bluuebunny/crossref_metadata_2025 huggingface dataset:
A Study on the Prognosis of Patients Relapsing after Autologous Stem Cell Transplantation (auto-SCT) for Follicular Lymphoma (FL): The Impact of Remission Duration.
Authors: Stumm Julia , Dreyhaupt Jens , Kornacker Martin , Hensel Manfred , Kneba Michael , D.Ho Anthony , Schmitz Norbert , Dreger Peter
Published: October 2019

Although auto-SCT has been in use for treatment of advanced FL since many years, little is known about the course of those who relapse after this procedure. Because these patients may be candidates for aggressive salvage approaches, we sought to study the outcome of patients with FL relapsing after auto-SCT with particular focus on factors predicting for survival.

Methods: Relapse cases were identified retrospectively from 244 patients autografted for FL between August 1990 and November 2002 in 3 institutions. Overall survival after relapse (OS) was calculated according to Kaplan-Meier and analyzed for the prognostic impact of pre-relapse variables as well as of post-relapse salvage treatment by univariate log rank comparisons and Cox regression analyses.

Results: With a median follow-up of 88 (5–186) months post auto-SCT, 104 relapses occurred, corresponding to a 10-year relapse probability of 0.47 (95%CI 0.4–0.53). Median age of relapsed patients was 48 (22–65) years. FLIPI score at diagnosis was low in 18%, intermediate in 58%, and high in 24%. In 51%, auto-SCT had been given as part of first-line treatment, and 45% had been in complete remission at auto-SCT. Myeloablation included total body irradiation (TBI) in 57% of the cases. Median time from auto-SCT to relapse was 19 (2–128) months, with only 2 relapses occurring later than 6 years post transplant. Transformed FL was present in 14% of those 87 patients who had relapse histology available. Rituximab-containing salvage therapy was given to 50% of the patients after relapse. With 45 (1–139) months of follow-up, median OS after relapse was 100 months. Log rank comparisons identified auto-SCT as part of salvage treatment, time to relapse <12 months, and salvage without rituximab as factors adversely influencing OS, while all other variables listed above had no impact. Cox analysis considering sex, age, salvage auto-SCT, TBI, time to relapse, and rituximab salvage confirmed a possible adverse impact of time to relapse <12 months (hazard ratio 2.58 (95%CI 0.99–6.82); p 0.055) but none of the other covariates on OS.

Conclusions: The prognosis of patients relapsing after auto-SCT for FL is surprisingly good. However, those whose disease recurs within the first post-transplant year tend to have a dismal outcome and might benefit from experimental salvage approaches, such as allogeneic SCT.
Arsenic Trioxide with Ascorbic Acid and High-Dose Melphalan for Autologous Hematopoietic Stem Cell Transplantation for Multiple Myeloma.
Authors: Qazilbash Muzaffar H. , Saliba Rima M. , Davis Marilyn S. , Mendoza Floralyn L. , Hosing Chitra , Couriel Daniel R. , De Lima Marcos , Kebriaei Partow , Weber Donna M. , Wang Michael , Alousi Amin M. , Matthes Steven G. , Jones Roy B. , Champlin Richard E. , Giralt Sergio A.
Published: October 2019

Backround: Arsenic trioxide (ATO) has been shown to be synergistic with melphalan both in vitro and in vivo. We conducted a phase I/II trial to determine the safety and efficacy of a combination of arsenic trioxide, melphalan and ascorbic acid (AA) as preparative regimen in patients undergoing high-dose therapy (HDT) and autologous hematopoietic progenitor cell transplantation for multiple myeloma (MM). We also assessed the impact ATO levels on melphalan pharmacokinetics (PK), engraftment and toxicity.

Methods: Forty-eight patients with secretory myeloma (23 females, 25 males; median age: 54, range: 3570) were treated between 4/04 and 8/05. All patient received melphalan 100 mg/m2 IV on days -4 and -3 and AA 1000 mg/day IV on days -9 to -3. Patients were randomized to 3 arms; no ATO (arm 1), ATO 0.15 mg/kg IV on days -9 to -3 (arm 2) and ATO 0.25 mg/kg IV on days -9 to -3 (arm 3). Twelve patients had disease progression or relapse after a prior autograft. Median CD34 cells dose infused was 4.5 x 106/kg (range 2.3–10.9).

Results: Patients in all 3 arms were evenly matched. With a median F/U of 14.0 months (range 6–25) post autograft, no dose-limiting toxicity or non-relapse mortality was seen. Toxicity was limited to grade I or II nausea, vomiting and diarrhea. Median ATO levels on day 0 in arms 1, 2 and 3 were 0.2, 26.3 and 46.2 ng/ml, respectively. Melphalan PK was not altered by ATO pretreatment. Median time to neutrophil engraftment (ANC >500/ dl) was 9 days. There were no engraftment failures or delays in the ATO arms. CR rate for the entire group was 23%, and total response rate (CR + PR) was 75%. 1-year Progression-free survival (PFS) and overall survival (OS) were 75% and 95%, respectively. There was no significant difference in CR, RR, PFS or OS between the 3 arms (p = 0.9, 0.9, 0.4 and 0.6, respectively). A prior autologous transplant (p = 0.02) and abnormal cytogenetics at transplant (p = 0.04) were associated with a significantly shorter remission.

Conclusions: ATO + melphalan + ascorbic acid is a safe, effective and well tolerated preparative regimen for patients with multiple myeloma undergoing an autotransplant. A longer follow up is needed to assess the impact of ATO on progression-free and overall survival.
Unrelated Allogeneic Stem Cell Transplantation for Severe Acquired Aplastic Anemia: Has Outcome Improved?.
Authors: Maury Sebastien , Balere-Appert M.-Lorraine , Chir Zina , Boiron J.-Michel , Galambrun Claire , Yakouben Karima , Bordigoni Pierre , Marie-Cardine Aude , Milpied Noel , Kanold Justina , Maillard Natacha , Bourhis J.-Henri , Fischer Alain , Lutz Patrick , Michallet Mauricette , Blaise Didier , Cordonnier Catherine , Raffoux Colette , Socie Gerard
Published: October 2019

We aimed to determine whether outcome of HSCT from an unrelated donor has improved over time for patients with severe acquired aplastic anemia (SAA) and, if so, to determine whether improvement resulted from changes in patient selection, changes in transplantation technique, or both. We thus analyzed the outcome of 89 patients (median age 17 years, range 0–52) who received such transplantations between 1989 and 2004. We compared two cohorts of patients transplanted within two successive time-periods (1989–1998 and 1999–2004) associated with probabilities (± 95% confidence interval) of 5-year survival significantly different: 29% ± 7% and 50% ± 7%, respectively (P<.01, Figure 1). Significant differences between the two cohorts concerned transplant-related, but not patient- and disease-related variables: the use of ATG and of fludarabine within conditioning and HLA matching at the allelic level for the 10 HLA-A, -B, -C, -DRB1 and -DQB1 loci (P=.0004) were more frequent in the recent 1999–2004 period. In multivariate analysis, the only 2 factors influencing survival were HLA allelic matching (P<.01) and younger age of recipient (<17 years, P<.0001). Of note, the impact of HLA matching disappeared in multivariate analysis when it was considered at the antigenic level for HLA-A, -B and -DR antigens, which underlines the importance of considering HLA matching at the allelic rather than the generic level for these transplantations. Survival reached 78% ± 11% at 5-year for the younger patients fully HLA-matched (n=14), which can be compared with survivals obtained for HLA-identical sibling transplants in similar young patients. Cumulative incidences of graft failure, grade II–IV or grade III–IV acute GVHD, and chronic GVHD were: 14% ± 4 % at 2 years, 50% ± 5 % and 24% ± 4 % at 100 days, and 28% ± 5 % at 2 years after HSCT (limited: 17% ± 4 %, extensive: 11% ± 3 %), respectively. In a competing risk analysis, allelic HLA matching - particularly incorporating HLA-C but not -DQB1 matching - but also a high cell-dose injected (> 2,6.108/kg nucleated cells, the median value of the cohort) and the use of fludarabine and/or ATG in the conditioning regimen were found as protective against graft failure and acute GVHD, respectively. Our results suggest that survival after unrelated transplantation for SAA has improved over the past 15 years, due to a better HLA matching at the allelic level for both HLA class I and class II antigens, but also to other transplant-related factors.

Figure 1 Figure 1.
Purified T Depleted Peripheral Blood and Bone Marrow Cd34 Transplantation from Haploidentical Mother to Child with Thalassemia.
Authors: Sodani Pietro , Erer Buket , Gaziev Javid , Polchi Paola , Roveda Andrea , Spitaleri Luca , Pagliai Francesca , Paciaroni Katia , Adorno Gaspare , Lanti Alessandro , Isacchi Giancarlo , Zinno Francesco , Lucarelli Guido
Published: October 2019

Approximately 60% of thalassemic patients can not apply to “gene therapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance established during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant.

We have employed a new preparative regimen for the transplant in fourteen thalassemic children aged 3 to 12 years (median age 5 years) using T cell depleted peripheral blood stem cell (PBSCTs) plus bone marrow (BM) stem cells. All patients received hydroxyurea (OHU) 60 mg/kg and azathioprine 3 mg/kg from day -59 until day-11, fludarabine (FLU) 30 mg/m 2 from day -17 to day -11, busulphan (BU) 14 mg/kg starting on day -10, and cyclophosphamide(CY) 200mg/kg, Thiotepa 10 mg/kg and ATG Sangstat 2.5 mg/kg, followed by a CD34 + t cell depleted (CliniMacs system), granulocyte colony stimulating factor (G-csf) mobilized PBSC from their HLA haploidentical mother. The purity of CD34+ cells after MACS sorting was 98–99%, the average number of transplanted CD34+ cells was 15, 4 x 10 6/kg and the average number of infused T lymphocytes from BM was 1,8 x 10 5/Kg.The patients received cyclosporin after transplant for graft versus host disease(GVHD) prophylaxis during the first two months after the bone marrow transplantation.

Results. Thirteen patients are alive. Four patients rejected the transplant and are alive with thalassemia One patients died six months after bone marrow transplant for central nervous system diffuse large B cell lymphoma EBV related. Nine patients are alive disease free with a median follow up of 30 months (range12–47). None of the seven patients showed AGVHD and CGVHD. This preliminary study suggest that the transplantation of megadose of haploidentical CD34+ cell from the mother is a realistic therapeutic option for those thalassemic patients without genotipically or phenotipically HLA identical donor.
Impaired Survival after Unrelated Hematopoietic Stem Cell Transplantation from KIR2DS1 and KIR2DS2 Positive Donors.
Authors: Giebel Sebastian , Czerw Tomasz , Nowak Izabela , Wojnar Jerzy , Kruzel Tomasz , Dziaczkowska Joanna , Markiewicz Miroslaw , Holowiecka-Goral Aleksandra , Karolczyk Agnieszka , Wylezol Iwona , Kusnierczyk Piotr , Holowiecki Jerzy
Published: October 2019

The function of NK cells is regulated by a balance between signals transmitted via actvating and inhibitory receptors, including killer-immunoglobulin-like receptors (KIRs). Activating KIRs (KIR2DS1 and KIR2DS2) are also present on subsets of T cells, which has recently been demonstrated to play a role in several autoimmune conditions. The goal of this analysis was to determine the relationship between donor activating KIRs genotype and outcome after unrelated donor hematopoietic stem cell transplantation (URD-HSCT).

Seventy five patients (age 30y; range 14–57) with hematological malignancies treated with URD-HSCT in a single centre between 2000 and 2006 were included in the analysis. Donors were selected using DNA-high resolution typing for both HLA class I and II alleles. KIR2DS1 and KIR2DS2 genes were examined in all donors. Additionally, in 16 cases a subpopulation of KIR2DS2/DL2/DL3-positive T cells in peripheral blood was monitored in donors as well as in recipients for 6 months after transplantation.

The conditioning regimen was myeloablative and based on chemotherapy alone (n=56) or TBI (n=19). Graft-vs-host disease (GVHD) prophylaxis was uniform and consisted of cyclosporin, methotrexate, and pre-transplant anti-thymocyte globulin. Twenty-five % of donors were positive for both KIR2DS1 and KIR2DS2 gene, 18% were positive for KIR2DS1 only, 25% - for KIR2DS2 only, and 32% were negative for both KIR2DS1 and KIR2DS2. In univariate and multivariate analysis, including other potential risk factors, the presence of both KIR2DS1 and KIR2DS2 genes of the donor was associated with decreased overall survival (0% vs. 80%, RR 3.2, p=0,01) and disease free survival (0% vs. 64%, RR 2.5, p=0,03), compared to the remaining subgroups. Furthermore, simultanous KIR2DS1 and KIR2DS2 positivity resulted in increased GVHD-related mortality (70% vs. 8%, p=0.01) in univariate analysis (incidence of both acute and chronic GVHD contributed to this effect). KIR2DS2/DL2/DL3-positive T cells were detected in 3/16 donors before URD-HSCT and in all recipients between day +28 and +180 after URD-HSCT. KIR-positive T cells were particularily frequent (32%–42% of all CD3 cells) in three patients who developed acute GVHD, and for whom the donors were KIR2DS2-positive.

We conclude that the simultaneous presence of both KIR2DS1 and KIR2DS2 in the donor is associated with decreased survival following URD-HSCT. The mortality may result from higher incidence of lethal GVHD. Preliminary observation suggests the role of KIR-bearing T lymphocytes in this effect.
Matched Unrelated Donor Stem Cell Transplantation for Relapsed or Refractory Mantle Cell Lymphoma. A Retrospective Analysis from the EBMT Lymphoma Working Party.
Authors: Avivi Irit , Canals Carme , Taghipour Goli , Finke Jurgen , Volin Liisa , Beguin Yves , Boogaerts Marc , Crawley Charles , Rieger Michael , Schmitz Norbert , Kobbe Guido , Milligan Donald , Niederwieser Dietger W. , Russell Nigel H. , Schwerdtfeger Rainer , Sureda Anna
Published: October 2019

High-dose therapy with allogeneic hematopoietic transplantation (allo-SCT) from matched sibling donors has been shown to induce durable remissions in some patients with relapsed / refractory mantle cell lymphoma (MCL). The aim of this retrospective analysis from the LWP of the EBMT was to investigate the outcome of patients with MCL treated with an unrelated donor allo-SCT (MUD-allo). From January 1994 to July 2005, 66 patients with MCL, 51 males and 15 females, with a median age of 50 years (range, 22 to 68) underwent a MUD-allo and were reported to the EBMT registry. The median time from diagnosis to MUD-allo was 34 months (range, 6–131). Thirty-five patients (53%) had previously failed an autologous procedure (ASCT). Forty-five patients (68%) had sensitive disease (including 22 patients in complete remission) at transplantation, whereas 21 patients (32%) were allografted with refractory disease. Reduced intensity conditioning regimens (RIC) were used in 44 patients (67%). Patients treated with RIC were older, more heavily pre-treated and had more frequently failed an ASCT (46% vs 30%, p = 0.01) than patients treated with a conventional conditioning protocol. Total body irradiation (TBI) was used in 68% of the patients receiving conventional protocols and low-dose TBI in 23% of the RIC patients. Grade II–IV acute graft versus host disease (GVHD) developed in 35% of the cases. The cumulative incidence (CI) of non-relapse mortality (NRM) was of 21% at 6 mo and 27% at 12 mo. The CI of relapse was 35% at 1 year and 45% at 2 years. After a median follow up of 15 months (1–73), 25 patients are alive without progression, with an estimated PFS and OS at 2 years of 28% and 42%, respectively. RIC protocols were not associated with a lower NRM or a better survival. Refractory disease at MUD-allo was an adverse prognostic factor for PFS (RR 1.7; p = 0.006). Patients allografted in sensitive disease presented a better 2-year PFS and OS (34% and 48%, respectively). In conclusion, although follow up is still short, MUD-allo is a feasible procedure in poor prognosis MCL patients, with almost one third of them being alive and progression-free in this series. RIC protocols do not seem to offer any advantage in terms of long-term outcome in relation to conventional conditioning regimens.
Unrelated Hematopoietic Blood Stem Cell Transplantation in Patients with Acute Myelogenous Leukemia (AML) in First Remission.
Authors: Ehninger Gerhard , Aulitzky Walter E. , Bodenstein Heinrich , Bornhaeuser Martin , Einsele Hermann , Faul Christoph , Finke Jurgen , Ganser Arnold , Gramatzki Martin , Haenel Mathias , Helm Gisela , Ho Anthony D. , Jakob Andreas , Knigge Owe , Link Hartmut , Mahlberg Rolf , Mahlknecht Ulrich , Neubauer Andreas , Schmitz Norbert , Schaich Markus A. , Schaefer-Eckart Kerstin , Soucek Silke , Wagner Thomas , Wandt Hannes , Zander Axel R.
Published: October 2019

Between 1996 and 2003, 994 patients aged under 60 years with de novo AML received a double induction chemotherapy consisting of MAV (mitoxantrone 10 mg/m2 day 4–8, Ara-C 100 mg/m2 continuous infusion day 1–8, etoposide 100 mg/m2 day 4–8) and MAMAC after an interval of at least 14 days (Ara-C 1 g/m2 q. 12 hrs day 1–5, amsacrine 100 mg/m2 day 1–5). A cytogenetic high risk profile (complex aberrations, −5/del(5q), −7/del(7q), hypodiploid karyotypes other than −5, −7, −X, −Y, abnl3q, abnl1q, abnl12p, t(6;9), t(9;22), t(9;11), +11, +13, +21, +22) was present in 252 patients (de novo AML [N=160], prior MDS [N=41], and secondary AML [N=51]). 119 out of 252 (47%) were in complete remission after 2 induction cycles. 35 (14%) additional patients showed complete remission after a further cycle. Patients with a HLA matched sibling donor (N=37) or an unrelated donor (N=34) received an allogeneic transplantation as consolidation therapy. The conditioning protocol consisted of 12 Gy TBI and cyclophosphamide. Cyclosporine and methotrexate were used as GvH prophylaxis. The actuarial survival at 5 years is 42% (CI: 26%–58%) in the related donor group and 53% (CI: 36%–70%) in the unrelated donor group. The disease free survival at 5 years is 40% (CI: 24%–56%) in the related group and 43% (CI: 26%–60%) in the unrelated group (for both, p=ns). In conclusion, the results after unrelated donor transplants are comparable with those after related donor transplants in patients with high risk AML. These data of the AML’96 study will be compared with the experience of our current upfront concept using allogeneic transplantation in aplasia after the 1st or 2nd induction cycle in high risk AML.
NK1.1+ Cells Are Involved in the Anti-Tumor Immunity Induced by the Rejection of Donor Hematopoietic Cells in Mixed Bone Marrow Chimeras.
Authors: Saito Toshiki I. , Sykes Megan
Published: October 2019

BACKGROUND

Surprisingly, some patients who reject donor marrow grafts following nonmyeloablative HCT sustain remissions of advanced hematologic malignancies (Br J Haematol2005, 128:351). In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced loss of donor chimerism and anti-tumor responses against host-type tumors (Blood2003, 102:2300) and that both CD4+ and CD8+ T cells are necessary to achieve maximum anti-tumor effects (J Immunol2005, 175:665). We now examined whether or not NK and/or NKT cells are required in the anti-tumor effect.

METHODS

Mixed chimerism was achieved in BALB/c (H-2d) mice conditioned with depleting anti-CD4 and CD8 mAbs on Day -5, cyclophosphamide 200 mg/kg on Day -1 and 7 Gy thymic irradiation on Day 0 prior to transplantation of 25x106 B10.BR (H-2k) or B10.RIII (H-2r) bone marrow cells. Some groups received RLI (3x107 BALB/c spleen cells) seven weeks post-BMT. A20 cells (5x105) were given i.v. one week after RLI. Some groups included Jα281-deficient BALB/c (Jα281 KO) mice that lack invariant NKT (iNKT) cells or NK1.1+ BALB/c mice whose NK and NKT cells can be depleted by anti-NK1.1 Ab, PK136. Some groups received a depleting dose of anti-NK1.1 antibody before and after RLI administration.

RESULTS

Untreated recipients of NK1.1-depleted RLI (n=9) showed similar tumor survival compared to recipients of NK1.1-sufficient RLI (n=9) (median survival time [MST] 52 versus 52 days respectively, p=0.68). NK1.1-depleted (with mAb in vivo) chimeras that received RLI (n=8) had shorter survival (MST 35.5 days) than NK1.1-sufficient chimeras that received RLI (n=10) (MST 68 days, P<0.01) and no anti-tumor effect was observed in the depleted group. Jα281 KO chimeric recipients receiving with Jα281 KO-derived RLI (n=12) had shorter survival than wild-type chimeric mice receiving wild-type RLI (n=11) (MST 41.0 vs 67.0 days, p<0.01) and no survival benefit of RLI was observed in Jα281 KO recipients (p=0.93).

CONCLUSION

When NK and NKT cells were depleted from NK1.1+ BALB/c recipients, no anti-tumor effect was observed. In contrast, no reduction in anti-tumor effect was observed in chimeras given NK1.1-depleted RLI. Invariant NKT cells are required to achieve anti-tumor effects in this model. Therefore, NKT cells and possibly NK cells in the recipient are essential for the anti-tumor effect of RLI, but NK and NKT cells in the RLI do not play a role. The requirement for both T cells and NK/NKT cells suggests an essential interaction between the innate and acquired immune systems.
Ex Vivo Fas Ligand Treatment Selectively Eliminates T Cells That Mediate GVHD.
Authors: Kashtan Osnat , Levitsky Hyam I. , Civin Curt I.
Published: October 2019

The addition of recombinant human soluble Fas ligand (sFasL) to a mixed lymphocyte reaction (MLR) resulted in selective reduction of the responder T cells during their activation against irradiated allogeneic stimulator cells, while sparing other T cells with anti-tumor reactivity, in studies using mouse models (Georgantas et al, Transplantation, In Press). To move our work toward reduction of clinical graft versus host disease (GVHD), we have extended these studies using a human model system. Peripheral blood mononuclear cell (PBMC) responders from a given individual (1st party) were stimulated in vitro with irradiated PBMC stimulators from a second person (2nd party), in the presence of sFasL. In control MLR cultures without sFasL, Fas expression began to increase on alloantigen-activated responder T cells by day 2. In 2–3 day MLRs done with carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled 1st party responder cells, there were greatly reduced numbers of CD25+CFSEhigh, CD25+CFSElow, and CD25−CFSElow activated responder cells in the sFasL-treated cultures. In addition, the numbers of CD4highCD25+CD38+HLADR+ activated T cells that otherwise appeared in response to alloantigen were severely reduced in cultures containing sFasL. Secondary MLRs, performed on cells harvested on day 2–3 of sFasL treatment in the first MLR, showed a marked depletion of anti-2nd party alloreactivity, but preservation of immune stimulation in response to cytomegalovirus and 3rd party antigens. These data indicate that ex vivo sFasL/MLR-mediated depletion of alloreacting anti-donor T cells was efficient and selective. The efficacy of this method is currently being tested in vivo using a xenogeneic GVHD human-immunodeficient mouse model.
Early CD30 Signaling Is Critical for Adoptively Transferred CD4+CD25+ Regulatory T Cells in Prevention of Acute Graft Versus Host Disease.
Authors: Zeiser Robert , Nguyen Vu H. , Hou Jing-Zhou , Beilhack Andreas , Zambricki Elizabeth A. , Buess Martin , Contag Christopher H. , Negrin Robert
Published: October 2019

CD4+CD25+ regulatory T cells (Treg) have been demonstrated to reduce the severity of acute graft-versus-host disease (aGvHD) in murine models of bone marrow transplantation. However, the surface molecules that are critical for suppression are unclear. The TNF-R superfamily member CD30 has been shown to be expressed on regulatory T cells that down-modulate nickel specific immune responses and to be relevant for Treg mediated protection from allograft rejection. Deficiency of the CD30 molecule (CD30−/−) is associated with impaired thymic negative selection and augmented T cell autoreactivity. Therefore, we investigated the role of CD30 signaling in Treg function in an aGvHD model. Treg derived from CD30−/ − animals were significantly less effective in preventing aGvHD lethality (wt vs. CD30−/ − p=0.002). Signal intensity derived from expanding luciferase expressing alloreactive conventional T cells (Tconv) was significantly higher if CD30−/ − Treg as compared to wt Treg (p=0.007) were transferred as assessed by bioluminescencent based imaging. Blockade of the CD30/CD153 pathway with a neutralizing anti-CD153 mAb during the early (days −2 to +4) but not late (days +4 to +10) phase of adoptive Treg transfer reduced Treg mediated protection from proinflammatory cytokine accumulation and apoptosis of donor-type CD4 and CD8 T cells. In vivo bioluminescence imaging demonstrated intact Treg homing, but reduced expansion when CD153 was blocked during the early phase after adoptive transfer. CD30 surface expression on Treg increased with alloantigen exposure and CD153 expression on recipient-type dendritic cells increased in the presence of an irradiation induced proinflammatory environment but not when T cell depleted bone marrow and Tconv were transferred into non-irradiated Rag 2−/ −γc−/ − recipients. These data are the first to demonstrate that early CD30 signaling is relevant for Treg mediated aGvHD protection after major MHC mismatch bone marrow transplantation.
A GMCSF & IL15 Fusokine Leads to Paradoxical Immunosuppression In Vivo Via Asymmetrical JAK/STAT Signalling through the IL15 Receptor Complex.
Authors: Rafei Moutih , Wu Jian Hui , Annabi Borhane , Lejeune Laurence , Francois Moira , Galipeau Jacques
Published: October 2019

Immune stimulatory cytokines can be exploited to treat human ailments including cancer. Amongst cytokines identified for such use, granulocyte-macrophage colony stimulating factor (GMCSF) has been under much scrutiny since it acts directly on the adaptive immune system by enhancing antigen presentation as well as costimulation. Furthermore, interleukin (IL)15 possesses overlapping activities with IL2 such as the activation of T-cells and the stimulation of natural killing as well as additive stimulatory effects on the immune system distinct from IL2. These features make IL15 an attractive companion to GMCSF as part of an immunotherapeutic fusokine. Therefore, we hypothesized that a GMCSF and IL15 fusion protein (GIFT15) would possess greater immune stimulatory properties than their combined use. Unexpectedly, tumor cells engineered to secrete GIFT15 protein led to enhanced tumor growth and suppression of natural killer (NK) and NKT-cell recruitment in vivo, suggesting an unheralded immune suppressive effect. We found that GIFT15 has pleiotropic effects on an array of immune competent cells. More specifically, peritoneal macrophages treatment with GIFT15 secrete de novo the tissue inhibitor of metalloproteinase-2 (TIMP-2); activated matrix metalloproteinase-2 (MMP-2); transforming growth factor-β (TGF-β) as well as vascular endothelial growth factor (VEGF). In terms of ligand-receptor interactions, we show by BIAcore analysis that the GIFT15 fusokine has increased affinity for the αchain component of the trimeric IL15R, which contributes to aberrant signalling through the β chain manifested by the hyperphosphorylation of STAT3 both in macrophages and splenocytes. In addition, GIFT15 and IL15R virtual interaction studies suggests that the GMCSF domain component of the GIFT15 fusokine may hinder the interaction of the IL15 domain component with the IL15Rγ chain, and thus leading to a downregulation of the JAK3/STAT5 pathway. We also show that GIFT15 leads to suppression of common γ chain-mediated STAT5 phosphorylation and blockade of the IL15-dependent IFN-γ response in mouse splenocytes We tested the utlity of GIFT15 as an immunosuppressor directly in vivo and demonstrated that it allowed engraftment of allogeneic B16F0 and human xenograft U87MG glioma cells in immunocompetent mice in a CD4-dependent maner. Thus, GIFT15 defines a new class of fusokine which mediates pro-angiogenic and potent immunosuppressive effects via aberrant signalling by the IL15R in lymphomyeloid cells. We propose that GIFT15 may serve as a novel pharmaceutical for tolerance induction of somatic allo or xenografts in mammals without requirement of toxic conditioning regimens.
Generation of Human T/NK Cell Precursors for Adoptive Transfer To Enhance Immune Reconstitution in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.
Authors: Chaudhury Sonali , Zakarzewski Johannes , Shieh Jae-Hung , van der Brink Marcel , Moore Malcolm A.S.
Published: October 2019

Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with significant post-transplant immunoincompetence which affects in particular the T cell lineage and results in an increased susceptibility to infections. Novel strategies to enhance immune recovery after HSCT could prevent malignant relapse and immune deficiency and improve the overall outcome of this therapy. We have established a serum free culture system using murine bone marrow stroma expressing the Notch ligand Delta-like 1 (DL1) to obtain high numbers of human pre-T cells from CD34+ cells. Human cord blood CD34+ cells were plated on OP9 DL1 stroma transduced with adenovirus expressing thrombopoietin (ad-TPO) at an MOI of 30. Media used was QBSF-60 (Serum free media prepared by Quantity Biologicals) supplemented with Flt-3 ligand and IL-7 (10ng/ml). At 4–5 weeks we obtained a 10 5–10 7 fold expansions of cultured cells of which about 70–80% were CD5, CD7 positive pre T cells (Fig 1). We then developed an optimal system to study human lymphohematopoiesis using mouse models (NOD/SCID/IL2rϒnull and NOD/SCIDβ2null) and established an adequate pre T cell number (4 × 10 6) and radiation dose (300 Rads). We injected CD34 and pre-T cells (CD45 +, CD4−, CD5+, CD7+) derived from OP9 DL1 cultures into these mice and achieved ~50%engraftment of NK in the bone marrow and spleen of the mice at 2 weeks following transplant. The thymus from the same mice showed evidence of about 12–15% CD7+ pre T cells. We are currently studying the function of the generated NK and T cells both in vivo and in vitro studies.

Figure Figure
Identification of a Single MiHA Specificity That Induces Resistance to MHC-Matched Allogeneic HCT.
Authors: Shatry Alwi M. , Levy Robert B.
Published: October 2019

T cell populations specific for transplantation antigens have been detected in sensitized individuals following multiple blood transfusions, marrow transplants as well as in multiparous females. Resistance to allogeneic hematopoietic cell transplant (HCT) in such sensitized individuals is consistent with the presence of a host memory T cell (TM) population specific for donor cell antigens. We hypothesized that a single donor minor histocompatibility (MiHA) epitope could elicit antigen-specific CD8 TM capable of resisting MHC-matched allogeneic hematopoietic cell engraftment. To address this question, CD8 TM were generated against a single MiHA epitope to determine if such cells could mediate resistance after ablative TBI conditioning. B6 mice were sensitized 2X to the H60 immunodominant MiHA epitope utilizing bone marrow-derived dendritic cells pulsed with the H60 (LTFNYRNL) peptide. Three weeks following booster sensitization, CD8 T cells were detected by tetramer staining in peripheral blood samples. These T cells exhibited a phenotype characteristic of memory cells (CD44hi, Ly 6C+). B6 (H2b) mice containing CD8+ H60+ T cells were subsequently conditioned with 9.0 Gy TBI and transplanted with 5 × 106 BALB.B (H2b) BM-TCD. One week post-transplant, naive recipients of BALB.B (H60+) or B6-H60 congenic TCD-BM contained >10-fold higher levels of circulating donor cells than the B6 dendritic cell/peptide sensitized recipients. Donor progenitor cells were also found to be significantly reduced in sensitized recipients of allogeneic TCD-BM at this time. Two weeks post-HCT, recipients of syngeneic marrow exhibited >10-fold greater frequency of circulating donor cells compared to recipients of MHC-matched allogeneic marrow (< 5% donor chimerism was detected). These findings demonstrate that host T cells with specificity against a single donor MiHA determinant are sufficient to induce resistance to MHC-matched allogeneic marrow engraftment. Such observations regarding the effector response of HVG contrast those by donor T cell responses post-transplant in which single MiHA differences fail to induce GVHD. Finally, heterologous immunity to virus has been reported to generate allo-reactive TM cells. Since such TM repertoires could include specificity for MiHA immunodominant epitopes, the presence of TM populations that can mediate resistance in “naive” recipients may be more prevalent than hitherto appreciated.
Impaired GvL Potential of Natural Killer Cells Early Reconstituting Following Haploidentical HSCT.
Authors: Vago Luca , Zino Elisabetta , Di Terlizzi Simona , Forno Barbara , Stanghellini Maria T. Lupo , Perna Serena K. , Mazzi Benedetta , Peccatori Jacopo , Rossini Silvano , Roncarolo Maria G. , Bonini Chiara , Ciceri Fabio , Bordignon Claudio , Fleischhauer Katharina
Published: October 2019

Alloreactive NK cells have been suggested to be important functional players in GvL activity after haploidentical HSCT for high risk leukemia. In this study we have characterized NK cells differentiating from purified haploidentical CD34+ cells after transplantation into 16 patients who did (n=8) or did not (n=8) suffer acute leukemia relapse in a long term follow-up (median 208 days). The incidence of relapse in these patients was not correlated with the presence (n=9) or absence (n=7) of predicted donor NK alloreactivity (p=0.94). NK cells in the first month after transplantation were, regardless of the occurence of relapse, NKG2A+ (>95%) and KIR− (13%), thus resembling CD56bright NK cells from healthy donors. However, in contrast to mature CD56bright cells, the patients’ NK cells expressed heterogeneous intensities of CD56, were only partly positive for the lymph node homing markers CD62L and CCR7, and expressed a higher amount of Fcγ receptor III (CD16). Importantly, in contrast to mature CD56bright cells, which constitrutively express the high affinity αβγ IL-2 receptor, thus releasing γ-IFN in response to low dose IL2, the patients’ NK cells lacked IL-R α (CD25) and did not release cytokines in response to low-dose IL2, nor, most importantly, when challenged with leukemic blasts. γ-IFN release induced by leukemic blasts could be restored by inhibition of NKG2A while cytotoxicity, which was consistently lower as compared to that of mature CD56+ cells, could not. Our data suggest that NK cells differentiating in patients from CD34+ progenitors after haploidentical HSCT have important phenotipical and functional differences from both subsets of mature NK cells, accounting for an impaired in vivo GvL potential.
Insertional Activation of MDS1/EVI1, PRDM16 and SETBP1 in a Successful Chronic Granulomatous Disease (CGD) Gene Therapy Trial.
Authors: Schwarzwaelder Kerstin , Schmidt Manfred , Deichmann Annette , Ott Marion G. , Stein Stefan , Glimm Hanno , Siler Ulrich , Hoelzer Dieter , Seger Reinhard , Grez Manuel , von Kalle Christof
Published: October 2019

The potential of gene therapy to correct genetic diseases of the lymphoid compartment has been demonstrated in ADA-SCID and X-linked SCID clinical gene therapy trials. The first successful correction of the myeloid compartment could be achieved in the latest chronic granulomatous disease (CGD) gene therapy trial. CD34+ bone marrow derived cells of 2 patients were transduced using a SFFV based retroviral vector encoding the therapeutic transgene gp91phox. After non-myeloablative conditioning the autologous cells were reinfused. 3 months post therapy the proportion of marked granulocytes was 20% in patient 1 and 10% in patient 2. 5 to 9 months after treatment the proportion of gp91phox expressing granulocytes expanded 4-fold in both patients. Until the latest time points analyzed, (P1: d820; P2: d560) the marking efficiency persisted at that level. In order to define the clonality of the corrected hematopoietic repopulation we accomplished linear amplification mediated PCR (LAM-PCR) on peripheral blood and bone marrow samples as well as sorted lymphoid and myeloid fractions derived from successive time points after therapy. To characterize the retroviral insertion site distribution, we carried out high throughput sequencing and mapping of the vector genome junctions. The hematopoietic repopulation in patient 1 was polyclonal up to day 542 after therapy. Subsequently the number of corrected cell clones and the activity of a predominant clone decreased up to 820 days post transplantation, when the patient succumbed to infectious complications. In this time frame, a different predominant clone appeared. The repopulation in patient 2 has been polyclonal until the latest time point analyzed. Identification of 435 integration sites from patient 1 and 330 insertion sites from patient 2 revealed the gene coding region of the zinc finger transcription factor homologues MDS1/EVI1 and PRDM16 as common integration sites (CIS) in both patients and the SETBP1 locus as a third CIS in patient 1. RT-PCR analysis demonstrated an activating influence of vector LTR on individual CIS genes. Our data show that prospectively studying insertions and stem cell contributions is feasible and that retroviral vector insertion may lead to an upregulation of genes causing an in vivo expansion of the affected cell clones, which can augment gene-corrected hematopoietic repopulation as an unexpected, thus far non-adverse side effect.
Transient Blockade of ICOS Co-Stimulatory Pathways Induces Long-Term Tolerance to Factor VIII Following Nonviral Gene Transfer of Factor VIII Plasmid into Hemophilia A Mice.
Authors: Peng Baowei , Ye Peiqing , Blazer Bruce R. , Ochs Hans D. , Miao Carol H.
Published: October 2019

Formation of inhibitory antibodies is a significant problem encountered in the treatment of hemophilia by replacement therapy. Nonviral gene transfer of a factor VIII plasmid into hemophilia mice induced strong humoral responses through predominantly TH2 signals. The plasmid-treated mice produced persistent, high-level inhibitory antibody specifically against FVIII, representing a unique and useful model for testing various immunomodulation strategies. It was previously demonstrated by our group that transient immunosuppression by CTLA4-Ig and anti-CD40L (MR1) can prevent inhibitory antibody formation following nonviral gene transfer of FVIII plasmid into hemophilia A mice. In this study, we tested if blockade of inducible costimulator (ICOS)-ICOS ligand (ICOSL) pathway in combination with or without agents blocking other co-stimulatory pathways can modulate the immune response following gene therapy treatment. Three groups of mice (n=5/group) were subjected to administration of FVIII plasmid via hydrodynamics-based tail-vein injection, and transient immunosuppressive regimens including anti-ICOS (8 treatment in 2 week period), combination of anti-ICOS (same dose) and CTLA4-Ig (2 treatment at day 0 and 2), and combination of anti-ICOS (same dose) and MR1 (5 treatment in 2 week period). 2 mice from anti-ICOS only group, 3 mice from combination treatment of anti-ICOS and CTLA4-Ig group, and 2 mice from combination treatment of anti-ICOS and MR1 group developed inhibitors at 2 weeks post treatment. The rest of the mice did not develop inhibitors. These results imply that neither synergistic nor additional modulation was achieved by combining CTLA4-Ig or MR1 with anti-ICOS compared to anti-ICOS alone. Subsequently a more frequent and longer anti-ICOS treatment (16 treatment in 4 week period) was administered in two separate groups of FVIII plasmid-treated mice (n=5 and 11 per group, respectively). All the treated mice did not produce inhibitory antibodies against FVIII and produced persistent, high-level (100–300 μg/ml) FVIII gene expression for at least 150 days (experimental period). The CD4+ T cells isolated from the spleen of tolerized mice did not proliferate in response to FVIII stimulation in vitro. Furthermore, higher population of CD4+CD25+ regulatory T cells were detected in peripheral blood in the tolerized mice compared to untreated and plasmid-treated mice. Adoptive transfer of CD4+ T cells isolated from tolerized mice is performed to test if these cells can protect the recipient mice from developing inhibitory antibodies against FVIII. Anti-ICOS treatment has the potential for a new immunomodulatory strategy for preventing the formation of inhibitory antibodies against FVIII following gene therapy.
Teaching Hematology to Second Year Medical Students: Results of a National Survey of Hematology Course Directors.
Authors: Broudy Virginia C. , Hickman Scot G.
Published: October 2019

An Association of American Medical Colleges position paper calls for a 30% increase in medical school enrollment by 2015. New faculty effort certification reporting requirements for NIH-supported investigators and increasing clinical productivity expectations at academic medical centers challenge the tradition of faculty volunteerism for medical student teaching. To better define the structure, content, and financial support of second year medical school hematology courses nationwide, in 2001/2002 we mailed a survey to the hematology course directors at 85 of the 125 accredited US medical schools. The 58 course directors who returned the survey represent all regions of the US, and both public and private medical schools. Ten of the hematology course directors subspecialized in areas other than adult or pediatric hematology or hematology/oncology. Median class size was 150 students (range 40–200), and some courses included a substantial proportion (up to 33%) of students other than medical students (dental students, graduate students, PA students). Median course hours was 33 hours (range 8 to 74, an almost 10-fold difference). Approximately 50% of the total teaching time was devoted to lecture (range 5% to 100%), but a wide variety of additional teaching approaches were also employed, including small group discussions, problem-based learning, and web-based teaching (used by 62% of course directors). The median number of faculty responsible for teaching the second year hematology course was 12 (range 1–36). The hematology course directors identified a number of obstacles, including difficulty recruiting teachers (experienced by 45% of course directors), the lack of well-defined content, and the very modest budget (less than $1500 for most courses). Only 3 of the course directors indicated that they received salary support for this role. The findings of this survey suggest that a national effort to define learning objectives for the hematology courses and to share teaching materials among medical schools is warranted. Of note, it was estimated in 1997 that the total educational costs for one medical student are $72,000–92,000 per year, and that the majority of these costs are instructional. In 2003 median medical school tuition nationally was $16,322 (for a state resident attending a public school) and $34,550 (private school tuition). The present results show that few of these funds are directed to support of faculty time to teach the hematology course, and compel the identification of a funding stream to pay faculty for teaching medical student required courses.
Using Geographical Information Systems (GIS) To Examine Associations between Characteristics of Males with Hemophilia and Geographic Distance to Hemophilia Treatment Centers (HTCs).
Authors: McClellan Ann C. , Soucie J. Michael , Kulkarni Roshni
Published: October 2019

Hemophilia, an inherited bleeding disorder, is best treated at specialized healthcare facilities offering comprehensive healthcare services. For healthcare providers at the over 130 federally-funded HTCs across the U.S., information about where individuals with hemophilia live and distances to the HTC can be useful in studying barriers to care and health outcomes. For the analysis, we used data collected on males with hemophilia in the CDC-sponsored Universal Data Collection (UDC) surveillance program that includes demographic and clinical data on about 90% of patients using these HTCs for their care. Patient characteristics, including age, race/ethnicity, insurance status, hemophilia severity, treatment (prophylaxis vs. on-demand) and residential 3-digit zip code were obtained from the UDC database. We calculated distances between the HTC and the centroid (geographic center) of the patients’ zip codes using GIS software (sample map shown in figure 1). The independent associations between patient characteristics and distance to the HTC adjusting for regional variations were examined using a multivariate generalized linear model. The mean age of the patients was 21 years (median 16 years); 70% were white; 42% used a health maintenance organization (HMO), 27% had Medicare or Medicaid coverage, and 16% had commercial health insurance; 50% of patients had severe hemophilia; and 26% were on prophylaxis. The mean distance from patient residence to HTC was 68 miles (median 35 miles). Of the 12,397 patients living in the contiguous U.S., 10,164 (82%) lived within 90 miles of the HTC (53% within 30 miles, 31% from 31–60 miles, and 16% from 61–90 miles). Among patients who lived within 90 miles of the HTC, we found that patients >25 years old and those on prophylaxis lived closer to HTCs than their counterparts (p=0.04 for each). On the other hand, white patients and those using an HMO lived farther from HTCs than those of other races and those with other types of health insurance, respectively (p<0.001 for each). Information from this geographic analysis may be used to guide resource allocation and programmatic activities and may help during emergencies such as natural disasters and treatment product shortages.

North Carolina Hemophilia Treatment Centers Male UDC* Participants with Hemophilia North Carolina Hemophilia Treatment Centers Male UDC* Participants with Hemophilia