bluuebunny/crossref_metadata_2025 huggingface dataset:
Naive and Non Naive CD4+ and CD8+ T Cell Subsets Distribution in Blood and Marrow Grafts and Their Impact on Patients’ Outcome after Allogeneic Stem Cell Transplantation.
Authors: Yakoub-Agha Ibrahim , Saule Pasquine , Grutzmacher Claudine , Cracco Pascale , Magro Leo , Bauters Francis , Villard Florence , Depil Stephane , Jouet Jean-Pierre , Dessaint Jean Paul , Labalette Myriam
Published: April 2020

Peripheral G-CSF-mobilized blood stem cells (PBSC), use as alternative to marrow stem cells (MSC), is associated with enhanced engraftment and accelerated hematopoietic recovery after allo-SCT. However, despite an increased number of donor T cells infused, the incidence of acute GVHD with PBSC appears to remain identical or less than with MSC. Recent works on the heterogeneity of the human CD4+ and CD8+ T cells have individualized 4 subsets: namely, naive (CCR7+CD45RA+), central memory (TCM, CCR7+CD45RA−), effector memory (TEM, CCR7−CD45RA−), and CD45RA+ effector memory cells (TEMRA, CCR7−CD45RA+). To our knowledge, the T cell subsets proportions in MSC and PBCSP grafts remain unknown. The impact of the infused T cell subsets on patients’ outcome is still to be investigated. Between September 2003 and July 2004, 25 consecutive hematopoietic allo-SCT (12 MSC and 13 PBSC) were considered for this study. Multiple combinations of immunophenotyping analysis were used to prospectively examine immune parameters related to graft as well as to early reconstitution. Early post-transplant complications including acute GVHD, relapse and infections were assessed. As expected, T cells subsets, B cells and NK cells numbers were significantly higher in PBSC grafts (versus MSC). However, proportions of CD4+ and CD8+ T-cell subsets were comparable in the 2 types of graft and similar to normal values observed with peripheral blood of healthy adults. Table below summarize the distribution of T cell subsets within T CD4+ and T CD8+ in the two types of graft. The incidence of acute GVHD was significantly higher in pts receiving > 5% of CD8+ TEM cells among infused CD8+ cells (p<0.05) suggesting a detrimental role of this CD8+ subset. Pts who developed acute GVHD, compared to those who did not, had a faster expansion of CD4+ and CD8+ TEM cells (p<0.003), and CD8+ TEMRA cells (p<0.05) after transplantation. Conclusion: this report contributes to the establishment of reference values regarding the distribution of T cell subsets within PBSC and MSC graft. Although, the quantity of infused T cell does not seem to influence early complications after allo-SCT, the qualitative composition of graft regarding T cell subsets, appears to be of great importance in acute GVHD occurrence.

MSC PBSC CD4+ naive 45% 48% CD4+ TCM 25% 24% CD4+ TEM 20% 16% CD4+ TEMRA 5% 2% CD8+ naive 40% 50% CD8+ TCM 4% 7% CD8+ TEM 23% 14% CD8+ TEMRA 29% 22%
Excellent Long-Term Survival of Patients with Steroid-Refractory and Steroid-Dependent Acute Graft-Versus-Host Disease after Extracorporeal Photochemotherapy.
Authors: Greinix Hildegard T. , Knobler Robert M. , Worel Nina , Mitterbauer Margit , Schulenburg Axel , Schneeberger Armin , Karlhofer Franz , Hoecker Paul , Leitner Gerda , Rosenmayr Agathe , Kalhs Peter
Published: April 2020

Despite posttransplantation immunosuppressive therapy, acute graft-versus-host disease (GVHD) remains a major cause of sickness and death. Second-line therapies for steroid-refractory acute GVHD have been used with limited success. Extracorporeal exposure of peripheral blood mononuclear cells to the photosensitizing agent 8-methoxypsoralen and UV-A radiation (ECP) has been shown to be effective in the treatment of selected diseases mediated by T cells. We have reviewed the responses and long-term outcome of 59 hematopoietic stem cell transplant (HSCT) patients treated from 1996 to 2003 with ECP for steroid-refractory acute GVHD, defined as progression or no improvement of acute GVHD after a minimum of 4 (range, 4–49, median 17) days of treatment with prednisone (n=37) or steroid-dependent acute GVHD, defined as flare-up of GVHD during prednisone taper (n=22). Patients received HSCT from 17 related and 42 unrelated donors. In 28 cases an HLA-mismatch between recipient and donor was present. Prior to ECP, grade III–IV GVHD was observed in 23 patients (39%) and grade II GVHD in 36 (61%). Organs involved included skin in 97% of patients, liver in 39%, and GI tract in 17%. Treatment consisted of ECP on two consecutive days per week (=1 cycle) for a median of 7 (range, 1–45) cycles administered within a median of 3 (range, 0.5–31) months in addition to cyclosporine A and prednisone. Three months after initiation of ECP complete resolution of GVHD was achieved in 82% of patients with cutaneous, 61% with liver, and 61% with gut involvement. Complete responses were obtained in 86% of patients with grade II, 55% of patients with grade III, and 30% of patients with grade IV acute GVHD. Probability of transplant-related mortality (TRM) at 4 years after HSCT is 15% in patients with complete response to ECP compared to 88% in patients not responding completely. After a median follow-up of 46 (range, 9–45) months since discontinuation of ECP, 28 (47%) patients are alive including 22 without chronic GVHD. Probability of survival (OS) at 4 years after HSCT is 59% in patients with complete response to ECP compared to 11% in patients not responding completely. Besides response to ECP only organ involvement and grade of GVHD at start of ECP, and ability to timely taper steroids during ECP had a significant impact on both TRM and OS. Thus, ECP is an effective adjunct therapy for acute steroid-refractory and steroid-dependent GVHD. Our long-term results demonstrate durability of responses without adverse events.
IL6 Protection of Myeloid Leukemic Cells Against P53 Induced Apoptosis Is Mediated by Activation of P13kinase/AKT and Stabilization of C-FLIP.
Authors: Vesely Diana L. , Hoffman Barbara , Liebermann Dan A.
Published: April 2020

Myeloid leukemic cells (M1) proliferate continuously in culture unless induced by Interleukin-6 (IL6) to undergo a terminal differentiation program into macrophages, followed by apoptosis. M1 cells lack the tumor suppressor p53, which is a critical determinant of the cellular decision to either growth arrest and repair DNA damage or to undergo apoptosis. Activation of a temperature sensitive p53 protein (p53 val) at the permissive temperature in M1 cells results in rapid apoptosis. IL6 treatment blocks this p53-mediated apoptosis. Towards understanding the basis for this p53-mediated apoptosis and its abrogation by IL6, we have shown that at the permissive temperature p53 activates the pro-apoptotic Fas/CD95 pathway by up regulating the Fas/CD95 receptor and cleaving antiapoptotic c-FLIP. On the other hand, antagonistic Fas antibody protects against apoptosis. IL6 decreases Fas/CD95 ligand expression and prevents cleavage of FLIP. Treatment of M1p53ts cells with the specific Akt inhibitor Ly294002 abrogated IL6 protection and resulted in Flip cleavage, suggesting that IL-6 blocks apoptosis by phosphorylating and activating P13kinase/Akt, which in turn promotes FLIP stability. Inhibition of the ERK pro-survival pathway did not abrogate IL6 protection against p53 apoptosis like Akt inhibition caused. Nevertheless M1p53ts-Flip cell lines fail to completely protect against p53 mediated apoptosis, suggesting that FLIP is itself insufficient to protect against apoptosis. Therefore we examined what other proteins may synergize with FLIP to protect from apoptosis, and found that that the pro-survival bcl2 protein family member MCL-1 is strongly up regulated by IL6 in our M1p53 cell line. Generation of M1p53-FLIP-MCL1, M1p53-MCL1, and M1p53 -uncleavable FLIP cell lines is underway to determine whether there is synergy between MCL-1 and FLIP in escape from p53 mediated apoptosis of M1p53 cells. Thus far taken together these data support a model for leukemic progression where cells that acquire the ability to produce an autocrine survival factor, such as IL6, can bypass normal p53 surveillance function by targeting downstream inhibitors of apoptosis such Akt, c-Flip and MCL-1.
Cytokine Production and Hematopoiesis Supporting Activity of Cord Blood Derived Unrestricted Somatic Stem Cells.
Authors: Koegler Gesine , Radke Teja , Lefort Aurelie , Sensken Sandra , Fischer Johannes , Sorg Rudiger Volker , Wernet Peter
Published: April 2020

Particularly within the last several years cord blood (CB) has become a well established hematopoietic source for unrelated transplantation in children. Assuming, however, a threshold of 2x107 nucleated cells (NC)/kg body weight required for transplantation in adults over 70kg, an average of only 25% of the CB units presently stored in the major CB banks contain sufficient NC to safely engraft patients. Thus far the majority of studies have assessed selective cytokine driven expansion of progenitors for transplantation, which however may be associated with exhaustion of stem cells and an increase of GvHD in adult patients. Therefore, we assessed cytokine production and hematopoiesis supporting stromal activity of CB derived unrestricted somatic stem cells (USSC, Koegler et al. JEM2004: 200: 2: 1–13) in comparison to bone marrow mesenchymal stem cells (BMMSC) and hematopoietic progenitor expansion solely driven by recombinant cytokines. Initiation of USSC generation was initiated from fresh (n=325) and cryopreserved CB (n=66). In the presence of H5100 medium/10−7M dexamethasone, USSC cultures from fresh CB were initiated from 57.1% of CB samples (n= 52 out of 91 CB samples), whereas in DMEM-medium/10−7M dexamethasone the frequency was 31.6% (n=74 out of 234 CB samples). The generation frequency from frozen HES separated CB was only 19.5%. Cytokine production by USSC and BMMSC was determined qualitatively by cytokine mRNA expression array analyses or quantitatively by Luminex or ELISA analyses. To analyze hematopoiesis supporting activity, CB CD34+ cells were expanded in co-cultures with USSC, BMMSC or in the presence of Flt3-L, SCF, TPO. Expansion of CD34+ cells, total cells and colony-forming cells (CFC) were determined after 1, 2, 3 and 4 weeks of culture. USSC constitutively produced SCF, LIF, TGF-1ß, M-CSF, GM-CSF, VEGF, IL-1ß, IL-6, IL-8, IL-11, IL-12, IL-15, SDF-1a and HGF. When USSC were stimulated with IL-1ß, G-CSF was released. Production of SCF (p= 0.0104) and LIF (p= 0.046) was significantly higher in USSC compared to BMMSC. At 1, 2, 3 and 4 weeks, respectively co-cultivation of CD34+ cells on the USSC layer resulted in a 14.62±1.1-fold, 110.1±17.9-fold, 151.8±39.7-fold and 183.6±40.4-fold amplification of total cells and in a 30.56±4.4-fold, 101.4±27.5-fold, 64.67±15.8-fold and 29.4±3.1-fold amplification of CFC. Expansion achieved was significantly higher for USSC compared to BMMSC feeder layers. USSC produce functionally significant amounts of hematopoiesis supporting cytokines and are able to expand CD34+ cells from CB in significantly higher amounts compared to BMMSC. Therefore the USSC is a suitable candidate to direct ex-vivo expansion of hematopoietic CB cells for short term reconstitution.
Increased Adipocytogenesis and Hematopoiesis in Long-Term Bone Marrow Cultures from SMAD3−/− Mice.
Authors: Cao Shaonan , Zhou Shuanhu , Epperly Michael W. , Glowacki Julie , Shields Donna , Greenberger Joel S.
Published: April 2020

The abrogation of an intact TGFß/TGFß receptor signal transduction pathway in SMAD3−/− mice is associated with a decreased inflammatory response, and decreased acute and late response to ionizing irradiation damage. We tested the role of SMAD3 in bone marrow cellular capacity for hematopoiesis and in marrow stromal cellular potential for adipocytogenesis. Long-term bone marrow cultures (LTBMCs) were established from SMAD3−/−, +/−, and +/+ mice. Permanent clonal bone marrow stromal cell (MSC) lines were established from each mouse strain. The SMAD3+/+ and SMAD3+/− LTBMCs generated hematopoietic progenitor cells (by CFU-GEMM assay), and maintained active cobblestone island numbers and production of non-adherent cells for 22–23 weeks. In striking contrast, the SMAD3−/− LTBMCs demonstrated a significant increase in longevity of hematopoiesis as measured by each assay for over 34 weeks and non-adherent cells continued to form GEMM beyond 34 weeks. The number of CFU-GEMM per 5x104 cells (d 14) in SMAD3−/− cultures (188 ± 8.7) was 1250% greater (p<0.0001) than in SMAD3+/+ cultures (15.3 ± 2.3). Microscopic inspection at 27 weeks showed that more (16.5%, p<0.001) SMAD3−/− adherent cells contained lipid droplets (6.6% ± 0.7) compared with SMAD3+/+ adherent cells (0.4% ± 0.2). Because of that difference, we examined the regulation of adipocyte differentiation in MSC lines from those mice. First, there was more extensive (37-fold) adipocyte differentiation in SMAD3−/ − than SMAD3+/+ cultures. Second, there was a 7-fold attenuation of TGF-ß inhibition of adipocytogenesis in cells lacking SMAD3. RT-PCR evaluation showed greater expression of the adipocyte gene, ADIPSIN, and absence of its downregulation by TGFß in SMAD3−/− compared to SMAD3+/+. The data suggest that SMAD3 is a critical inhibitor of adipocyte differentiation. The data document pleiotrophic effects of interruption of the TGFß/TGFß receptor signal transduction cascade for both inhibition of hematopoietic support capacity and adipocyte differentiation of the bone marrow microenvironment.
Indian Hedgehog Stimulates Proliferation of Short-Term NOD/SCID-ß2mnull Repopulating Cells on Coculture with Human Stromal Cells.
Authors: Kobune Masayoshi , Kawano Yutaka , Kato Junji , Ito Yoshinori , Chiba Hiroki , Kuribayashi Kageaki , Takimoto Rishu , Matsunaga Takuya , Hamada Hirofumi , Niitsu Yoshiro
Published: April 2020

Hematopoietic stem cells (HSCs) represent a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However, the mechanisms involved in controlling the differentiation and self-renewal of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has been shown to play an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in postnatal hematopoiesis using coculture system between CD34+ cells and human stromal cells. Ihh gene transfer into hTERT-stromal cells enhanced the expression of BMP4, angiopoietin-1 and Wnt5A, and their hematopoietic supporting potential was elevated compared with control stromal cells, as indicated by the colony-forming units in culture (CFU-C, 26±2 vs. 59±3-fold of the initial cell number; CFU-Mix, 63±37 vs. 349±116). Engraftments of NOD/SCID-ß2mnull repopulating cells (RCs) at 8 weeks post-transplantation expanded on Ihh-stromal cells were significantly higher compared with control coculture results and engraftments were neutralized by addition of an anti-hedgehog antibody. Limiting dilution analysis indicated that SCID RCs proliferated more efficiently on Ihh-stromal cells than those on control stromal cells. The degree of expansion on Ihh-stromal cells was estimated to be 6.6-fold greater than that of control stromal cells, according to the Poisson predicted frequency. However, engraftment of NOD/SCID-ß2mnull RCs was decreased by 13 weeks post-transplantation of hematopoietic cells that had been expanded on either Ihh-stromal cells or control stromal cells, although engraftment of human hematopoietic cells that were expanded on Ihh-stromal cells is higher than that for cells expanded on control stromal cells (%CD45+ cells: 3.28±0.60 vs. not detected). These results suggest that Ihh-stromal cells are quite potent to support human short-term RCs in NOD/SCID-ß2mnull mice. Thus, hedgehog signaling is considered to play an important role in the hematopoietic microenvironment.
Identification of Tenascin-C as a Key Molecule Determining the Stromal Cell Dependent Erythropoiesis.
Authors: Seki Masanori , Kameoka Junichi , Takahashi Shinichiro , Harigae Hideo , Sasaki Takeshi , Obinata Masuo
Published: April 2020

Stromal cells in the bone marrow provide an optimal microenvironment for hematopoiesis through cell-to-cell interaction as well as cytokine production. We have previously established 33 bone marrow stromal cell lines from SV40 T-antigen transgenic mice, and demonstrated the lineage-specific activity of supporting hematopoiesis (J Cell Physiol1995: 164; 55). Of the 33 cell lines, 27 clones showed stimulatory abilities to support large erythroid colony formation in the presence of erythropoietin, while 6 cell lines did not. Since these activities were not dependent on the amount of produced cytokines, the existence of unknown molecules responsible for the selective activity of erythropoiesis has been implicated. To identify molecules expressed on bone marrow stromal cells which are responsible for determining the stromal cell dependent erythropoiesis, we compared the gene expression profiling by cDNA microarray between cell lines which support erythropoiesis (E+; TBR 9, 184, 31-2) and cell lines which do not (E-; TBR 17, 33, 511). Out of 7226 genes examined, 138 genes were up-regulated 3-fold or more, and 282 genes were down-regulated 3-fold or more, in E+ cell lines. Among these genes, we have selected one of the up-regulated genes, tenascin-C, as a candidate for erythroid progenitor supporting genes, and examined its expression and function in detail. First, we confirmed the expression of tenascin-C by real time quantitative RT-PCR. The expression of tenascin-C adjusted by the expression of GAPDH in the three E+ cell lines were all higher than the three E- cell lines with a mean of 3.6 times. Next, we examined the effect of suppressing tenascin-C expression by small interfering RNA (siRNA) on the erythroid colony formation. siRNAs were synthesized by in vitro transcription method, and were transfected into TBR cell lines by lipofection. At 24 hours after the transfection of tenascin-C siRNA, the expression of tenascin-C was reduced to 10 to 40% of the control siRNA as determined by real time quantitative RT-PCR. The number of erythroid colony dependent on TBR 9 and TBR 184 in the presence of tenascin-C siRNA was significantly lower than that in the presence of control siRNA (13.0±6.5 versus 0.75±0.95 colonies/104 fetal liver cells; p<0.05). These results suggest that tenascin-C plays an important role in the stromal cell dependent erythropoiesis.
Biochemical Characterization and Gene Expression Profiling of Cytarabine Treated Stromal Fibroblasts Reveal Regulatory Mechanisms Affecting Hyaluronan and Heparan Sulfate Proteoglycans.
Authors: Zweegman Sonja , Kessler Floortje L. , Kerkhoven Ron , Heimerikx Mike , Huijgens Peter C. , Drager Angelika M. , van den Born Jacob
Published: April 2020

High dose chemotherapy and radiation, as applied in stem cell transplantation procedures, have been found to impair the hematopoiesis-supportive capacity of bone marrow stroma. However, the molecular mechanisms involved are far from elucidated yet. As bone marrow stromal glycosaminoglycans (GAGs) play an important role in various regulatory steps in hematopoiesis, we examined the effect of chemotherapy on the synthesis and composition of GAGs and gene expression profiles of the hematopoiesis supportive M210B4 fibroblastic stromal cell line. To this end GAGs were metabolically labeled with 3H-glucosamine and 35S-sulfate, and analyzed by conventional biochemical techniques.

Cytarabine treatment resulted in a pronounced increase (1.6±0.3-fold increase) in hyaluronan (HA) and a reduction in the content (47±7% reduction) and in the extent of sulfation (3H-glucosamine to 35S-sulfate ratio increased from 2,1 versus 2,6) of heparan sulfate proteoglycans (HSPG). Gene expression analysis showed a corresponding increase in hyaluronan synthase 1 (Has 1) gene expression and a decrease in hyaluronidase 2 (Hyal 2) gene expression. The decrease in HSPG was found to correspond with downregulation of the Exostosin 1 (Ext 1) gene, encoding a bifunctional glycosyltransferase, required for the biosynthesis of HS. Finally, there was a trend towards decreased expression of the N-deacetylase/N-sulfotransferase-1 (Ndst1) gene, which might explain the decrease in sulfation of HSPG after cytarabine treatment, as deacetylation is required for sulfation of HSPG to occur.

The functional consequence of the decrease in HSPG was investigated by Stromal Derived Factor-1α (SDF-1α) binding. SDF-1α was added to either untreated or treated M210B4 cells and allowed to bind for 90 minutes. After washing, bound SDF-1α was quantified by staining with the anti-SDF-1α antibody K15C. Cytarabine treatment resulted in a 31.1 ± 11.8% decline in K15C binding, indicating a decline in GAG-mediated binding of SDF-1α (n=6, Mann Whitney test, p<0.05). In conclusion, cytarabine treatment alters the expression of genes involved in GAG synthesis and degradation, thereby affecting the amount and structure of stromal GAGs. Accordingly, the chemokine-binding capacity of HSPG was negatively affected. In view of the critical role of SDF-1α in migration of stem cells and its presumed participation in the creation of the stem cell niche, our results suggest that chemotherapy-induced changes in stromal GAGs might affect homing and hematopoiesis. In addition, because of known functional capacities of HA, malignant hematopoiesis as well as tumor growth and invasion may be promoted.
Hodgin’s Lymphoma in Elderly Patients: A Comprehensive Analysis of the German Hodgkin Lymphoma Study Group (GHSG).
Authors: Engert Andreas , Ballova Veronika , Haverkamp Heinz , Pfistner Beate , Diehl Volker
Published: April 2020

Background: With improved prognosis of patients with Hodgkin’s lymphoma (HL), interest increasingly focuses on high-risk groups such as elderly patients. We thus performed a retrospective analysis of the German Hodgkin Lymphoma Study Group (GHSG) database to determine clinical presentation, toxicity of treatment and outcome of elderly HL patients.

Methods: A total of 4251 patients included in the GHSG studies HD5-9 were analysed of whom 372 (8.8%) were 60 years or older and 3879 (91.2%) were younger than 60 years. Patient characteristics, treatment results, toxicity, failure free survival (FFTF) and overall survival (OS) were compared.

Results: The median age of the two cohorts was 65 and 31 years, respectively. Statistically significant differences in patient characteristics were as follows: elderly patients had more frequently mixed cellularity subtype (35% vs. 19%), B-symptoms (50% vs. 43%) and elevated ESR (59% vs. 51%). Less frequently observed in elderly patients were nodular sclerosis subtype (41% vs. 66%), large mediastinal mass (9% vs. 20%), bulky disease (49% vs. 60%) and Karnofski index <8, (11% vs. 3%). Acute toxicity during chemotherapy was higher in elderly patients (87% vs 79%). The most obvious differences were observed for infections (17% vs 6%). The significantly higher rate of infections was due to more severe leukopenia in elderly patients (grade IV: 41% vs 25%). As a result, significantly fewer elderly patients received at least 85% of intended chemotherapy dose (74% vs. 90%). At a median follow-up of 65 months, 38% and 11% of patients respectively died. The survival analysis showed a statistically significant poorer prognosis for elderly patients in terms of 5-year OS (65% vs. 90%), FFTF (60% vs. 79%) and Hodgkin-specific FFTF (73% vs. 82%).

Conclusion: Elderly patients have a poorer risk profile compared to younger HL patients and experience more severe treatment-associated toxicity. Higher mortality during treatment as well as lower dose-intensity are the major factors explaining the poorer overall outcome of elderly Hodgkin lymphoma patients.
Fertility in Male Patients with Hodgkin’s Disease - Results from the German Hodgkin Lymphoma Study Group (GHSG).
Authors: Sieniawski Michal , Josting Andreas , Breuer Kai , Diehl Volker , Engert Andreas
Published: April 2020

Background:

Treatment results in Hodgkin Disease (HD) have improved tremendously over the last two decades. Therefore long term sides effects of therapy as infertility are of growing importance. However, patients (pts) with HD have increased risk for inadequate semen quality even prior to treatment. To investigate the influence of disease and therapy on the fertility status in pts with HD we performed semen and hormone analysis before and after treatment.

Patients and Methods:

All analysis were evaluated in pts with first diagnosis of HD enrolled into trials of the GHSG between 1988 and 2002, in 40 centers in Europe. Pts had no history of chemotherapy or radiotherapy. All analysis were evaluated according to WHO-guidelines.

Results:

We included 243 male pts with a median age of 26,5 years (range 16–57,5 years). At first diagnosis 138 pts were in clinical stage (CS) I/II, 105 in CS III/IV, and systemic symptoms were present in 106 pts. 202 pts underwent fertility screening before therapy: normospermia was diagnosed in 40 (20%) pts and 162 (80%) had inadequate semen quality. Normal values of FSH, LH, and testosterone were found in 140/151 (93%), 99/125 (79%), and 84/101 (83%) of pts respectively. 112 pts underwent at last one fertility screening after therapy. Treatment consisted of chemotherapy alone in 10 pts, in 9 pts of radiotherapy alone and in 93 pts of combined modality. In 40 (36%) pts a recovery of spermatogenesis was observed. Normal values of FSH, LH, and testosterone after treatment were found in 32/63, 40/51, and 36/40 pts respectively. Table 1 shows time of onset of spermatogenesis and normal values of fertility hormones after the end of therapy.

Table 1

1st year 2nd year 3rd year after 3rd year Spermatogenesis (40/112) 7 (18%) 9 (24%) 10 (26%) 14 (32%) Normal FSH (32/63) 8 (25%) 12 (38%) 6 (19%) 6 (19%) Normal LH (40/51) 13 (33%) 16 (40%) 6 (15%) 5 (13%) Normal testosterone (36/40) 17 (47%) 12 (33%) 1 (3%) 6 (17%)

Recovery from azoospermia was observed in 8 (89%) pts treated with radiotherapy, in 1 (10%) pt treated with chemotherapy alone and in 31 (33%) pts treated with combined modality. Recovery rate from azoospermia was lower in pts treated with alkylating agents, BEACOPP chemotherapy, with systemic symptoms and elevated BSG.

Conclusions:

We confirmed that HD pts had inadequate semen quality even prior to treatment. The majority of pts had azoospermia after treatment, but recovery of spermatogenesis was observed, in general 2 years after the end of therapy. Hormone values usually recovered in the first two years. Patient treated with radiotherapy alone recovered in higher frequency. A detailed analysis (including multivariate analyses) will be presented at the annual meeting.
Bleomycin Pulmonary Toxicity Does Impact on Hodgkin’s Disease Outcome.
Authors: Martin William G. , Ristow Kay M. , Colgan Joseph P. , Habermann Thomas M. , Ansell Stephen M.
Published: April 2020

Background: Bleomycin-containing chemotherapy regimens have become the mainstay of treatment in Hodgkin’s Disease (HD). The risk of bleomycin pulmonary toxicity (BPT) ranges from 0–46% and mortality rates range from 0–27%. Although there is no excepted standard treatment for BPT, corticosteroid treatment, withholding bleomycin from subsequent chemotherapy, and proceeding with a non-bleomycin containing regimen is the most common approach. We reviewed our experience to better delineate outcome and appropriate treatment in these patients.

Methods: We reviewed all patients managed for HD at Mayo Clinic, Rochester, from January 1986 to February 2003. BPT was defined by the presence of pulmonary symptoms, bilateral interstitial infiltrates, and no evidence for an infectious etiology.

Results: A retrospective review identified 141 patients with HD treated with a bleomycin-containing regimen. The mean age of patients at diagnosis was 34 years. Fifty four percent were males. The histology was nodular sclerosing in 85%. The Hasenclever index was 0 in 15%, 1 in 35%, 2 in 24%, 3 in 16%, 4 in 9%, and >5 in 1%. Frontline chemotherapy included ABVD in 57%, MOPP-ABV(D) in 33%, COPP-ABV in 8%, BEACOPP in 3%, and Stanford V in 2%. Forty one percent of patients received radiation. The 5-year overall survival (OS) and progression free survival (PFS) for all patients were 87% and 79% respectively. BPT was observed in 18% of patients (25/141). There was a significant decrease in OS, 63% in patients with BPT versus 90% in non-BPT patients at 5 years (p=0.0013). The mortality from BPT was 4.2% (6/141) in all patients and 24% (6/25) in patients who developed the pulmonary syndrome. These 6 patients all died within 9 months of their HD diagnosis from BPT. The Hasenclever index was 3 or less in 5 of these 6 patients. CR rates were equal at 91% and 93% in BPT and non-BPT patients respectively (p=0.299). Twenty two percent (31/141) of patients, either symptomatic or asymptomatic, had bleomycin omitted at any time from their regimen with no difference in OS or PFS, 83% versus 88% (p=0.369) and 82% versus 78% (p=0.853) respectively. The mean bleomycin dose was 84 mg/m2 (range of 20–180 mg/m2). Bleomycin dose had no impact on OS or PFS. In BPT patients subsequent non-bleomycin therapy included AVD 44%, MOPP-AV 31%, or no further chemotherapy 25%. The five-year OS was equal between the AVD and MOPP-AV groups at 91%. However in patients who received no further chemotherapy 5-year OS was 30% (p=0.0001).

Conclusion: Bleomycin pulmonary toxicity (BPT) results in a significant decrease in OS survival at 5 years in patients being treated for Hodgkin’s Disease. In patients who do not die acutely from pulmonary toxicity both OS and PFS appear equal despite the omission of bleomycin.
Preliminary Results of a Phase I/II Trial of BBR-2778 (Pixantrone) in Combination with Fludarabine, Dexamethasone, and Rituximab (FPD-R) in the Treatment of Patients with Relapsed/Refractory Indolent Non-Hodgkin’s Lymphoma (NHL).
Authors: Fayad Luis , Liebmann James , Modiano Manuel , Cohen Gary I. , Pro Barbara , Romaguera Jorge , Davite Cristina , Lymboura Margarita , Comis Silvia , Laffranchi Bernard L.
Published: April 2020

Background: Fludarabine-mitoxantrone-dexamethasone-rituximab (FND-R) is a combination chemotherapy with significant activity in untreated and relapsed indolent B-cell lymphoma patients (pts). Pixantrone is a novel aza-anthracenedione that has no delayed cardiotoxicity in animal models and has single agent activity in NHL. We conducted an outpatient phase I/II trial, to define the recommended dose (RD) of pixantrone when substituting for mitoxantrone in the FND-R regimen, and the safety and efficacy of this regimen in pts with relapsed or refractory indolent NHL.

Methods: Pts received pixantrone with fludarabine (25mg/m2/day, days 1–3), dexamethasone (20mg/day, days 1–5), and rituximab (375mg/m2/day on day 1) in a 28 day regimen (FPD-R). Based on previous single agent clinical studies, the starting dose of pixantrone was defined as 80mg/m². The RD was established to be 120mg/m2 and the protocol was amended to treat patients in the phase II part.

Results: 9 pts (6 males) with a median age of 65 years (range 41–78) were included in the dose-finding part of the study. Following the protocol amendment, 23 pts (11 males) of WHO performance status 0–1, median age 61 (range 32–78) have been enrolled in the currently ongoing phase II part. Pathology included SLL/CLL, follicular grade I, follicular grade II, MALT, marginal cell, and other indolent B-cell lymphomas. All pts had received a prior anthracycline-containing regimen (median number 1, range 1–4). The study regimen was well tolerated; median number of courses received was 5 (range 2–8). Grade 4 toxicities were neutropenia in 10 pts, leukopenia in 5 pts, and thrombocytopenia in 1 pt. No clinically significant cardiac events or decreases in LVEF ≥20% were noted. However 4 pts went below 50% LVEF including 1 pt who was below 50% at baseline. Non-hematologic adverse events were primarily grade 1 or 2 in severity. Response rate was 75% for the 20 pts evaluable for response, with 11 (55%) complete remission [7 confirmed (CR), 4 unconfirmed (CRu)], and 4 (20%) partial remission (PR). Two responders went onto bone marrow transplant (BMT): one had a CRu and one a PR. A third patient was pending BMT after CR. At a median follow-up of 345 days, the median duration of response has not been reached, as only two pts have progressed: the first progression occurred at 113 days, and the second pt progressed at 699 days. The remaining pts are still in remission.

Conclusions: The RD of pixantrone in the FPD-R regimen is 120mg/m2. The primary toxicity is hematologic. The regimen can be given on an outpatient basis, is associated with major responses, and is very well tolerated in relapsed and refractory indolent NHL pts.
Immunodeficiency Associated with a STAT-5b Mutation and Growth Hormone Insensitivity.
Authors: Cleary Aileen M. , Hwa Vivian , Tu Wenwei , Dionis Kira , Bezrodnik Liliana , Teper Alejandro , Gaillard Maria , Heinrich Juan J. , Rosenfeld Ron G. , Lewis David B.
Published: April 2020

We recently identified a homozygous mutation in the STAT-5b (signaling transducer and activator of transcription 5b) gene in a female patient with growth hormone insensitivity. The patient had significant growth retardation (height -7.5 SD), despite normal levels of growth hormone (GH) and GH binding protein. Analysis of the GH receptor signaling pathways indicated that the mutation (Ala630 to Pro630) resulted in a protein that could not be activated by GH, and subsequent dysregulation of STAT-5b-dependent genes, such as IGF-I and IGFBP-3. This abstract details an immunodeficiency due to a lack of functional STAT5b. Clinically, the patient had chronic diarrhea as an infant, and suffered several severe infections, including persistent lymphocytic interstitial pneumonia (LIP), a condition associated with children who are HIV-positive or immunodeficient. Laboratory studies showed hypergammaglobulinemia, T-cell lymphopenia with a normal CD4:CD8 T-cell ratio, and total B and NK cells within the normal range. Immunologic analysis showed decreased interleukin (IL)-2 receptor α expression in response to IL-2 on both CD4 and CD8 T cells, despite normal proliferation to mitogens, and normal anti-CD3 proliferation by CFSE analysis. IL-2 mediated γc (common gamma chain) upregulation, induction of CD40-ligand and interferon-γ production by intracellular cytokine analysis in response to either the bacterial superantigen SEB or anti-CD3/anti-CD28 mAb stimulation were normal, suggesting that STAT5b is not necessary for these functions. Importantly, perforin expression in response to IL-2 was decreased in CD8 T cells suggesting that both decreased CD8 number and function contribute to this immunodeficiency. Interestingly, NK cells failed to increase adhesion molecules CD54 or CD58 in response to IL-2 suggesting impaired NK cell function. Lastly, EBV transformed B cell lines were unable to phosphorylate STAT5b in response to IL-2 and IL-7 suggesting an intrinsic B cell defect that may affect immunoglobulin production. Thus, the STAT5b −/ − mutation appears to limit the effects of important γc-containing cytokines on CD4, CD8, NK and B cells, resulting in increased susceptibility to infection.
Tumor Localization and Quantitation of Adoptively Transfered T Lymphocytes in a Murine Model.
Authors: Hokland Marianne , Petersen Mikkel S. , Fleischer Charlotte C. , Stødkilde-Jørgensen Hans , Hansen Søren B. , Skands Ulrik , Gundersen Hans J.G. , Agger Ralf
Published: April 2020

Tracking adoptively transferred antigen-specific T lymphocytes is an important prerequisite for devising better protocols for cellular therapy. To this end we have developed a highly sensitive method for “in situ” visualization of labelled lymphocytes in vivo by combined PET and magnetic resonance imaging (MRI) to monitor the distribution of adoptively transferred tumour-specific T cells in a mouse model system. Moreover, quantitation of the adoptively transferred cells in tumor was performed by flow cytometry.

C57BL/6J mice carrying subcutaneous tumours of the ovalbumin (OVA)-expressing malignant melanoma cell line B16-OVA were adoptively transferred with OVA-specific CD8+ T cells labelled with 124IdU. Five days after transfer of T cells, mice were killed and subjected to PET and MR imaging. Using a newly developed method for co-registration of the two image modalities, the anatomical localisation of the transferred cells was visualised and the amount of radioactivity in various anatomical locations very accurately determined.

For quantitation of tumor infiltrating non-labelled OVA-specific CD8+ T cells by flow cytometry (using AbsoluteCount Beads), tumors were removed from mice day 1 until day 8 following adoptive transfer (6 mice/group) and prepared for single cell suspension before labeled with anti-CD8-FITC and SIINFEKL-Tetramer-PE.

Results showed a clear tumor localization of the adoptively transferred OVA-specific T cells in the tumours. In two independent experiments comprising 12 and 13 evaluable mice, respectively, we found a mean value of 0.909 +/− 0.468 Bq and 0.926 +/− 0.553 Bq in the tumours, and only 0.182 +/− 0.479 Bq and 0.026 +/− 0.480 Bq in the corresponding contralateral control volumes. The difference in activity between the tumour regions and the control regions was statistically highly significant with 2p-values of 0.002 and 0.006 for the two experiments. Using flow cytometry it was shown that the number of OVA specific T lymphocytes accumulating in tumor gradually increased until day 5 after transfer when an average of 3.3 million SIINFEKL-specific cells per gram tumor tissue was found. From day 5 until day 8 the number of SIINFEKL-specific cells per gram tumor tissue fluctuated at a fairly constant level.

This method presented for tracking adoptively transfered tumor specific T lymphocytes represent a significant advancement for studies of adoptively transferred specific T cells, and could potentially be developed for diagnostic purposes. Moreover, since these studies show that tumor-specific T cells home to subcutaneous tumours in substantial numbers, we suggest that these migrating cells could be employed in a new form of therapy as carriers of toxic substances to tumors.
Efficient and Reproducible Retroviral Infection of Primary Human Natural Killer Cells.
Authors: Becknell Brian , Trotta Rossana , Yu Jianhua , Ding Wei , Mao Hsiaoyin C. , Hughes Tiffany , Marburger Trent , Caligiuri Michael A.
Published: April 2020

Molecular characterization of human natural killer (NK) cells will require targeted gene delivery to inhibit and activate specific signaling pathways, yet to our knowledge, an effective means to deliver such products for long-term gene expression without disrupting normal cellular processes has not been described. In this study we have developed a retroviral strategy to effectively express gene products in the NK cell, whereby its effector functions of cytotoxicity and cytokine production remain intact. Using an EBV/retroviral hybrid vector PINCO, we demonstrate infection of human peripheral blood NK cells with simultaneous expression of a marker for infection - the enhanced green fluorescent protein (EGFP) - along with various genes of interest. This technique results in successful infection of the CD56dim NK population that predominates among human peripheral blood NK and is the effector of antibody-dependent cellular cytotoxicity (ADCC) and natural killing. In addition, we demonstrate infection of the CD56bright NK subset as well as the NK-92 and NK-L cell lines. Finally, we modify PINCO to express a cytoplasmically truncated murine CD8 molecule in place of GFP. The resulting vector enables us to transduce NK cells with multiple genes of interest simultaneously and provides an alternative purification method to FACS by using magnetic beads. In summary, we have devised an efficient and highly reproducible methodology for the targeted delivery of gene products to human NK cells that should now provide opportunities to dissect the molecular processes critical to normal NK cell physiology and to genetically manipulate NK cell populations prior to their administration in cancer therapy.
In Vitro Induction of Myeloid Leukemia Specific CD4 and CD8 T-Cells by CD40 Ligand Activated B Cells Gene-Modified to Express Primary Granule Proteins.
Authors: Fujiwara Hiroshi , Melenhorst Joseph J. , El Ouriaghli Frank , Kajigaya Sachiko , Grube Matthias , Sconocchia Giuseppe , Rezvani Katayoun , Price David A. , Hensel Nancy F. , Douek Daniel C. , Barrett A. John
Published: April 2020

The closely-related primary granule proteins are a source of multiple antigens with immunotherapeutic potential for myeloid leukemias. To further explore the possibility of generating leukemia-specific T cell responses, DNA vectors encoding the primary granule proteins proteinase 3 (PR3), human neutrophil elastase (HNE) and cathepsin-G (CathG) were transfected into autologous CD40-activated B (CD40-B) cells and used to stimulate T cell responses from peripheral blood T cells of five patients with myeloid malignancies and three ALL patients three months post-bone marrow transplantation (BMT), and one patient with CML-BC pre-transplant. T-cell responses were measured by flow-based intra-cytoplasmic IFN-g assay and cytotoxic assay. CD8 and CD4 cell response to PR3 and HNE were observed in CD8+ and CD4+ T-cells in 6/6 patients with myeloid leukemias with various degrees. T-cell response against CathG were found in both myeloid and lymphoid leukemias. We conclude that gene-transfected CD40-activated B cells are fully capable of expanding functionally-competent primary granule protein-specific CD4 and CD8 T cells, making this approach a valuable means of expanding leukemia antigen-specific T cells for adoptive immunotherapy.

PGP-DNA primed T cells showed CTL activity to CML cell (case#5, post transplant)

CTL activity % ( E:T ratio ) CML cell; recipient BM cell, normal BM; HLA identical DNR BM cells Effector cells Target 50:1 25:1 12.5:1 PR3-primed CML cell 63.8 54.6 30.4 normal BM 15.5 13.1 5.9 HNE-primed CML cell 43.9 38.3 32.1 normal BM 0 4.1 2.3 CathG-primed CML cell 43.1 29.1 27.5 normal BM 0 0 1.7 mock-primed CML cell 33.3 34.2 29.5 normal BM 5.5 12.9 0
Cotransfection of Dendritic Cells with RNA Coding for a Tumor Antigen and Costimulatory Molecule 4-1BBL Increases the Induction of Her-2/neu Specific Cytotoxic T-Cells.
Authors: Grünebach Frank , Kayser Katrin , Appel Silke , Weck Markus M. , Müller Martin R. , Reichardt Volker L. , Brossart Peter
Published: April 2020

Dendritic cells are professional antigen-presenting cells (APC) with the unique capacity to initiate primary immune responses. Recently, it was demonstrated that transfection of DC with RNA coding for specific antigens can elicit an effective T cell response in vitro and in vivo.

4-1BB (CD137) is a member of a family of receptors found on the surface of cells of the immune system. It plays an important role in the costimulation of T-cells: it enhances T-cell proliferation, cytokine production and cytotoxic effector functions and suppresses the induction of apoptosis. It is predominantly expressed on activated T-cells, and its ligand 4-1BBL is expressed on activated APC including DC.

In the present study we analyzed the effects of 4-1BBL on the stimulatory capacity of DC. Monocyte derived DC were electroporated with in vitro transcribed RNA encoding the tumor-associated antigen Her-2/neu. Additionally these cells were cotransfected with in vitro transcribed RNA coding for the human 4-1BBL with the aim to enhance the costimulatory functions of the cells and therefore the induction of Her-2/neu specific cytotoxic T-cells (CTL).

Standard 51Cr-release assays performed after two restimulations showed specific cytotoxic activity of the induced CTL against autologous DC transfected with Her-2/neu RNA and against the HLA-matched allogeneic Her-2/neu+/HLA-A2+ renal cell carcinoma cell line A498 whereas DC transfected with irrelevant RNA as well as Her-2/neu-/HLA-A2+ CROFT cells and the Her-2/neu+/HLA-A2- cell line SK-OV-3 were spared. There was no lysis of K562 cells, thus excluding a NK-cell mediated killing. In this experimental setting the specific lysis rate of target cells could nearly be doubled by the coadministration of Her-2/neu RNA and an equal amount of 4-1BBL RNA (50% vs 90% specific lysis at effector to target ratio of 30:1). Interestingly, overexpression of 4-1BBL had no effect on the stimulation of allogeneic T cells by electroporated DC in a mixed lymphocyte reaction, indicating that 4-1BBL increases the induction of antigen specific T cell responses rather than the unspecific stimulation of bulk T lymphocytes.

Furthermore, FACS analysis of DC transfected with 4-1BBL plasmid DNA or in vitro transcribed 4-1BBL RNA showed higher expression of the costimulatory molecules CD80, CD86, CD40 and the T-cell adhesion molecule CD54 as compared to the controls indicating that 4-1BBL signaling can mediate activation of DC and thus increase their stimulatory capacity. In the context of immunotherapies for cancer patients the findings of our study could contribute to optimise the in vitro manipulation of DC for the induction of antigen-specific CTL responses.
Transduction of Tolerogenic Dendritic Cells by Foamy Virus and Lentivirus Vectors.
Authors: Su RuiJun , Josephson Neil C.
Published: April 2020

The induction and maintenance of antigen specific peripheral tolerance is critical for the prevention of autoimmunity and maintenance of immune homeostasis. Antigen presentation by tolerogenic dendritic cells (DCs) initiates the process through the stimulation of regulatory T cells and other mechanisms that may include peripheral deletion and anergy induction of autoreactive T cells. In contrast to DCs that mediate immune priming, tolerogenic DCs have a resting phenotype. They lack expression of co-stimulatory molecules and MHC class II. Recently, a method for identifying murine tolerogenic DCs (lin−, CD45RBhigh, CD11clow) and expanding them ex vivo has been described (Wakkach et al. Immunity18:605, 2003). Directed antigen presentation by viral vector transduction of these cells has the potential to produce antigen specific tolerance for therapeutic purposes. To generate tolerogenic DCs, we cultured lin− murine bone marrow in complete RPMI medium in the presence of 10 ng/ml GM-CSF, 2.5 ng/ml TNF-α and 10–100 ng/ml IL-10 for 6–7 days. Cells were transduced overnight at different time points (day 0, day 3, day 6), with foamy virus vectors and lentivirus vectors containing either the GFP or alkaline phosphatase reporter genes. Transductions were performed both in the presence or absence of fibronectin fragment CH-296. For day 0 transductions, we also evaluated whether stem cell stimulatory cytokines (50 ng/ml TPO, 100 ng/ml SCF, 100 ng/ml FL) improved gene transfer efficiency. On days 6–7 the CD45RBhighCD11clow cell population was flow sorted and then further analyzed. The percentage of CD45RBhighCD11clow cells harvested on days 6–7 varied from 10% to 30% and was increased by IL-10 in a dosage dependent manner. As expected for tolerogenic DCs, these cells were morphologically immature by Giemsa-Wright staining and phenotypically negative to low for the expression of co-stimulatory molecules CD80 and CD86, and MHC class II, even after exposure to lipopolysaccharide. At an MOI of 0.5–1, transduction rates of CD45RbhighCD11clow cells were 30%–37% for foamy virus vectors and 23%–24% for lentivirus vectors. Most importantly, vector transduction did not induce expression of co-stimulatory molecules, suggesting that these two viral vectors do not activate DCs. There was no difference in transduction efficiency at the three different time points tested. Furthermore, transduction efficiencies were not affected by either stem cell stimulatory cytokines or CH-296. In summary, cells with the phenotype of tolerogenic DCs can be efficiently transduced by foamy virus and lentivirus vectors with overnight exposure to vector stocks. In vivo studies in mice are in progress to evaluate if transduced CD45RBhighCD11clow cells are able to induce antigen specific tolerance. These studies have potential clinical implications for the treatment of autoimmune diseases, the induction of tolerance after stem cell transplantation, or the prevention of immune responses triggered by specific transgenes in gene therapy.