bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of Phospholipase C γ 2 in Lymphoma Cells.
Authors: Huynh Minh Q. , Wuendisch Thomas , Ramaswamy Annette , Neubauer Andreas
Published: April 2020

The Nf-κB signaling pathway is important for lymphoma survival and cell proliferation. In recent immunohistological studies, the B-cell receptor (BCR), Src kinases and PLC γ 2 were shown to be highly expressed in primary mediastinal B-cell lymphoma and lymphocyte predominant Hodgkin lymphoma, but not in classical Hodgkin lymphoma. Activation of PLC γ 2 is a crucial step in regulating Nf-κB through protein kinase C (PKC). The PLC-PKC cascade also activates the Ras-Raf-Erk signaling pathway by recruiting GRP3 to the cell membrane. These signaling cascades are well studied in physiological B-cells, but there is little data about the role of PLC γ 2 in lymphomas. Our study compared the PLC γ 2 expression in low-grade gastric MALT lymphoma tissue and chronic gastritis tissue from the same patient by quantitative PCR. In 8 out of 10 cases PLC γ 2 was overexpressed in MALT lymphomas, but not in gastritis. We performed functional studies with the PLC-inhibitor U73122 and PLC γ 2 specific siRNA in two lymphoma cell lines (SSK41, Raji). Proliferation of lymphoma cells was significantly reduced by inhibition of PLC γ 2 on protein level and by knocking down PLC γ 2 mRNA by transfection with a specific siRNA. Our results indicate that proliferation and survival of lymphoma cells depend, at least in part, on the activated PLC-PKC signaling cascade and that it is possible to reduce lymphoma survival by the “small molecule inhibitor” U73122. PLC γ 2 could be a new target for lymphoma treatment.
3′ Distal Regulatory Elements Required for Human CD34 Expression in Transgenic Mice.
Authors: Levantini Elena , Okuno Yutaka , Zhang Pu , Koschmieder Steffen , Radomska Hanna S. , Fenyus Maris , Guiney Maureen , Tenen Daniel G.
Published: April 2020

CD34 is the best-defined human hematopoietic stem cell (HSC) marker, however the regulation of its gene expression is still largely unknown. Therefore, unraveling the elements that regulate human CD34 expression would be an invaluable tool for a broad range of studies, including the establishment of models of leukemia in mice, which require targeting of the transgene to stem and/or early progenitor cells. Moreover, identification of such regulatory elements will provide important insights into the transcriptional agenda of stem and progenitor cells and most importantly will prove useful for gene therapy protocols. Studies from our laboratory demonstrated that human CD34 transgenes are expressed in murine repopulating HSCs, which resembles the expression of the CD34 gene in human hematopoiesis, thus indicating the mouse model as an excellent way to study the expression of human CD34. Using P1 derived artificial chromosome (PAC) clones encompassing the human CD34 gene to generate transgenic mice, we showed that 90kb of upstream and 26kb of downstream flanking sequences were capable of regulating human CD34 expression in murine transgenic lines. Successive deletions of this larger construct were then performed to identify the important control regions. Deletion of the 5′ region from −90kb to −18kb did not result in any loss of activity. PAC54A19, a clone extending from −18kb to +26kb, expressed RNA in various tissues in a manner similar to that of larger fragments. In contrast, deletions creating a construct spanning from −10kb to +17kb led to complete loss of expression in transgenic animals, indicating that critical distal elements are located between −18kb to −10kb and/or +17kb to +26kb. In order to facilitate identification of important regulatory elements present in the upstream (−18kb to −10 kb) and/or downstream (+17kb to +26kb) regions of human CD34, we created further deletions of PAC54A19 using rare-cutting restriction enzymes, and studied the effects of the deletions on human CD34 expression in transgenic mice. Interestingly, we did not detect any human CD34 mRNA and protein expression in bone marrow and HSCs from transgenic mice carrying a construct spanning from −18kb to +17.4kb. In contrast, we observed expression of human CD34 transcripts in the bone marrow of transgenic mice containing a PAC spanning from −12.8kb to +26kb. Furthermore, HSCs from this latter group of mice presented the human CD34 antigen on their surface, as detected by FACS. Taken together, these data are highly suggestive that critical cis regulatory element(s) required to drive human CD34 in vivo expression are located in a 8.6kb fragment placed between +17.4kb and +26kb downstream of the human CD34 gene. Our current efforts focus on identifying the element(s) within the 8.6kb 3′ region that might be required to achieve human CD34 expression in HSCs.
Immunomodulation of Transgene Responses Following Naked DNA Transfer of Factor VIII into Hemophilia A Mice.
Authors: Ye Peiqing , Rawlings David J. , Thompson Arthur R. , Ochs Hans D. , Miao Carol H.
Published: April 2020

Naked DNA transfer of liver-specific, high-expressing plasmid pBS-HCRHPI-FVIIIA in Rag2(−/ −) SCID mice produced persistent high-level gene expression of human factor VIII (hFVIII) (Miao, Hum. Gene Ther. 2003). However, in immunocompetent hemophilia A mice, a robust humoral immune response against FVIII that followed gene transfer led to complete inhibition of circulating FVIII activity (Ye, Mol. Ther. 2004). Transient immunomodulation strategies were explored to prevent the formation of inhibitory antibody formation. Eight groups of mice (n=8) were treated by naked DNA transfer of plasmid pBS-HCRHPI-FVIIIA. Each group were subjected to treatment with single or combined immunosuppressive regimen: CyclosporineA (CSA) daily for 14 days; Rapamycin daily for 14 days; Mycophenylate mofetil (MMF) daily for 14 days; combination of CSA and MMF; combination of Rapamycin and MMF; a monoclonal antibody (MR1) against murine CD40 ligand on days -1, 1, 2, 7, & 14; recombinant murine Ctla4Ig on days 1 & 2; and combination of MR1 and Ctla4Ig. Combination regimens were given using the same combined schedule and dosages. All animals treated with immunosuppression had delayed or no immune responses against hFVIII except the group treated with CSA only. The most effective treatment was observed in animals treated with the combination of Ctla4Ig and MR1. Seven of 8 animals failed to develop detectable inhibitors. One animal developed transient low-titer antibodies. This group of animals produced persistent, therapeutic levels of hFVIII gene expression for over 6 months. Tolerized animals were subsequently challenged by the T dependent antigen, bacteriophage Φx174, and exhibited a normal primary and secondary response including amplification and isotype switch. These results strongly suggest that transient immunomodulation strategies to disrupt B- and T- cell interactions at the time of plasmid injection is effective to promote long-term immune tolerance that is specific for FVIII without altering subsequent immune responses to other T cell dependent antigens.
Antigen-Dependent Suppression of Graft Versus Host Disease by Foxp3-Induced Regulatory T Cells in Transplantation.
Authors: Albert Michael H. , Liu Yan , Anasetti Claudio , Yu Xue-Zhong
Published: April 2020

Adoptive transfer of polyclonal CD4+CD25+ regulatory T cells (Tregs) can tolerize transplantation alloresponses and prevent lethal acute graft-versus-host disease (GVHD). For optimal suppressive function, Tregs need to be activated via their T-cell receptors (TCR), but the antigen specificity of wild type Tregs remains elusive, and therefore controlling potency and duration of Treg activity in the transplantation setting remains not feasible. In this study, we used a murine lethal acute GVHD model system to test the hypothesis that specifically activated, antigen-specific Tregs induced by foxp3 transduction could suppress the response of T effector cells to alloantigens in vitro and prevent GVHD in vivo more effectively than polyclonal Tregs.

We found that the suppressive potential of TCR transgenic (Tg), antigen-specific CD4+CD25+ Tregs was much greater than that of polyclonal Tregs in vitro and in vivo. When activated by their specific peptide antigen, Tg Tregs protected 95% of recipients from lethal GVHD even at ten times lower doses than polyclonal Tregs. To facilitate the acquisition of larger numbers of antigen-specific Tregs, we transduced naive CD4+CD25- cells with foxp3, and observed that these foxp3-induced Tregs also suppressed alloresponses in vitro and prevented GVHD in vivo as effectively as naturally derived CD4+CD25+ Tregs. To enhance translational feasibility, we then used an antigen-specific CD4 Th1 T-cell clone as a source of Tregs after transduction with foxp3, and found those Tregs to effectively prevent GVHD in 90% of recipients. We further found that prevention of GVHD via foxp3-induced Tregs was also dependent on their activation by either a specific alloantigen expressed on recipient cells or by immunization with a specific peptide antigen.

The findings of this study provide a basis for the concept that the onset and potency of alloresponse suppression in GVHD can be regulated by using Tregs with known antigen specificity. The novel evidence that antigen-specific T cell clones can be used as the cell source for foxp3-induced Tregs further improves the feasibility of using Tregs for modulating immune responses in vivo. These data suggest a novel approach to control induction of tolerance using Tregs as an adoptive immunotherapy in allogeneic transplantation.
Early Treatment with CD4+CD25+ Regulatory T Cells Provides Prolonged Suppressive Effects Which Control Evolving but Not Established Graft-Versus-Host-Disease.
Authors: Nguyen Vu H. , Zeiser Robert , Dasilva Daniel , Contag Christopher N. , Negrin Robert S.
Published: April 2020

CD4+CD25+ Regulatory T cells (Treg) reduce the incidence and severity of acute graft-versus-host disease (GvHD) in murine models of allogeneic bone marrow transplantation (BMT) when given at the time of transplantation at a 1:1 ratio with donor effector T cells (Tcon). In our previous study with Treg trafficking, bioluminescene imaging (BLI) indicated a persistence of signal consistent with a prolonged survival of Treg in vivo following allogeneic BMT. In the current study, we evaluated the duration of Treg suppression and the impact of Treg on evolving and established GvHD. Lethally irradiated Balb/c (H2d) hosts received 5x106 T-cell depleted bone marrow (TCD-BM) cells from wild-type FVB mice (H2q) on day 0. On day 2, 3x106 splenocytes, or 1x106 purified CD4+/CD8+ T cells (Tcon) from luciferase+ transgenic mice (FVB) were infused to induce GvHD. Treg, purified from wt-FVB mice, in a 1:1 dose ratio with Tcon, were infused either on day 0, 2, 9, or 23 post-transplantation. Bioluminescence imaging was used to localize and quantitate the proliferation of Tcon in the absence or presence of Treg. Signal intensity, measured by photons/second/mouse, was significantly decreased in animals which received Treg at day 0, 2, or 9 (p-value < 0.05). More importantly, the greatest reduction in signal intensity occurred when Treg were given prior to the induction of GvHD by Tcon. This reduction was associated with a significantly lower clinical score for GvHD. Studies in which Treg are given up to 10 days prior to the addition of Tcon show similar findings. At day 23, when clinical GvHD was fully established in mice which received Tcon, the addition of Treg did not alter the increasing BLI signal level or the clinical course such that all animals died of GvHD (p-value=0.38). Lymphoid reconstitution was not affected by the addition of Treg prior to the induction of GvHD. In dose titration studies whereby Treg are given two days prior to the induction of GvHD, a 10-fold dose reduction in Treg was sufficient to significantly reduce Tcon proliferation and suppress clinical GvHD. We next assessed the duration of Treg suppressive effect by inducing GvHD on day 7, 14, 19 with luc+ Tcon following the infusion of Treg on day 0 of allogeneic BMT. Treg provided protection from the Tcon challenge at all 3 time points, leading to improved survival (p-value < 0.05). We conclude that Treg provide prolonged protection due to their ability to proliferate in vivo in an allogeneic setting. The capacity of Treg to proliferate in vivo permits a significant reduction in the number of Treg needed for adoptive transfer to induce a clinical response, a practical finding given the rarity of Treg. In addition, we conclude that Treg suppress the early proliferation of Tcon, allowing them to prevent and control evolving but not established GvHD.
Donor CD4+ T and B Cells in Transplants Induce Autoimmune-Like Chronic Graft Versus Host Disease.
Authors: Zhang Chunyan , Todorov Ivan , Zhang Zhifang , Liu Yinping , Kandeel Fouad , Forman Stephen , Strober Samuel , Zeng Defu
Published: April 2020

Chronic graft-vs-host disease (GVHD) is a major morbidity and mortality of long-term survivors of allogeneic hematopoietic cell transplantation (HCT). Chronic GVHD can have features of an autoimmune collagen-vascular disease with clinical manifestations similar to autoimmune scleroderma and systemic lupus erythematosus (SLE). However, the pathogenesis of chronic GVHD is poorly understood. It is unclear how autoreactive T and B cells are generated in chronic GVHD recipients. We have recently developed a new autoimmune-like chronic GVHD model by transplantation of donor DBA/2 (H-2d) spleen cells into MHC-matched but minor antigen-mismatched sublethally irradiated BALB/c (H-2d) recipients, as well as athymic BALB/c nu/nu and adult thymectomized BALB/c recipients. Both euthymic and athymic BALB/c recipients developed high-levels of serum IgG autoantibodies, sclerodermatous skin damage and glomerulonephritis. Disease induction required both donor CD25−CD4+ T and B cells in transplants. In contrast, donor CD25+CD4+ T regulatory (Treg) cells prevented the disease induction. These results indicate that host thymus is not required for induction of autoimmune-like chronic GVHD, and that quiescent autoreactive T and B cells in transplants from non-autoimmune donors may be activated and expanded to cause autoimmune-like chronic GVHD in allogeneic recipients, and donor Treg cells can suppress this process.
Pathological Anti-β2-Glycoprotein I Antibodies Disrupt the Anticoagulant Activity of Annexin A5: A Possible Explanation for the Lupus Anticoagulant Paradox.
Authors: de Laat Bas , Wu Xiao-Xuan , van Lummel Menno , Derksen Ronald H.W.M. , de Groot Philip G. , Rand Jacob H.
Published: April 2020

One of the most enigmatic features of the antiphospholipid (aPL) syndrome is the paradoxical correlation between thrombosis and the in vitro lupus anticoagulant (LAC) effect. It has recently been shown that only LAC that are due to β2-glycoprotein I (β2GPI) antibodies correlate with thrombosis. It has also been reported that aPL antibodies can disrupt the formation of an anticoagulant crystal shield that is formed by annexin A5 (AnxA5) over phospholipids bilayers. This disruption can be assayed by measuring resistance to AnxA5 anticoagulant activity. We therefore investigated whether the presence of these β2GPI-dependent LACs might correlate with the disruption of AnxA5 anticoagulant activity. We performed a double blind study with 30 patients divided into three groups; Group A: plasma of patients that show a β2GPI-dependent LAC; Group B: plasma of patients that show a β2GPI-independent LAC; Group C: plasma of SLE patients without a LAC. As previously demonstrated, we were able to discriminate between a β2GPI-dependent and a β2GPI-independent LAC by titrating cardiolipin into the plasma sample. A β2GPI-dependent LAC could be normalized by the addition of cardiolipin in contrast to a β2GPI-independent LAC which was prolonged by cardiolipin. To investigate the effects on AnxA5, we performed an APPT-based coagulation assay. In this assay we added AnxA5 to the plasma of patients and measured the prolongation in clotting time. We calculated the ratio of the clotting time divided by the clotting time with AnxA5 added to the plasma (=AnxA5 ratio). This assay represents the resistance against AnxA5 and gives an indication of its effect on the anticoagulant shield formed by AnxA5. Eleven patients displayed a β2GPI-dependent LAC (group A, all thrombosis), that gave an AnxA5 ratio of 151.3% (± SD 14.5). For 9 patients that displayed a β2GPI-independent LAC (group B, thrombosis n=3) we found a ratio of 225.4% (± SD 29.4). Ten patients did not have LAC (group C, thrombosis n=2) that gave a ratio of 219.2% (± SD 31.0). The annexin A5 ratio of group A was significantly lower than the ratio of group B (P=0.0002) and group C (P=0.0001). There was no difference in AnxA5 ratio between group B and group C (P=0.7802). β2GPI-dependent LAC correlate strongly with both thrombosis and AnxA5 resistance. The results therefore suggest that anti-β2GPI antibodies that cause LAC, in contrast to aPL antibodies with other specificities, are capable of disrupting the anticoagulant effect of AnxA5 on phospholipids. The disruption of the AnxA5 anticoagulant shield by LAC-causing anti-β2GPI antibodies may be a mechanism for thrombosis in APS and offers a potential explanation for the LAC paradox.

Figure Figure
Syndecan-1 Is Expressed on a Subset of Hematopoietic Stem Cells and Affects Their Function and Susceptibility to Malignant Transformation.
Authors: Ranheim Erik A. , McDermott Sean P. , Alexander Caroline M.
Published: April 2020

Syndecan-1 (CD138) is a heparan-sulfate proteoglycan (HSPG) known to be involved in cytokine signaling and cellular migration in the extracellular matrix. Knockout mice lacking CD138 expression exhibit a profound resistance to Wnt-1 induced mammary carcinoma that may be secondary to changes in mammary progenitor cell number or function. To determine whether this represented a more global phenomenon, we assessed the effects of CD138 expression on tumorigenesis induced by IP administration of the chemical carcinogen, DMBA, to post-natal mice. CD138−/− mice were highly resistant to DMBA-induced lung, liver, and ovarian carcinomas with an 80% reduction in tumor incidence compared to CD138+/+ mice. Absence of CD138 also protected from DMBA-induced hematopoietic tumors, with 4/58 (6.9%) KO versus 11/56 (19.6%) WT animals developing hematopoietic proliferations. Moreover, upon histologic and phenotypic analysis, the tumors arising in WT animals all proved to be CD4+CD8+ T-lymphoblastic lymphomas, which was seen in only a single KO animal. The other CD138−/− mice instead developed a myeloproliferative disorder with extensive splenic extramedullary hematopoiesis. We hypothesized that differences in tumor susceptibility between WT and KO mice may be secondary to abnormalities in tissue specific stem cell numbers and/or function. Therefore, we examined the expression and function of CD138 on mouse hematopoietic stem cells (HSC) and found that CD138 is expressed on approximately one third of mouse long-term HSC (LT-HSC), as defined by the Kit+Thy1.1loLin−Sca+ (KTLS) phenotype. Knockout mice lacking CD138 expression have mildly reduced numbers of HSC in the bone marrow with Lin−Sca+Kit+ cells making up 0.073±0.017% of marrow cells in CD138−/− mice versus 0.098±0.019% in WT mice (p = 0.038, n=6). While apparently adequate for hematopoiesis throughout the first 18 months of life, these HSC are functionally defective in competitive reconstitution assays in wild-type recipients compared to syngeneic, CD138+/+ LT-HSC. CD45.2+Kit+Flk2-Lin−Sca+ LT-HSC from CD138−/− or control CD138+/+ C57/Bl6 mice were flow sorted and 200 purified LT-HSC were injected into lethally irradiated CD45 congenic recipients along with 3 x 105 syngeneic whole bone marrow cells. Peripheral blood was monitored for reconstitution of T, B, and myeloid cells by analysis of CD45 allele expression; and as shown in the figure, LT-HSC from CD138−/− (KO) donor mice gave significantly less total leukocyte (p = 0.03 at 12 wks.) and myeloid (CD11b+ cells) reconstitution versus WT donor LT-HSC. These data suggest that CD138 plays a critical role in HSC function and provide a potential mechanism for the tumor resistant phenotype of CD138−/− mice. We are currently assessing function (in vivo and in vitro) of CD138 positive and negative WT KTLS-defined LT-HSC to determine whether CD138 may further subdivide this heterogeneous population.

Figure Figure
Induction of Adipocyte Differentiation and Reduction of Hematopoesis-Supporting Activity of Mouse Stromal Cells by Suppression of Transcription Factor GATA-2.
Authors: Okitsu Yoko , Harigae Hideo , Seki Masanori , Fujiwara Toru , Takahashi Shinichiro , Yokoyama Hisayuki , Kameoka Junichi , Minegishi Naoko , Sasaki Takeshi
Published: April 2020

(Introduction) Aplastic anemia (AA) is characterized by peripheral pancytopenia and fatty bone marrow. An immunological attack to hematopoietic stem cells has been thought to be responsible for the development of the disease. Previously, we reported the expression of transcription factor GATA-2 is significantly decreased in CD34 positive cells in AA. Together with the phenotypes of hematopoietic stem cells in GATA-2 hetero-knockout mice, GATA-2 down-regulation may play a role in the reduction of a stem cell pool observed in AA. On the other hand, GATA-2 has been shown to be essential for the maintenance of immaturity of preadipocytes. If a pathological immune response in AA decreases the level of GATA-2 expression in not only hematopoietic stem cells but also stromal preadipocytes, it may accelerate the maturation of preadipocytes, leading to the formation of fatty bone marrow. To explore this possibility, the phenotypic change of stromal preadipocytes by suppression of GATA-2 was examined in this study.

(Method) The GATA-2 expression level was suppressed by using siRNA for GATA-2 in mouse stromal preadipocyte cell lines, TBR9 and TBR343. After the treatment with siRNA, the adipocyte differentiation was induced by the incubation with insulin and dexamethasone for 7days. Then, the maturation level was examined by oil drops formation judged by oil red staining, and by the expression level of adipcin and PPAR-γ mRNA. Supporting activity of hematopoietic colony formation was also evaluated by using mouse fetal liver cells after siRNA treatment.

(Results) By using designed siRNA, the GATA-2 expression was suppressed to 30% of control, whereas the expression level of GATA-3, which is co-expressed in preadipocytes, was unchanged. When GATA-2 was suppressed by siRNA, the oil drop formation and adipocyte-specific gene expression was significantly accelerated in both of stromal cells. Furthermore, the number of fetal liver hematopoetic colonies was significantly decreased by suppression of GATA-2, suggesting that GATA-2 down-regulation in stromal preadipocytes results in not only the acceleration of the maturation but also the reduced supporting activity of hematopoietic colony formation

(Conclusion) These results suggest that suppression of GATA-2 in hematopoietic tissues induces the characteristic features of AA, i.e., decreased the number of hematopoietic stem cells and increased number of mature adipocytes.
Expansion of Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells Requires Rac-Mediated Interaction with Stromal Cells.
Authors: Schuringa Jan Jacob , Rozenveld-Geugien Marjan , Baas Inge , van Gosliga Djoke , Vellenga Edo
Published: April 2020

We have studied Rac signal transduction in human cord-blood (CB) and acute myeloid leukemia (AML) CD34+ cells and determined that Rac proteins are critically involved in the interaction between human stem/progenitor cells and stroma. Constitutive activation of Rac signaling was achieved by retroviral introduction of Rac1-V12 into CB-derived CD34+ cells, while inhibition of Rac signaling was established by retroviral introduction of dominant negative Rac1-N17 or by utilizing the Rac inhibitor NSC23766. Inhibition of Rac signaling resulted in a proliferative disadvantage of CB-CD34+ cells when cultured on MS5 stromal cells. Cells were severely disturbed in their migration towards and direct association with MS5 stroma when Rac signaling was inhibited. The Long Term Culture-Initiating Cells (LTC-ICs) migrated underneath the stromal MS5 layer within 24 hrs after plating, and similar results were obtained for about 50% of the Colony Forming Cells (CFCs). However, transient inhibition of Rac signaling for 24–72 hrs resulted in a shift of LTC-ICs and CFCs to the suspension phase as determined by colony assays and CAFC week 5 enumeration. When Rac signaling signal transduction was inhibited during the 5 week coculture period a dramatic decrease in LTC-IC frequency from 0.6% to 0.15% was observed. Many of these phenotypes were reversed in the presence of activated Rac1-V12, including improved migration towards and association with MS5 cells and elevated LTC-IC frequencies. Importantly, in CAFC assays using AML cells (n=8) that were enriched for leukemic stem cells on the basis of a CD34+/CD38low phenotype we observed a dramatic decrease in leukemic CAFC formation as well as strongly diminished clonal expansion in the presence of the Rac inhibitor NSC23766. Taken together, our data indicate that Rac signal transduction is required for the maintenance and expansion of both normal as well as leukemic stem/progenitor cells by mediating their interaction with stromal cells.
T-Cell Depleted PBSCT Mitigates Acute GVHD, but Preserves Protective Chronic GVHD. Long Term Follow up of 138 Patients.
Authors: Montero Aldemar , Savani Bipin N. , Mielke Stephan , Solomon Scott R. , Childs Richard , Barrett A. John
Published: April 2020

Between 1997 and 2004, 138 patients with hematological malignancies had an HLA-identical sibling peripheral blood stem cell transplant (PBSCT). Preparative regimen was TBI (12–13.6Gy), cyclophosphamide 120mg/kg +/− fludarabine 125mg/m2. T-cell dose was adjusted to 0.2-1 x 105 CD3+ cells/kg. CD34 dose was 2.7 – 15.9 x 106/kg (median 5.9). Patients without ≥ grade 2 acute GVHD received 1 or 2 donor lymphocyte infusions (DLI) of 107 CD3+ cells/kg between days 45 and 100. For GVHD prophylaxis, all patients received cyclosporine (CSA) following the DLI for a minimum of 6 months. In addition 72 patients received CSA in the first 6 weeks post PBSCT and 66 received no CSA immediately post-transplant. Patients were aged between 10–56 (median 34 years). Seventy-seven patients with CML-CP (42), acute leukemia in first remission (22) or MDS RA (10) were designated as standard risk (SR) for transplant outcome. Sixty-one patients with more advance or refractory disease were designated as high risk (HR) for transplant outcome. Seven patients, distributed in both groups had CLL, NHL, MM and CMML. Overall survival, relapse and TRM were 58 ± 4.5%, 40.2 ± 5.5% and 18.4 ± 3.4% respectively after a median follow up of 4 years. Twenty-two patients did not receive DLI, 70 received one and 46 received two DLI. Twenty-three patients had TRM, 2 from graft failure, 5 from acute or chronic GVHD. Fifty two (37%) developed ≥ grade II, and 21 (15%) patients grade III-IV acute GVHD. Forty two (30%) had limited and 30 (22%) extensive chronic GVHD. In univariate Kaplan-Meier analyses standard risk disease, cGVHD, single DLI and day 30 lymphocyte count (LC30) above median, favorably affected overall survival (DLI p=0.002; LC30 p=0.0002, all other variables p<0.0001). Risk status was the strongest factor predicting relapse (28.8 ± 8% and 58.4 ± 7% in SR and HR patients respectively p<0.0001). Fludarabine, degree of T-cell depletion, age, cyclosporine schedule and CD34 dose were not significant.

In a Cox regression analysis, only cGVHD (RR, 4.268; p=0.001; 95% CI=1.77–10.25) and standard risk (RR, 0.146; p<0.001; 95%CI=0.06–0.35) remained as independent predictive factors for outcome (Figure A). On landmark analysis after day +120, cGVHD was associated with better OS (p<0.0001) and DFS (p=0.0006) (Figure B), and relapse (21.7± 6% and 51.5 ± 11% in patients with and without cGVHD p= 0.002).

Aggressive T-cell depletion, combined with relatively high doses of CD34+ cells had proven to be a key strategy to minimize TRM and severe aGVHD. Similar to unmanipulated PBSCT this approach appears to provide a protective effect of chronic GVHD, but with low late mortality.

Figure Figure
Polymorphisms of NOD2/CARD15 Are Associated with Clinical Outcome in T-Cell Depleted HLA-Identical Sibling Allogeneic Stem Cell Transplantation.
Authors: Granell Miquel , Urbano-Ispizua Alvaro , Arostegui Juan Ignacio , Fernandez-Aviles Francesc , Martinez Carmen , Rovira Montserrat , Gaya Anna , Navarro Alfons , Talarn Carme , Carreras Enric , Monzo Mariano , Yague Jordi , Montserrat Emili
Published: April 2020

Three single nucleotide polymorphisms (SNP) in NOD2/CARD15 have been associated with the incidence and severity of acute GVHD following allogeneic stem cell transplantation (allo-SCT). The potential clinical influence of SNP in NOD2/CARD15 in a model of allo-SCT with a low incidence of GVHD, such as T-cell depleted transplants (TCD), has not been studied. We analyzed the association of SNP in NOD2/CARD15 with the outcome of 84 patients undergoing allo-SCT with TCD by means of CD34+ selection. Results were compared with a concurrent group of 94 patients receiving an allo-SCT with unmanipulated grafts (Non-TCD). Transplants were performed in a single institution, donors were HLA identical siblings and all patients received myeloablative-conditioning regimen. Median follow-up was of 30.6 months (range, 0.2–112). SNP 8, 12 and 13 were analyzed by RT-PCR allelic discrimination method in all patients and in 137 donors. SNP in NOD2/CARD15 were detected in 13.8% of patients and in 10.7% of donors. Results of the association of patient’s NOD2/CARD15 genotype and outcome are given in the table. In TCD transplants, independent risk factors associated with event free survival in a multivariate analysis were: patient’s NOD2/CARD15 genotype (RR 2.7, p=0.02), patient’s age (RR 2.3, p=0.03) and conditioning with radiotherapy (RR 2.7, p=0.03). The only association of donor’s NOD2/CARD15 genotype with clinical outcome in the multivariate analysis was with acute GVHD (RR 3.3, p=0.03). In conclusion, polymorphisms in NOD2/CARD15 have a strong association with clinical outcome in TCD transplants, which is independent of GVHD.

Overall group (n=178) SNP vs. WT P* TCD (n=84) SNP vs. WT P* Non-TCD (n=94) SNP vs. WT P* WT, wild type; N.S, non significant; * Log-rank test Acute GVHD II–IV 27 vs. 22 N.S 22 vs. 40 N.S 33 vs. 12 N.S Chronic GVHD 61 vs. 35 0.1 29 vs. 14 N.S 100 vs. 44 0.005 Event free survival 21 vs. 47 0.03 17 vs. 48 0.04 22 vs. 31 N.S Overall survival 30 vs. 59 0.01 33 vs. 56 0.04 30 vs. 48 N.S
IL-15 Plays a Critical Role in CD8+ T-Cell Recovery after Allogeneic Transplantation.
Authors: Hakim Frances T. , Allen Kenton , Carson Jesse M. , Boyiadzis Michael , Memon Sarfraz A. , Dean Robert , Odom Jeanne , Wayne Alan , Fowler Daniel , Bishop Michael R. , Gress Ronald E.
Published: April 2020

In severely lymphopenic hosts, CD4+ and CD8+ T cell populations increase rapidly through peripheral homeostatic expansion, a process in which IL-7 has been found to play a key role. Because of the marked differences in the kinetics of CD4+ and CD8+ T cell repopulation following hematopoietic stem cell transplants (HSCT), we have investigated the roles of additional cytokines in early repopulation. Interleukin-15 (IL-15) supports the proliferation, terminal differentiation, and survival of NK, NKT and memory CD8+ T-cell populations, all of which increase disproportionately in the early transplant period. We therefore investigated the role of IL-15 in post-transplant CD8+ T cell recovery by assessing plasma IL-15 levels, IL-15 receptor expression and IL-15-induced proliferation by BrdU incorporation. In patients undergoing non-myeloablative HLA-matched allogeneic HSCT for hematological and non-hematological malignancies, IL-15 levels in the plasma increased concurrent with the loss of lymphocytes during each cycle of inductive chemotherapy, and peaked at a 50-fold increase over pretreatment levels at day of transplant, a time when CD8+ T cell levels were usually less than 1 cell/μL. Plasma IL-15 levels fell rapidly in the first two weeks, during the rapid recovery of NK and CD8+ T cell populations, returning to pretransplant levels by 1–2 months. Overall, during the cytoreductive transplant and for the first year post transplant, the IL-15 levels were inversely proportional to the level of CD8 T cells (P < 0.0001; r = −0.73). In the first weeks after transplant, CD8+T-cells expressed elevated levels of CD122, but had little or no expression of CD25, the IL-2Ralpha chain. Levels of CD122 remained elevated for several months, while expression of CD127, IL-7Ralpha, was reduced. In vitro BrdU incorporation assays demonstrated that CD8+ T-cells from both normal donors and transplant recipients responded primarily to IL-15, not IL-7 or IL-2. CD4+ T cells, in contrast, responded primarily to IL-7. A higher proportion of CD28+CD45RA− memory and CD28−CD45RA+/− effector-memory CD8+ T-cells incorporated BrdU than did naive CD28+CD45RA+ CD8+ T cells. Finally, IL-15, not IL-2 or IL-7, was found to maintain survival and support expansion in culture of the CD28−CD57+ terminal effector cells that dominate post transplant CD8+ T-cells populations. These data, describing an inverse relationship between the levels of free plasma IL-15 and CD8+ T cells, elevated expression of IL-15 receptor chain and strong responsiveness of post transplant CD8+ T cells to IL-15, suggest that IL-15 serves as a critical homeostatic cytokine post transplant, supporting the initial rapid generation CD8+ T cells and maintaining elevated levels of memory/effector CD8+ T-cell populations in the post transplant period.
Inverse Correlation of Early Regulatory T Cell Reconstitution Post-Transplant and Acute and Chronic Graft-Versus-Host-Disease.
Authors: Rezvani Katayoun , Mielke Stephan , Kilical Yasemin , Savani Bipin , Keyvanfar Keyvan , Hensel Nancy , Kurlander Roger , Barrett A. John
Published: April 2020

Data on the impact of regulatory T cells (Tregs) on GVHD following SCT in man is conflicting, possibly due to differences in techniques used to characterize them. We studied reconstitution of CD4+CD25+Foxp3+ T cells in the first 150 days following a T-depleted allogeneic SCT by surface phenotyping, Foxp3 gene expression and functional assays to characterize their post-transplant origin and pattern of recovery relating these findings to GVHD. Although there was a direct correlation between Foxp3 gene expression and CD4+CD25+ T cells in healthy donors the ratio between CD4+CD25+ T cell numbers and Foxp3 gene expression was more variable after SCT. In a longitudinal post-transplant study in 12 patients with leukemia, the Foxp3 copy number and CD4+CD25+ T cells were strongly correlated in 5. In 4 patients CD4+CD25+ and CD4+CD25+CD69+ T cells were disproportionately increased compared with Foxp3 gene expression, corresponding temporally with the development of acute GVHD and suggesting expansion of effector T cells. In 3 patients who did not develop clinically significant GVHD, the ratio of Foxp3 gene expression to CD25 fraction of CD4+ T cells was disproportionately high suggesting that Treg may have expanded or upregulated Foxp3 gene copy numbers. Treg reconstitution was slow in the first 45 days after SCT, lagging behind CD3 recovery but reaching normal levels by day 120. High day 30 Foxp3 gene expression inversely correlated with grade II–IV aGVHD (p = 0.02) and with severity of cGVHD. Chimerism analysis was performed on sorted CD4+CD25+ and CD4+CD25− T cell fractions from 7 patients on day120 samples. In most patients, the expanded Treg population was enriched in cells of donor origin. These results support strategies to prevent GVHD by increasing post-transplant Treg recovery by adoptive transfer of ex-vivo expanded Treg. Conversely, the absence of Treg together with lymphopenia-driven T cell expansion may offer a window of opportunity for boosting GVL effects by vaccination in the first few weeks after transplantation.

Figure Figure
Decrease of FoxP3 Gene Expression Precedes and Inversely Correlates with Onset of Acute GVHD.
Authors: Tabellini Laura , Haeusermann Peter , Damodaran Anand C. , Hansen John A.
Published: April 2020

Background: Regulatory CD4+CD25+FoxP3+ T-cells are hypothesized to play a role in modulating the severity of acute GVHD and induction of non-responsiveness or tolerance after hematopoietic cell transplantation. Recent studies in patients post transplant demonstrated association between decrease of FoxP3 and development of acute GVHD. However sequential changes of FoxP3 expression prior to onset of clinical GVHD have not been reported.

Methods: Thirteen patients who received myeloablative-conditioning regimen for hematological malignancies and cyclosporine and methotrexate for GVHD prophylaxis have been studied on two or more occasions prior to onset of clinically significant grade II-IV acute GVHD. Six of these patients developed GVHD by day 30 (range, 29–31 days), 4 patients by day 37 (range, 35–39 days), and 3 patients showed no evidence of GHVD during the first 100 days post-transplant. Sequential measurements of FoxP3 gene expression by real time PCR were made at day 21, day 28 and day 35 post-HSCT. All samples analyzed were collected before initiation of systemic steroid therapy. The 6 patients with onset by day 30 and 1 patient without GVHD were studied at days 21 and 28. RNA was extracted from white blood cells and cDNA templates were synthesized using 1μg RNA. The standard curve method was used for relative quantitation of Foxp3.

Results: Using the paired t test, we found a significant decrease of FoxP3 gene expression between day 21 (median 0.3; range 0.01–1.9) and day 28 (median 0.13; range 0.003–0.321) (p=0.05). Additionally, a clear trend for a decreasing expression of Foxp3 was found between day 28 and day 35 (median 0.1; range 0.06–0.54). Two of the 3 patients who remained GVHD free the first 100 days showed either an increase or no change in FoxP3 expression between days 21, 28 and 35 and between days 21 and 28 respectively.

Conclusion: These preliminary results indicate a pattern of decreasing FoxP3 gene expression in patients preceding the development of acute GVHD independent of the day of onset of this disease. These studies are being extended in a new cohort of transplant patients, and monitoring of patients without GVHD is continuing to determine if there are any patterns of FoxP3 expression associated with the onset of chronic GVHD. The available preliminary data suggest that sequential measurements of FoxP3 may predict onset of clinically significant acute GVHD.
Expression of Interleukin-7 Receptor and Activation Markers on Central Memory CD8 Cells in HIV-Infected Individuals Treated with Antiretrovirals.
Authors: Routy Jean Pierre , Boulassel Med-Rachid , Mercier Francois , Grignon Chantal , Young Mavis , Legault Mario , Tremblay Cecile , Cheynier Remi , Sekaly Rafick-Pierre
Published: April 2020

We have recently shown that high levels of interleukin-7 (IL-7) predict poor virological response in HIV-1-infected patients. To understand the underlying mechanisms associated with increase in IL-7 levels, we assess the expression of IL-7 receptor and activation markers on different CD8 subsets in there well defined groups of patients including successfully treated (ST; n=8), untreated (UT; n=6), failing HAART due to drug resistance (FT; n=12) and healthy subjects (n=5).

The expression of IL-7/IL-2 receptors (CD127/CD25) and activation markers (CD38/HLA-DR) on CD8 naïve cells (CD45RA+CCR7+CD27+), central memory (TCM; CD45RA−CCR7+CD27+), pre-terminal effector memory (TPEM; CD45RA−CCR7−CD27−) and terminal effector memory cells (TTEM; CD45RA+CCR7−CD27−) was measured in fresh blood samples using 8-color flow cytometry. Mann Whitney U test and Spearman correlation were used.

Although there was a trend toward a decrease in naïve, TCM, TPEM and TTEM cells from ST to UT and then to FT, this did not reach significance (P=0.09). FT and UT subjects showed higher number of CD8 activated cells than ST or controls (22%, 20%, 3% and 4% respectively; P=0.07). Similarly, the percentages of TCM activated cells were significantly higher in FT and UT than ST or controls (28%, 27%, 7% and 0.07%; P=0.02), whereas naïve and TTEM activated cells were similar in all groups. Interestingly, the percentage of TCM cells expressing CD127+CD25+ was lower in FT compared to UT or ST (0.7%, 2.1 and 7.4%; P=0.02). A strong positive correlation was observed between viral load and CD8+ or TCM activated cells (r=0.46, P=0.02; r=0.65, P=0.004 respectively) but not with naïve CD8+ cells. These data suggest that a substantial proportion of TCM but not naïve, TPEM and TTEM cells expressing immune activation markers with lower IL-7 receptor expression is present in subjects with persistent antigen stimulation, thus providing an explanation for increasing circulating IL-7 levels.
Successful Allogeneic HSCT in a Patient with Chronic Granulomatous Disease and McLeod Phenotype after Sensitization to Kx- and K- Antigens.
Authors: Hoenig Manfred , Schulz Ansgar , Schuetz Catharina , Freihorst Joachim , Baumann Ulrich , Flegel Willy A. , Debatin Klaus-Michael , Friedrich Wilhelm
Published: April 2020

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by the defective generation of active oxygen metabolites by phagocytic cells. CGD can be associated with a condition called McLeod phenotype if a large X-chromosomal deletion includes the genes for gp91phox and the XK-protein. The absence of XK on erythrocyte membranes concomitantly leads to a reduced expression of Kell antigens. If transfused with red cells expressing these proteins, patients with CGD and McLeod phenotype become sensitized which may limit future transfusions. We present a patient with CGD and McLeod phenotype, who had received Kx- (the antigen of XK) and K- positive red cells at the age of 10 months; 9 years later he was given granulocyte transfusions following lung surgery because of pulmonary aspergillosis. Shortly thereafter he required a transfusion of red cells, to which he responded with a severe hemolytic adverse reaction. Antibodies against Kx- and K- antigens were detected. Hematopoietic stem cell transplantation (HSCT) is potentially curative in CGD and was planned for this boy at the age of 14 years because of progressive aspergillosis. Transfusions in this situation can only be given from Kx- and K- antigen negative donors suffering from McLeod syndrome themselves. Resources for these products are extremely limited. Furthermore any transplant would contain Kx- antigen and could potentially boost immunological reactions inducing graft rejection. To cope with these potential complications the following strategy was used: before HSCT autologous red cells as well as bone marrow for an autologous rescue therapy in case of graft rejection were cryopreserved. B-cells were depleted in the patient by two infusions of Mabthera (anti-CD20 antibody). Total body irradiation (12Gy), fludarabine and ATG were given for conditioning. No blood products were necessary during conditioning and before HSCT from a HLA- matched unrelated donor. Transfusions of platelets and red cells from Kx- antigen positive and K- negative donors were well tolerated 11 days after HSCT. No GvHD occurred on prophylaxis with CSA. Hematological and immunological reconstitution with complete donor chimerism was uneventful except a transient episode of immune mediated thrombocytopenia at 5 months after HSCT which responded promptly to steroids. After 10 months of follow up the patient is currently fully engrafted, in an excellent clinical condition and back to school. Conclusion: In patients with X- linked CGD screening for McLeod phenotype needs to be performed prior to transfusion of blood products. Even if sensitization to Kx- and K- antigens has occurred, HSCT can be curative as demonstrated for the first time in this report.
Secondary Cytogenetic Abnormalities and Outcome in Children with TEL-AML1-Positive Acute Lymphoblastic Leukemia.
Authors: Abla Oussama A. , Hitzler Johann , Ye Charles , Abdelhaleem Mohamed , Grant Ronald , Nie Gloria , Kangesan Shanthini , Teshima Ikuko , Shago Mary
Published: April 2020

The t(12;21) translocation, which results in the fusion of the TEL (ETV6) and AML1 (RUNX1) genes, is present in ~25% of pediatric B-precusor acute lymphoblastic leukemia (ALL) patients. Although initially thought to be a favorable prognostic indicator, the t(12;21) was associated with a similar rate of relapse as t(12;21)-negative pre-B ALL in subsequent studies. While secondary cytogenetic abnormalities are often present, their prognostic significance is unknown. The objective of this study is therefore to examine the type, frequency and prognostic impact of secondary cytogenetic abnormalites in t(12;21)-positive blasts of children with ALL. We studied retrospectively 56 patients diagnosed with t(12;21)-positive ALL at the Hospital for Sick Children between 2000–2005. The mean age at diagnosis was 4.9 years (range <1 to 12 years). Nine patients (16%) presented with a white blood cell count of greater than 50 × 109/L. Cytogenetic studies consisted of a combination of FISH, G-banding and spectral karyotyping. The most frequent secondary cytogenetic changes in patients with t(12;21)-positive ALL were: the deletion of the non-translocated TEL allele (38%), an extra copy of the TEL-AML1 fusion gene (14%). Additional numerical abnormalities were seen in approximately one-third of the cases, with the most common being a gain of chromosome 21(13%). Recurrent deletions of chromosomal regions 6q and 11q were observed (14%). A surprising degree of karyotypic complexity was noted. Three or more additional chromosome abnormalities, two abnormal clonal lines, and complex structural rearrangements involving TEL-AML1 were detected in 28%, 21%, and 9% of patients, respectively. Five children (9%) in this cohort have developed a relapse of ALL. The 5-year event-free survival (EFS) was 83±7%. The 5-year EFS for patients with a deleted second TEL allele or an extra TEL-AML1 fusion was not statistically different. Of note, 4 of the children who relapsed had high-risk features at presentation (NCI criteria). All were treated with chemotherapy protocols developed for lower risk ALL. The realization that TEL-AML1 does not always predict excellent prognosis indicates that an on-going study of patients and their survival, with detailed analysis of complex genetic changes, is necessary for a reliable assessment of the prognostic value of the t(12;21) in pediatric ALL.