A. , Liebman Howard A. , Slovick Frank T. , Kelly Reggie , Guo Matthew , Nichol Janet
Published: April 2020

AMG 531 is a novel thrombopoiesis-stimulating peptibody that acts by stimulating the thrombopoietin (TPO) receptor. We report efficacy data in splenectomized patients from a randomized, double blind, placebo-controlled Phase 3 study designed to evaluate the efficacy and safety of AMG 531 in patients with chronic ITP. Sixty-three splenectomized patients were enrolled (placebo, 21; AMG 531, 42), with a median age of 52 years (range 26 to 88) and a mean baseline platelet count 14.7x109/L. Subcutaneous AMG 531 or placebo was administered weekly for 24 weeks at a starting dose of 1μg/kg, and adjusted to maintain a target platelet count of 50–200x109/L. The primary study endpoint was the incidence of a durable platelet response, defined as a platelet count ≥50x109/L for ≥6 weeks during the last 8 weeks of the 24 week treatment period in the absence of rescue medications. Sixteen of the 42 splenectomized patients (38.1%) receiving AMG 531 achieved a durable platelet response compared to 0/21 (0.0%) receiving placebo (p=0.0013). Overall response, defined as either durable or transient platelet response (≥4 weekly platelet responses), was observed in 33/42 (78.6%) patients receiving AMG 531, compared to 0/21 (0%) placebo patients. The mean number of weekly platelet responses (platelet count ≥50x109/L) was significantly greater in patients receiving AMG 531 (12.3/24 weeks, 51%) compared to placebo (0.2/24 weeks, 1%) (p<0.0001). AMG 531 reduced the proportion of patients requiring ITP rescue medications, defined as either increase from baseline in dose of concurrent medication or use of new medication to increase platelet counts. Twelve of 21 (57.1%) placebo-treated patients received rescue medications compared to just 11/42 (26.2%) AMG 531-treated patients (p=0.0175). In addition, 12/12 (100.0%) AMG 531-treated patients compared to 1/6 (16.7%) placebo-treated patients either discontinued or reduced by >25% their concurrent ITP medications. AMG 531 was well-tolerated. There were 2 treatment-related serious adverse events; 1 patient with elevated bone marrow reticulin that returned to baseline 3 months after withdrawal of AMG 531, and 1 patient experienced thrombosis that was successfully treated allowing study continuation. No patient developed neutralizing antibodies against either AMG 531 or endogenous TPO. In summary, AMG 531 was well-tolerated, and effectively increased and sustained platelet counts in splenectomized patients with ITP. AMG 531-treated patients required less frequent rescue medications in comparison to those receiving placebo, and were able to reduce their use of concurrent therapies.
Double Cord Blood Transplantation in Bone Marrow Failure Syndromes.
Authors: de Latour Regis Peffault , Rocha Vanderson , Robin Marie , Rodrigues Celso A. , Rea Delphine , Laghero Jerome , Traineau Richard , Ribaud Patricia , Ferry Christelle , Devergie Agnes , Gluckman Eliane , Socie Gerard
Published: April 2020

The outcome of severe aplastic anemia, refractory to immunosuppressive therapy or observed in case of Fanconi anemia (FA), is usually poor in the absence of Hematopoietic Stem Cell Transplantation (HSCT). Umbilical cord blood is an alternative stem cell source for patients without matched related or unrelated donors. However, single cord blood unit is associated with high transplant related mortality due to the low cell dose infused in previous highly transfused patients. We have driven the hypothesis that double cord blood transplantation (dCBT) could circumvent the cell dose problem. For the purpose of this study, we have studied 13 patients with bone marrow failure syndromes given 2 partially matched dCBT from 2004 to 2007. The diagnoses were FA (n=9), SAA (n=4) and PNH (n=1). Among those patients, 5 (39%) received a dCBT as a rescue of previous rejected transplants (2 SAA and 3 FA). All patients received a fludarabine-based regimen, with TBI (2 Gy) for 4 patients. Cord blood units were a 4/6 or 5/6 HLA A, B and DR match with the patient except one which was 3/6. Graft versus host disease (GVHD) prophylaxis consisted in CSA+MMF. Steroids were given from day 7 to day 14 and stopped in case of no GVHD. Five male (39%) and 8 female (61%) with a median age of 16 years (range 7–31) were treated. The cell doses infused were a median of 5.0 × 107 NC/Kg (4–9) and 5.3 × 105 CD34+ cells/Kg (2–8). Graft rejection was seen in 5 patients (2 previously allotransplanted). Among those patients, one displayed a temporary mixed chimerism before rejection and another presented an autologous reconstitution. Among the remaining 8 patients, the median time to an absolute neutrophils count > 500 was 25 days (range 14–42) and the median time to a platelet count > 20,000 was 39 days. In these last patients, we observed a complete donor chimerism with one cord blood unit during 100 days after dCBT. Acute GVHD grade II–III was scored in 9 patients (69%) (7 grade II, 2 grade III). No patients presented acute GVHD grade IV. Four patients out of 8 developed Chronic GVHD (3 limited and 1 extensive). Four patients died (1 GVHD, 2 fungal infection, 1 thrombotic microangiopathy). With a median follow-up of 13 months (range 1 to 19 months), the overall survival was 55% (±15%) for all patients. The median survival of patients who were transplanted twice was 50% (±25%). In conclusion, dCBT seems to be an option to treat patients with bone marrow failure syndromes and without a suitable compatible HLA donor. Those results need to be established on a large number of patients to warrant the inclusion of dCBT in the treatment strategy of diseases with high risk of rejection.
Unrelated Cord Blood Cell Transplantation for Acquired Severe Aplastic Anemia: The Report from the Japan Cord Blood Bank Network.
Authors: Kojima Seiji , Yoshimi Ayami , Taniguchi Shuichi , Hara Junichi , Matsui Toshimitsu , Kato6 Shunichi
Published: April 2020

Treatment approaches for patients with severe aplastic anemia (SAA), who failed immunosuppressive therapy and lack a bone marrow donor remains a great challenge. Unrelated cord blood transplantation (UCBT) has not been recommended for SAA because of historical poor outcome with high rate of engraftment failure. To evaluate the current feasibility of UCBT in SAA, we retrospectively analyzed the outcomes of 31 patients (median age 28 years old; ranged 0.9–72.3 years old) with SAA, who received UCBT as the first graft between 1998 and 2006 in Japan. Median disease duration before UCBT was 337 day (31–5063 days). By serology, HLA loci were matched in 4 recipient-donor pairs and mismatched (1–2 loci) in 27 patients. A minimum cell count of 2 × 107 nucleated cells/kg body weight was infused in all patients. Engraftment was observed in 17 of 24 evaluable patients. The median times to achieve a neutrophil count ≥ 0.5 × 109/l and a platelet count ≥ 50 × 109/l were 19 days (range 12–35 days) and 59 days (range 39–145 days), respectively. The results of chimerism analysis were available in 9 of them and all of them showed complete donor chimerism (>99%) except one with autologous recovery. Late rejection was seen in one patient. Acute GVHD (≥ grade II) was observed in 5 of 18 evaluable patients (grade II; n=4, grade III; n=1) (cumulative incidence =17.1%) and chronic GVHD was observed in 4 of 14 evaluable patients (extensive: n=1, limited: n=3) (cumulative incidence =19.7%). Currently, 13 patients are alive, having survived for median 22.5 months (ranged 3 to 77 months) after UCBT (overall survival at 2 years=40%). Causes of death of 18 patients were following: graft failure (n=7), bacterial/fungal infections (n=3), hepatic veno-occlusive disease (n=3), and others (n=5). The conditioning regimen appeared to be the most important factor for the outcome and low dose total body irradiation (2–4 Gy) + fludarabine (90–250/ mg/m2) and cyclophosphamide (50–100 mg/kg or 2250/mg/m2) (n=5) gave the best outcome with 80% of survival. The GVHD prophylaxis with single agent (cyclosporine or tacrolimus) related with a better engraftment rate than 2 or more agents (84.4% vs 47.3%, p=0.02). These results suggest that UCBT can be a salvage treatment for patients without a bone marrow donor and warrant further evaluation in prospective studies. Optimization of conditioning regimen will improve the engraftment and outcome of UCBT.
Unrelated Cord Blood Transplanation for Malignant Lymphoma: A Retrospective Study from the Japan Cord Blood Bank Network (JCBNW).
Authors: Takita Morihito , Yuji Koichiro , Yamaguchi Takuhiro , Matsumura Tomoko , Kami Masahiro , Sakamaki Hisashi , Kouzai Yasushi , Okada Masaya , Osugi Yuko , Kobayashi Ryuji , Inoue Masami , Takahashi Satoshi , Murata Makoto , Kai Shunro , Kato Koji , Nagamura Tokiko , Taniguchi Shuichi , Kato Shunichi
Published: April 2020

Unrelated cord blood transplantation (u-CBT) may provide a treatment option for patients with malignant lymphoma (ML) who have failed other conventional therapies and do not have a compatible donor available. Japan Cord Blood Bank Network (JCBNW) has established a registry for u-CBT as a quality management and promotion of CBT. We have analysed 271 patients with ML (aggressive lymphoma=189, indolent lymphoma=52) transplanted from 1999–2006 and reported to JCBNW. The median follow-up was 19 months (1–85) and the median age was 47 years (1–75). At transplant, 83% of the patients are non-CR and 23% had received previous autologous transplants. The conditioning regimen varied according diasease and centers: reduced intensity conditioning regimens (RIC) were used in 49% and ATG/ALG was added in 5% of the cases. GVHD prophylaxis consisted of CsA alone (23%), CsA/sMTX (28%), TAC alone (18%), TAC/sMTX (11%) and others (20%). The median nucleated cell dose infused was 2.6 × 10^7/kg and the graft was HLA identical (6/6) (HLA A and B low resolution and DRB1 allelic typing) in 37 cases, 5/6 in 110, and 4/6 in 122. Median time to neutrophil recovery (>500/mm3) was 22 days (0–61) and 39 days for platelets recovery (>20.000/mm3). The probability of neutrophil recovery was 80%. Grades II–IV aGVHD was observed in 50%. Treatment-related mortality (TRM) was observed in 107 patients (39%), and 61% of them were related to infection. The overall survival (OS), disease-free survival (DFS), and cumulative incidences of disease progression at 2 years after u-CBT were 28%, 21%, and 11%, respectively. In a multivariate analysis, chemoresistance, bacterial infection, and cGVHD were identified as adverse prognostic factors for overall survival. The development of grades II–IV aGVHD was associated with higher rate of DFS, which suggested the existence of a graft versus lymphoma effect. Althouth u-CBT can be an effective therapeutic option for patients with refractory ML, more work is still needed to decrease TRM.
Immunological Responses in CLL Patients Vaccinated with Autologous Dendritic Cells Loaded with Apoptotic Bodies (Apo-DC).
Authors: Choudhury Aniruddha , Palma Marzia , Hansson Lotta , Widen Karin , Eriksson Ingrid , Nasman-Glaser Barbro , Adamson Lars , Osterborg Anders , Mellstedt Håkan
Published: April 2020

We have previously established the suitability of dendritic cells that have endocytosed apoptotic B-CLL cells (Apo-DC) as an antigen presentation platform to stimulate autologous antileukemic immune responses. The safety as well as immunological and clinical efficacy of Apo-DC vaccination was tested in a phase I/II clinical trial. CLL patients (Rai stage 0–2) who did not require concurrent therapy were leukapheresed and CD14+ monocytes as well as CD19+ B-CLL cells were purified immunomagnetically. Dendritic cells were generated ex vivo with GM-CSF and IL-4. Apoptotic bodies were obtained by irradiating B-CLL cells and overnight culture. DC were allowed to endocytose the apoptotic bodies and further matured with TNFα. The first cohort of 5 patients received 4 doses of the vaccine 2 weeks apart and a 5th dose at week 14. Each vaccine dose comprised of 107 autologous Apo-DC. The second cohort of 5 patients received the vaccine in a similar schedule with the addition of 75 μg of GM-CSF (Leukine™, Berlex Laboratories, Richmond VA) with every vaccine administration. Patients are scheduled to be monitored for a minimum of 52 weeks which has been completed for all of the patients in cohort 1 and is ongoing for cohort 2 The vaccine was well tolerated and occasional, low-grade toxicity was associated with the administration of GM-CSF. 4/5 patients in cohort 1 and 3/5 patients in cohort 2 responded immunologically in terms of increase in T cell proliferation or γ-IFN ELISPOT against autologous leukemic targets compared to prevaccination levels. Analysis of T cell responses at week 8 and week 16 following vaccination did not demonstrate an obvious augmentation of immunity by GM-CSF. One patient in cohort 1 demonstrated a transient decrease in WBC counts but no significant clinical responses have been noted thus far. Our results demonstrate that it is feasible to enrich monocyte precursors and generate Apo-DC for vaccinating patients with high count B-CLL. No significant toxicity was associated with this approach to vaccination therapy. The clinical efficacy of Apo-DC vaccination in CLL remains to be determined at the termination of the clinical trial.
Autoantibodies Against p53 Are Associated with p17 Deletions in CLL.
Authors: Messmer Bradley T. , Nour-Omid Talia S. , Ghia Emanuela , Rassenti Laura , Kipps Thomas J.
Published: April 2020

The TP53 gene located on the short arm of chromosome 17 (17p) encodes p53, which is a transcription factor that regulates the cell cycle and the apoptotic response to genotoxic stress, such as that caused by most anti-cancer drugs. Loss of p53 function in chronic lymphocytic leukemia (CLL) typically is associated with deletions in 17p, which is associated with a poor response to many anti-cancer drugs and poor survival. Although only 5–10% of CLL patients at diagnosis have deletions in 17p by interphase fluorescence in situ hybridization (FISH), the frequency of cases that have 17p- increases in patients with disease that is refractory to chemotherapy. Assays that can screen for loss of p53 function in CLL could help identify patients who most likely will not respond well to most common forms of chemotherapy. Autoantibodies against p53 protein have been observed in several cancers and are, in some cases, linked to p53 protein accumulation in the tumor cells owing to loss of p53-induced expression of the p53-repressor MDM2. We examined the serum samples derived from platelet-free plasma of patients with CLL for anti-p53 autoantibodies by solid-phase ELISA. Anti-p53 autoantibodies were detected in 19 out of 31 plasma samples from patients with known 17p deletions defined by interphase FISH. In contrast, we detected anti-p53 autoantibodies in only 1 out of 27 plasma samples from patients with 11q22–23 deletions and in none of the 29 samples from patients with normal cytogenetics. Detectable levels of soluble p53 protein were identified in only 5 of the 31 patients with 17p deletions, and 3 of those had detectable anti-p53 antibodies. There was no correlation between anti-p53 autoantibody levels and the total plasma IgG. Samples from multiple time points were available for 22 of the patients with 17p deletions. In only one case did the p53 autoantibody status change (from positive to negative). The mechanism that leads to the generation of anti-p53 autoantibodies is unknown. One model suggests that constitutive or over expression of p53 protein by the leukemic cells may lead to a break in tolerance, perhaps if coupled with necrotic or otherwise immunogenic death of the cells. Studies are currently underway to address the status and function of the p53 protein in those patents with high autoantibody levels. This work suggests that auto-antibodies may be useful as a surrogate marker for p53 dysfunction and, more broadly, raises the possibility that auto-antibody profiles may provide clinically relevant insight into the biology and pathophysiology of CLL.
Gene Expression Signature of B-Cell Chronic Lymphocytic Leukemia with Trisomy 12.
Authors: Porpaczy Edit , Bilban Martin , Kroemer Elisabeth , Heinze Georg , Gruber Michaela , Vanura Katrina , Heintel Daniel , Hilgarth Martin , Hilgarth Bernadette , Le Trang , Hubmann Rainer , Schnabl Susanne , Chott Andreas , Schwarzinger Ilse , Hauswirth Alexander , Scrabs Cathrin , Stilgenbauer Stephan , Wagner Oswald , Fonatsch Christa , Jaeger Ulrich , Shehata Medhat , Gaiger Alexander
Published: April 2020

The prognosis of patients with B-CLL is largely determined by the karyotype of the malignant clone. Microarray technology has facilitated linkage between chromosomal aberrations and gene expression signatures. We have investigated the gene expression profile associated with trisomy 12 (+12). Expression data were obtained by microarray analysis of mRNA from unselected PBMNC of 4 patients with +12 and compared with 16 B-CLL controls. 146 genes were at least 2-fold over- or underexpressed in samples with +12. Five of the 16 genes showing the strongest correlation with +12 were selected for further analysis (HIP1R FC=3,43; MYF6 FC=3,92; P2RY14 FC=−9,59; RASGRP3 FC=−3,85; SLC2A6 FC=2,13) and validation by real time PCR: HIP1R located on chromosome 12q24, with a fold change (FC) of 3,43, MYF6 (chromosome 12q21, FC=3,92), P2RY14 (chromosome 3q21–q25, FC-9,59) RASGRP3 (chromosome 2p25.1–p24.1, FC=−3,85). SLC2A6 (chromosome 9q34, FC=2,13). Quantitative PCR was performed with mRNA from 61 patients (29 with +12, 32 B-CLL controls) and 2 healthy donors. Only 3 genes were significantly associated with +12 compared to the B-CLL-controls in this evaluation: HIP1R (3,486; p<0,0001), MYF6 (1,498; p=0,005), P2RY14 (1,216; p=0,013). (Table1). Two of these genes (HIP1R, MYF6) are located on chromosome 12 indicating a “gene dosage effect”, while P2RY14 is localized on a different chromosome suggesting trans-acting processes. We have used expression of HIP1R as a surrogate marker for trisomy 12. The predicted sensitivity was 79,3% and the predicted specifity was 90,6. Analysis of CD19+ selected B-CLL and normal B-cells revealed that MYF6 is exclusively expressed in normal or malignant B-cells in peripheral blood. We confirmed that MYF6 is highly specific for skeletal muscle, however strong expression was found in normal tonsils, DLBCL, and other B-cell malignancies. Our data link a specific gene expression signature with trisomy 12. 3 novel marker genes were identified, which could be used as diagnostic tools. The linkage with P2RY14 suggests that +12 influences the expression of genes from other chromosomes.

Table 1 Mean + 12, N=29 Mean B-CLL controls, N=32 p-value Locus Fold change microarray HIP1R 0,7819 0,2243 0,000 12q24 3,43 MYF6 37,55 25,06 0,005 12q21 3,92 P2RY14 −0,2873 0,3494 0,013 3q21–q25 −9,59 RASGRP3 0,8404 1,0774 0,055 2p25.1–p24.1 −3,85 SLC2A6 29,78 22,37 0,080 9q34 2,13
The Development of Severe Anti-HPA 1a-Related Neonatal Alloimmune Thrombocytopenia Is Influenced by the Maternal ABO Type.
Authors: Ahlen Maria T. , Killie Mette K. , Husebekk Anne , Kjeldsen-Kragh Jens , Olsson Martin L. , Skogen Bjorn
Published: April 2020

About 100 000 pregnant women were included in a screening and intervention program aimed to reduce morbidity and mortality related to neonatal alloimmune thrombocytopenia (NAIT). NAIT due to HPA 1a immunization have many features in common with hemolytic disease of the newborn (HDN) caused by RhD immunization. Platelets express blood group A and B antigens, and as described in HDN, ABO incompatibility between mother and fetus could possibly protect against immunization against HPA 1a. The aim of the study was to describe the ABO distribution among the immunized mothers and examine whether ABO status of the mother influenced the severity of thrombocytopenia in the newborn. ABO typing of HPA 1a immunized women was routinely performed by serological methods during the screening. ABO genotyping resolving the major alleles (A1, A2, B, O1, O1v or O2 alleles) of the women and their newborns was performed by PCR-RFLP methods. The ABO distribution among the immunized HPA 1a negative women was found to be similar to the normal distribution in the Norwegian population, which may indicate that the ABO type does not influence the immunization mechanism. However, we find that HPA 1a immunized women of blood group A have a higher risk of delivering a child with severe NAIT (platelet count < 50×109/L) than women with blood group O. Twenty percent of the immunized women with blood group O gave birth to children with severe NAIT, compared to 46% among the immunized blood group A mothers, resulting in a relative risk of 0.43 (95% CI 0.25–0.76). The ABO type of the newborn was not found to influence development of severe NAIT. The O1/O1v allele distribution among the immunized women with blood group O resembles the distribution reported for a Swedish population. However, only 2/22 (9.1%) pregnancies among the O1v negative blood group O mothers resulted in a newborn with severe NAIT, compared to 10/34 (29.4%) among the O1v-positive blood group O women, resulting in a relative risk of 0.31 (95% CI 0.07–1.28). The observation that the immune response against HPA 1a may have different consequences depending on the ABO blood group of the mother is interesting. O1v, also termed O02, constitutes a separate but ancient allelic lineage at the ABO locus and we are now in the process of examining if our observation is related to linkage disequilibrium between the ABO gene and one or more pregnancy-related immunoregulatory genes.
Bleeding Complications and Pregnancies in an Established Plasminogen Activator Inhibitor, Type-1 (PAI-1) Deficient Population.
Authors: Mehta Rakesh P. , Simmons Molly , Heiman Meadow , Shapiro Amy D.
Published: April 2020

In 1992, a young Amish girl with marked post-procedural bleeding was found to have a PAI-1 deficiency with a homozygous frame shift mutation in exon 4 of the PAI-1 gene that results in a non-functional protein. This mutation was tracked within the ancestors of this Amish community and two individuals were identified that likely introduced this gene into the community. In 1997, seven children (including the proband) from two families within the kindred were described with symptoms consistent with a mild bleeding disorder. Two further homozygous children, one from a third family within the kindred, have subsequently been identified. Presently, over 300 members of this kindred have been tested with nearly 100 identified heterozygotes, none of whom have experienced bleeding complications. The homozygotes all have had bleeding problems with injury a common cause. Five of the nine homozygotes have had head trauma, resulting in significant bleeding complications. ε-aminocaproic acid (EACA) initiated early controls bleeding. One child had a recurrence of his subdural hematoma when he discontinued the EACA upon discharge from the hospital. But the bleeding was subsequently controlled with reinstitution of EACA, and the child had a full recovery. Another common bleeding complication is menorrhagia with all homozygous females experiencing this symptom, and two even requiring red blood transfusions due to severe anemia. Currently, these women require concurrent EACA with onset of menses to control bleeding. Of interest PAI-1 and the plasminogen activation system have been shown to play an important role in embryo implantation, and it was unknown whether homozygous PAI-1 deficient women would be able to conceive and maintain a pregnancy. We now report 2 of these homozygous deficient females have become pregnant; one remains pregnant at 28 weeks gestation, and one delivered at 32 weeks. This child represents that first documented birth to a woman with complete PAI-1 deficiency. The mother experienced abnormal cervical bleeding beginning at 27 weeks requiring EACA therapy which was continued until delivery and for about 7 days post-partum without abnormal bleeding at delivery or in the post-partum period, and without ill effect on the fetus. This cohort is the largest group of PAI-1 deficient patients ever documented. The molecular defect is well defined so that it is simple to differentiate normals from heterozygotes and homozygotes. Continued follow-up will allow further understanding of the role of PAI-1 in human physiology.
Histone Deacetylase Inhibitor SAHA Reduces Cytokine Storm and Facilitates Induction of Chimerism When Combined with Anti-CD3 mAb in Conditioning of Recipients.
Authors: Li Nainong , Zhao Dongchang , Zhang Chunyan , Lin Chia-Lei , Kirschbaum Mark , Kandeel Fouad , Forman Stephen , Zeng Defu
Published: April 2020

Donor T cell-mediated graft versus leukemia (GVL) and graft versus autoimmunity (GVA) plays a critical role in the treatment of hematological malignancies and refractory autoimmune diseases using allogeneic hematopoietic cell transplantation (HCT). However, graft versus host disease (GVHD) remains a major obstacle in classical HCT, where recipients are usually conditioned with total body irradiation or high dose chemotherapy. We have recently reported a radiation-free and GVHD preventive anti-CD3-conditioning regimen, which allows donor CD8+ T cells to facilitate engraftment and mediate GVL without causing GVHD (PNAS, 2007 and J. I. 2007). In order to promote the clinical application of the anti-CD3-conditioning regimen, we need to overcome the cytokine storm that causes flu-like syndrome in patients after anti-CD3 mAb injection and reduce the required donor bone marrow cell dose. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, was reported by others to induce apoptosis of human T cell lymphoma cell lines and to reduce serum levels of pro-inflammatory cytokines in HCT recipients. Therefore, we tested whether conditioning with a combination of anti-CD3 and SAHA can reduce the cytokine storm caused by anti-CD3 and reduce the resistance of engraftment mediated by residual host T cells. Accordingly, titrated dose of SAHA (0.125–8 μM) were added to cultures of BALB/c spleen cells stimulated with plate-bound anti-CD3. We found that SAHA augmented apoptosis of anti-CD3 activated T cells in a dose-dependent manner. Although low-dose SAHA (0.125–0.25 μM) did not augment apoptosis, it rendered the residual live T cells partially unresponsive. At the same time, SAHA significantly reduced the pro-inflammatory cytokines (IL-2, IFN-γ, TNF-α, and IL-6) in the culture supernatants. Similarly, in vivo treatment with anti-CD3 and low-dose SAHA (40 μg/g) led to significant reduction of serum levels of those cytokines and partial unresponsiveness of the residual host T cells. Finally, conditioning with combined anti-CD3 and SAHA (40 μg/g) induced complete chimerism without GVHD in 12/12 of old BALB/c as well as in 8/8 of old autoimmune lupus NZB/NZW F1 recipients after two injections of BM cells (2 ×106/g) and CD4+ T-depleted spleen cells (4 ×106/g), although conditioning with anti-CD3 alone did not induce any chimerism (0/8). The chimeric NZB/NZW F1 recipients showed complete reversal of autoimmune glomerulonephritis and proteinuria. These results indicate that SAHA can not only reduce cytokine storm but also facilitate engraftment when combined with anti-CD3 for conditioning of recipients. This radiation free and GVHD preventive conditioning regimen may provide a novel approach for clinical HCT.
CD6-Depletion in the Dog Model: New Insights into Chimerism and Tolerance.
Authors: Zorn Julia , Herber Martin , Schwamberger Sabine , Adler Heiko , Kolb Hans-Jochem
Published: April 2020

Adoptive immunotherapy with donor lymphocyte infusion (DLI) following bone marrow transplantation may produce a graft-versus-leukemia (GVL) effect after transplantation of T-cell-depleted bone marrow. However, this method is limited by a high risk of GvHD. We studied the occurrence of graft-versus-host disease (GVHD) and chimerism after DLI at various times after CD6-depleted bone marrow transplantation in a canine model of DLA-homozygous donor and DLA-heterozygous littermate recipient. Using a cross reactive mouse-anti-human antibody which recognizes the canine CD6 antigen, a simple and economical depletion method could be established, which makes use of the ability of the CD6 antibody MT-606 to activate the complement cascade. In vitro experiments using MT-606 and rabbit complement showed the effective depletion of CD6-positive cells whereas hematopoietic progenitor cells remained unaffected at the same time. For in vivo studies, DLA heterozygous recipients with one DLA haplotype being identical to the donor were treated with 10 Gy total body irradiation (TBI) and received CD6-depleted bone marrow of their DLA-homozygous littermate donors. In the control group 7 dogs given unmanipulated marrow died of GVHD within 28 days of transplantation. Two dogs showed sustained engraftment without the occurrence of GvHD after the infusion of 1,8 · 108/kg – 2,0· 108/kg CD6-depleted mononuclear marrow cells. The establishment of tolerance to the bone marrow donor was shown by the transplantation of skin from the donor to the host. Transfusion of donor lymphocytes (1.0 · 108 MNCs/kg body weight) on day 3, 7 or 14 induced GvHD in the host. However, with DLI on day 20, only 2 out of 4 dogs developed GvHD, but DLI always converted mixed into complete chimerism. We conclude from this study that the cross reactive CD6 antibody MT606 effectively depletes T cells in the presence of rabbit complement and induces GVH tolerance without jeopardizing engraftment, and the risk of GVHD after DLI is reduced with time after transplantation.
Host Interferon gamma Production and STAT-1 Signalling Is Crucial for Minor Antigen Mediated Rejection of High Grade Lymphoma.
Authors: Sukumar Madhusudhanan , Wilke Andrea , Jaeger Eva , Mautner Josef , Ellwart Joachim , Kolb Hans-Jochem , Bornkamm Georg W. , Gerbitz Armin
Published: April 2020

To date mechanisms of T-cell mediated immunity and immune-escape in high grade lymphomas are poorly understood. Using a transgenic mouse lymphoma model, where the proto-oncogene c-myc is driven in parts of the immunoglobulin lambda locus representing a t(8;22) translocation as found in Burkitt’s lymphoma, we developed a syngeneic model to investigate the anti-lymphoma activity of lymphoma specific T-cells. By retroviral transduction of a lymphoma specific antigen (chicken ovalbumin-IRES-GFP vector) into primary cell lines from c-myc transgenic lymphomas we established a model that would allow us to investigate the contribution of interferon gamma signalling in rejection of high grade lymphoma. All lymphomas established displayed low MHC class I and II levels on the surface when compared to wildtype B-cells. This expression could be enhanced by treatment with interferon gamma (100U/ml) up to 10 fold. When retrovirally transduced lymphoma cells were injected into wildtype or GFP transgenic C57BL/6 recipients, animals displayed a significant delay in lymphoma growth compared to IRES-GFP transduced control cell lines. 50% of the recipient mice rejected OVA containing lymphomas whereas we observed a 100% lymphoma growth of IRES-GFP transduced lymphomas in GFP transgenic recipients. Developing OVA containing lymphomas displayed a loss of GFP expression indicating a selection for non transduced cells. In spleens from mice successfully rejecting OVA-containing lymphomas we found up to 1.5% (±0.12%) SIINFEKL specific T-cells. To gain mechanistic insights of lymphoma rejection, we transferred OVA transduced lymphoma cells to Stat1−/− and IFNg−/− recipients. Lack of STAT1−/− on the recipient side or inability to secrete interferon gamma was associated with fast lymphoma progression and was not different when compared to IRES-GFP transduced cell lines injected into GFP transgenic hosts. Although we found 1.6% (±0.52%) SIINFEKL T cells in spleens of lymphoma bearing STAT1−/− animals, interferon gamma production was significantly decreased. We could also show that in wild type recipients, OVA containing lymphomas displayed high MHC class I and II expression which is completely absent in lymphomas from Stat1−/− recipients. Our results suggest that rejection of high grade lymphoma by a specific minor antigen is possible and Stat1 signaling and interferon gamma production by the host is crucial for lymphoma rejection.
CD62L Is Not Critical for T Regulatory Cell-Mediated Protection from Lethal Acute GVHD.
Authors: Carlson Michael J. , Siedel Hans E. , Serody Jonathan S.
Published: April 2020

INTRODUCTION: Graft-versus-host disease (GVHD) is a major complication following allogeneic bone marrow transplantation. Despite advances in understanding the etiology of GVHD it remains a formidable obstacle to the widespread application of BMT. T regulatory (Treg) cells have been shown to inhibit GVHD while preserving the beneficial graft-versus-leukemia (GVL) effect. Published studies by numerous groups, including our own, have shown the importance of Treg homing molecule expression in preventing GVHD. Of note, the Treg population that expressed high levels of the adhesion molecule L-Selectin (CD62L) was shown to be capable of promoting BM engraftment and inhibiting lethal acute GVHD, whereas the CD62Llo population was not. While these studies provided evidence that the phenotype of the CD62Lhi Treg population was responsible for inhibition of GVHD they did not directly assess the role of CD62L.

METHODS: To investigate the importance of CD62L in Treg mediated protection from GVHD we utilized a well-described parent into F1 model. We transferred Tregs (2×106) from CD62L deficient mice (CD62L−/−) or wild type (WT) C57BL/6 mice with WT naïve T cell effectors (5×106) into lethally irradiated B6D2 recipients. Our results demonstrated similar survival of recipient mice receiving CD62L−/− Tregs (40% survival) compared to those receiving WT Tregs (60% survival: p =0.66). In addition fluorescence microscopy revealed no difference in GFP+ T cell effector migration/accumulation in GVHD target organs (liver, lung, GI tract) or secondary lymphoid organs (spleen, mesenteric lymph node, inguinal lymph node, Peyers Patches) in the presence of CD62L−/− or WT Tregs. Also, we tracked the migration of GFP+ CD62L−/− Tregs in vivo by fluorescence microscopy and found they were able to efficiently migrate to secondary lymphoid organs and GVHD target organs. Surprisingly, GFP+ CD62L−/− Tregs were also able to home to peripheral lymph nodes.

CONCLUSIONS: These results suggest CD62L is dispensable for Treg mediated suppression of GVHD and that the function of the L-selectinhi Treg fraction may be due to the difference in the expression of other homing/migration molecules necessary for Treg migration into lymphoid and GVHD target organs.

Figure Figure
Cbl Mutants Interact with c-Kit and Lead to Factor Independent Growth and Transformation of a Myeloid Cell Line 32D.
Authors: Sargin Bülent , Bandi Srinivasa Rao , August Benjamin , Rensinghoff Marion , Choudhary Chunaram , Gonchi Pavan Nagaruri , Berdel Wolfgang E. , Müller-Tidow Carsten , Serve Hubert
Published: April 2020

The Cbl proto-oncogene products have emerged as important components of the signal transduction cascades downstream of both non-receptor and receptor tyrosine kinases (RTKs). By regulation of receptor trafficking and degradation, they have been shown to tightly regulate the intensity and amplitude of RTK activation. c-Kit belongs to the family of the class-III RTKs and plays an important role in the pathogenesis of acute myeloid leukemia (AML). So far, very little is known about the role of c-Cbl mutants in the role of c-Kit signaling. We analyzed the interaction of c-Cbl with c-Kit and the functional relevance of this interaction in the IL-3-dependent murine myeloid progenitor cell line 32Dcl3. We analyzed the effect of c-Cbl and two different dominant negative mutants of c-Cbl (Cbl-70Z and Cbl-R420Q) on c-Kit-ligand-activated internalization. The transfection of c-Cbl mutants, but not of wildtype c-Cbl, significantly inhibited receptor internalization, as analyzed by FACS analysis. Expression of Cbl-70Z in 32Dcl3 cells severely inhibited apoptosis induced by growth factor deprivation, as has been described before. However, when coexpressed with wildtype c-Kit, 32Dcl3 cells also rapidly proliferated in the absence of any exogenously added growth factors. We furthermore analyzed SCF induced c-Kit ubiquitination in the presence and absence of c-Cbl mutants. SCF induced rapid ubiquitination of c-Kit that was strongly reduced in the presence of Cbl-70Z and abolished by Cbl-R420Q. Also, the Cbl mutants altered the amplitude and changed the quality of c-Kit dependent signaling events. In colony assays we were able to show ligand independent colony growths in methyl cellulose only in the presence of wildtype c-Kit together with a c-Cbl mutant. Our results indicate that c-Cbl has an important role in c-Kit signal mitigation. Furthermore, they demonstrate that disturbed mechanisms of c-Kit internalization have important implications for its transforming potential, possibly in the development of AML.
Reciprocal Regulation of DSCR1 (Down Syndrome Critical Region 1) Expression and NFAT (Nuclear Factor of Activated T Cells) Activation in Megakaryocytes.
Authors: Kyttaelae Satu , Habermann Ivonne , Bornhaeuser Martin , Ehninger Gerhard , Kiani Alexander
Published: April 2020

NFAT (Nuclear Factor of Activated T cells) is a family of calcium-induced, calcineurin-dependent transcription factors, well characterized as central regulators of inducible gene expression in T lymphocytes but now known to function also in several other cell types in various adaptation and differentiation processes. Activation of NFAT by the phosphatase calcineurin is counteracted by several inhibitory kinases and can be completely blocked by the immunosuppressant Cyclosporin A. The Down syndrome critical region 1 (DSCR1; also termed CSP1, MCIP1 or RCAN1) gene belongs to the calcipressin family of endogenous calcineurin inhibitors and is expressed in several isoforms, one of which (isoform C, coded by exons 4–7) has been described to be a transcriptional target for NFAT in striated muscle, endothelial, and neural cells. The DSCR1 gene is located within the Down syndrome critical region of human chromosome 21 and is, together with 200–300 other genes, overexpressed about 1.5-fold in patients with Down syndrome (DS). Previously, dysregulation of NFAT signaling by overexpression of DSCR1 has been implicated in causing various of the pathophysiological features observed in DS patients. Children with DS also suffer from an about 500-fold increased incidence of acute megakaryocytic leukemia; the respective roles of NFAT or DSCR1 in megakaryocytes of either normal individuals or those with DS, however, has not yet been established. Here we show that DSCR1 is upregulated during megakaryocytic differentiation in a lineage-specific manner, and in mature megakaryocytes is further strongly induced by calcineurin stimulation. DSCR1 expression in megakaryocytes is regulated by NFAT, since overexpression of NFATc2 enhances, while overexpression of the specific inhibitor of NFAT activation, VIVIT, suppresses expression of the gene. We further demonstrate that DSCR1 does not only represent an NFAT target in megakaryocytes, but itself acts an inhibitor of NFAT signaling in these cells. Overexpression of DSCR1 in CMK cells as well as in primary megakaryocytes by retroviral transduction profoundly suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFATc2, as well as transactivation of an NFAT-dependent promoter construct. Finally, overexpression of DSCR1 in megakaryocytes markedly downregulated both the constitutive and induced expression of Fas Ligand, a pro-apoptotic gene recently established as a NFAT target in megakaryocytes. Together, these results suggest that DSCR1 acts as an NFAT-induced NFAT inhibitor in megakaryocytes and, when overexpressed, interferes with the expression of NFAT-dependent megakaryocytic genes.
A Human Lymphoid Progenitor Capable of Circulating and Seeding the Thymus.
Authors: Six Emmanuelle M. , Bonhomme Delphine , Monteiro Marta , Beldjord Kheira , Garrigue Alexandrine , Dal Cortivo Liliane , Rocha Benedita , Fischer Alain , Cavazzana-Calvo Marina , André-Schmutz Isabelle
Published: April 2020

Identification of a thymus-seeding progenitor originating from human bone marrow constitutes a key milestone in understanding and correcting defects in T-cell development. Here, we report the characterization of a novel human bone marrow lymphoid-restricted subpopulation which is part of the lineage-negative CD34+CD10+ progenitor population and which can be distinguished from B-cell-committed precursors by the absence of CD24 expression. We demonstrated that these Lin-CD34+CD10+CD24- progenitors lack myeloid and erythroid potential but can generate B, T and NK lymphocytes following culture on MS5 or OP9-hDelta1 stroma. The gene expression profile of this population, analyzed by a multiplex RT-PCR assay, revealed co-expression of RAG1, TdT, PAX5, CD3ε and IL-7Rα. These progenitors are not only present in the bone marrow but also in the blood throughout life, suggesting an ability to circulate. Moreover we showed that the Lin-CD34+CD10+CD24- cells also correspond to the most immature population of the thymus which gives rise to Lin-CD34+CD7+ T-cell precursors. Taken as a whole these findings unravel for the first time the existence of a postnatal lymphoid-restricted population which is capable of migrating from the bone marrow to the thymus.
Disruption of the Osteoblast Niche by G-CSF Induces Hematopoietic Stem Cell Quiescence and Loss of Long-Term Repopulating Activity.
Authors: Gopalan Priya K. , Christopher Matthew J. , Link Daniel C.
Published: April 2020

There is evidence that hematopoietic stem cells (HSC) are physically localized to specialized areas in the bone marrow termed the vascular and osteoblast niches. It is not clear if there are differences in the capacity of these niches to support HSC function. We and others previously showed that G-CSF treatment suppresses both osteoblast number and function, effectively eliminating the osteoblast niche. In contrast, G-CSF treatment has no apparent effect on the microvasculature in the bone marrow, suggesting that the vascular niche is intact. In this study, we utilized this system to assess the capacity of each niche to support HSC function. We previously reported that the competitive repopulation capacity of bone marrow isolated from G-CSF treated mice is markedly reduced. This is not due to a simple loss of HSC in the bone marrow, as the number of HSC, phenotypically defined as lineage-CD41-CD48-CD150+ (SLAM) or lineage-Kit+Sca+CD34- cells, was comparable to control mice. Moreover, the long-term repopulating activity of sorted SLAM cells from G-CSF treated mice was reduced. This repopulating defect is not secondary to impaired homing to the bone marrow, as direct intrafemoral injection of G-CSF treated bone marrow cells failed to rescue the engraftment defect. Since G-CSF is able to stimulate HSC proliferation, we predicted that the defect in repopulating activity might be secondary to loss of HSC quiescence. Contrary to our prediction, the percentage of quiescent HSC in the bone marrow was actually increased in G-CSF treated mice. Whereas 28.0 ± 3.4% of control SLAM cells were labeled after treatment with BrdU for 48 hours, only 7.5 ± 0.8% of SLAM cells isolated from G-CSF mice were labeled (p < 0.008). Moreover, the percentage of SLAM cells in G0, as determined by Hoechst and pyronin staining, was increased in G-CSF treated mice (80.3 ± 5.0% versus 65.5 ± 6.8% in untreated mice, p=0.104). To elucidate the molecular mechanisms by which disruption of the osteoblast niche leads to a loss of HSC activity, we performed RNA profiling experiments on SLAM cells sorted from G-CSF or saline-treated mice. Consistent with the quiescent phenotype, a significant increase in the expression of the cell cycle inhibitor, Cdkn1a (p21waf1), was observed in G-CSF treated SLAM cells. Collectively, these data show that the osteoblast and vascular niches are not functionally redundant and suggest that it is the osteoblast niche that is key to maintaining long-term repopulating activity of HSC.
Loss of Gfi1 Impedes Development of T-Cell Lymphoma upon Exposure to N-ethyl-N-nitrosourea but Predisposes to Severe Myleodysplastic Changes.
Authors: Khandanpour Cyrus , Duehrsen Ulrich , Möröy Tarik
Published: April 2020

Exogenous toxic substances often cause the initiation and development of leukemia and lymphoma by acting as mutagens. N-ethyl-N-nitrosourea (ENU) is a paradigmatic example for such a substance, which introduces point mutations in the genome through DNA damage and repair pathways. ENU is widely used to experimentally induce T-cell lymphomas in mice. We have used ENU to investigate whether the hematopoietic transcription factor Gfi1 is required for lymphomagenesis. The Gfi1 gene was originally discovered as a proviral target gene and a series of experiments with transgenic mice had suggested a role of Gfi1 as a dominant oncogene with the ability to cooperate with Myc and Pim genes in the generation of T-cell lymphoma. In addition, Gfi1 deficient mice showed a defect in T-cell maturation but also aberration in myeloid differentiation and an accumulation of myelomonocytic cells. ENU was administered i.p. once a week for three weeks with a total dose of 300mg/kg to wild type (wt) and Gfi1 null mice. Wild type mice (12/12) predominantly developed T-cell tumors and rarely acute myeloid leukemia, as expected. However, only 2/8 Gfi1 −/− mice succumbed to lymphoid neoplasia; they rather showed a severe dysplasia of the bone marrow that was more pronounced than in wt controls. These changes in Gfi1 null mice were accompanied by a dramatic decrease of the LSK (Lin-, Sca1- and c-Kit+) bone marrow fraction that contains hematopoietic stem cells and by a higher percentage (18%) of bone marrow cells, not expressing any lineage markers (CD4, CD 8, Ter 119, Mac1, Gr1, B220, CD3). In particular, we found that the LSK subpopulation of Gfi1 deficient mice showed a noticeable increase in cells undergoing apoptosis suggesting a role of Gfi1 in hematopoietic stem cell survival. In addition, Gfi1−/− bone marrow cells and thymic T-cells were more sensitive to DNA damage such as radiation and exposure to ENU than their wt counterparts pointing to a role of Gfi1 in DNA damage response. Our results indicate that Gfi1 is required for development of T-cell tumors and that a loss of Gfi1 may sensitize hematopoietic cells and possibly hematopoietic stem cells for programmed cell death. Further studies have to show whether interfering with Gfi1 expression or function might represent a tool in the therapy of leukemia.