Two Distinct Co-Culture Systems, Designed to Mimic the Tumor Microenvironment, Induce Remarkably Similar Phenotypic Changes in Primary CLL Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2362-2362
Author(s):  
Andrea Gail Sherman Buggins ◽  
Laurence Pearce ◽  
Piers EM Patten ◽  
Liam Morgan ◽  
Paul Brennan ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Model System ◽  
Model Systems ◽  
Microvascular Endothelial Cell ◽  
Phenotypic Changes ◽  
Culture Systems ◽  
Tissue Microenvironment ◽  
Cell Counts

Abstract Abstract 2362 Poster Board II-339 There is growing evidence that interactions in the tumour microenvironment promote the survival, proliferation and drug resistance of primary chronic lymphocytic leukaemia (CLL) cells. Furthermore, progressive CLL is frequently associated with lymphadenopathy, pointing to a crucial role for signals emanating from the tissue microenvironment in the accumulation of malignant B-cells. Proliferation of CLL cells appears to be confined to specialized structures called pseudofollicles, which contain a number of cell types including CLL cells, T-cells, vascular endothelial cells and stromal cells. Using two separate model systems designed to mimic this microenvironment, we characterised the phenotypic changes induced in primary CLL cells and assessed both viability and proliferation when compared with corresponding control cultures. In the first model system we co-cultured CLL cells with mouse embryonic fibroblasts transfected with human CD40L supplemented with soluble IL-4. In the second model system we utilised the microvascular endothelial cell line HMEC-1 with and without the addition of stimulated autologous T-cells. Microarray analysis of this cell line confirmed that it expresses high levels of the CD38 ligand CD31 as well as the cytokines IL-2, IL-4, IL-6 and the chemokines CXCL1 and CXCL2. Both model systems resulted in enhanced CLL cell survival when compared to control cultures (P<0.0001). Furthermore, these conditions induced significant cell division as evidenced by increasing absolute cell counts, significant reductions in mean fluorescence intensity (MFI) of CFSE-loaded cells beyond day 4 (P<0.0001) and the presence of mitotic cells on cytospins. Morphologically, the CLL cells showed a marked increase in size together with cytoplasmic projections. In the CD40L expressing fibroblast model this was associated with a modest increase in CD11c expression (P = 0.02) but no increase in CD103 or the plasmacytoid marker CD138 (P = 0.23 and P = 0.45 respectively). We showed a consistent increase in CD38 MFI in both culture systems (P = 0.0003) and this was further increased by the addition of activated T-cells (P = 0.007). Importantly, in the CD40L transfected fibroblast system, this increase in CD38 expression was strongly associated with an increase in the expression of ZAP-70 (r2 = 0.43) adding further weight to the suggestion that these parameters are in some way biologically linked. Extended immunophenotyping revealed marked increases in the expression of CD69 and CD44 (P<0.0001 and P = 0.0005 respectively). Both of these molecules are NF-κB regulated genes suggesting a role for this transcription factor in generating the altered phenotype observed. Taken together, we have demonstrated that two very different co-culture systems induce remarkably similar changes in primary CLL cells. It seems likely that the further characterisation of these model systems will reveal important new information about the nature of the key interactions between CLL cells and accessory cells in the tissue microenvironment. They also represent a more realistic, clinically relevant, drug testing platform. Disclosures: No relevant conflicts of interest to declare.

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Gisele Olinto Libanio Rodrigues ◽  
Julie Hixon ◽  
Hila Winer ◽  
Erica Matich ◽  
Caroline Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals CAL-1 as Cellular Model System to Study CCR7-Guided Human Dendritic Cell Migration

2021 ◽  
Vol 12 ◽  
Author(s):  
Edith Uetz-von Allmen ◽  
Guerric P. B. Samson ◽  
Vladimir Purvanov ◽  
Takahiro Maeda ◽  
Daniel F. Legler
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Neoplastic Cell ◽  
Model System ◽  
Model Systems ◽  
Cellular Model ◽  
Gm Csf ◽  
Endogenous Expression ◽  
Dendritic Cell Migration ◽  
Spatio Temporal

Dendritic cells (DCs) are potent and versatile professional antigen-presenting cells and central for the induction of adaptive immunity. The ability to migrate and transport peripherally acquired antigens to draining lymph nodes for subsequent cognate T cell priming is a key feature of DCs. Consequently, DC-based immunotherapies are used to elicit tumor-antigen specific T cell responses in cancer patients. Understanding chemokine-guided DC migration is critical to explore DCs as cellular vaccines for immunotherapeutic approaches. Currently, research is hampered by the lack of appropriate human cellular model systems to effectively study spatio-temporal signaling and CCR7-driven migration of human DCs. Here, we report that the previously established human neoplastic cell line CAL-1 expresses the human DC surface antigens CD11c and HLA-DR together with co-stimulatory molecules. Importantly, if exposed for three days to GM-CSF, CAL-1 cells induce the endogenous expression of the chemokine receptor CCR7 upon encountering the clinically approved TLR7/8 agonist Resiquimod R848 and readily migrate along chemokine gradients. Further, we demonstrate that CAL-1 cells can be genetically modified to express fluorescent (GFP)-tagged reporter proteins to study and visualize signaling or can be gene-edited using CRISPR/Cas9. Hence, we herein present the human CAL-1 cell line as versatile and valuable cellular model system to effectively study human DC migration and signaling.

Resistance to the Novel Translation Inhibitor Silvestrol Is Mediated by Elevated Mcl-1 Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1737-1737
Author(s):  
David M. Lucas ◽  
Ellen J. Sass ◽  
Ryan B. Edwards ◽  
Li Pan ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Multi Drug Resistance ◽  
Parental Line

Abstract Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 < 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

CMV Specific T Cells Prevent Progression From Low to High Level Viremia In D+R+ but Not D-R+ Patients.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4532-4532
Author(s):  
Mette Hoegh-Petersen ◽  
Lina Roa ◽  
Yiping Liu ◽  
Feng Zhou ◽  
Alejandra Ugarte-Torres ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Spontaneous Resolution ◽  
Cell Transplant ◽  
Low Level ◽  
Peak Viremia ◽  
Cell Counts ◽  
High Level ◽  
Ifnγ And Tnfα

Abstract Abstract 4532 Background. Cytomegalovirus (CMV) reactivates (becomes detectable in blood) in most seropositive hematopoietic cell transplant (HCT) recipients. In some reactivating patients, low level viremia (<50,000 DNA copies/ml plasma, ie, less than our institutional threshold for preemtive therapy) progresses to high level viremia or CMV disease, which is potentially fatal. We hypothesized that low level viremia progresses in patients with low specific T cell counts and spontaneously resolves in patients with high specific T cell counts. Methods. In 30 CMV seropositive HCT recipients monitored weekly for reactivation by real-time PCR, blood was drawn for specific T cell counts within 4 days from the first episode of low level viremia. Fourteen patients received grafts from seropositive donors (D+R+), and 16 patients from seronegative donors (D-R+). Mononuclear cells were stimulated overnight with CMV lysate, pp65 overlapping peptides, no stimulus (negative control) or staphylococcal enterotoxin B (positive control). T cells producing IFNγ, TNFα and/or IL2 were enumerated by flow cytometry. Results. Among D+R+ patients, counts of CMV lysate and pp65 specific CD4 T cells producing IFNγ and TNFα (and not IL2) were higher in patients with spontaneous resolution than patients with progression (p=0.02 for CMV lysate, p=0.004 for pp65). Also, there was an inverse correlation between pp65 specific CD8 T cells producing IFNγ and TNFα and peak viremia (r=-0.94, p=0.005) in D+R+ patients who progressed to high level viremia/disease. In contrast, among D-R+ patients, CMV lysate and pp65 specific T cell counts were similar in patients with spontaneous resolution and patients with progression, and there was no correlation between specific T cell counts and peak viremia. Conclusion. CMV specific T cells play a role in preventing progression from low to high level CMV reactivation/disease in D+R+ patients. Other immune mechanisms (eg, NK cells?) play the role in D-R+ patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Model System for Activation-Induced Alternative Splicing of CD45 Pre-mRNA in T Cells Implicates Protein Kinase C and Ras

2000 ◽  
Vol 20 (1) ◽  
pp. 70-80 ◽  
Author(s):  
Kristen W. Lynch ◽  
Arthur Weiss
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
Alternative Splicing ◽  
T Cell ◽  
Protein Kinase ◽  
Cell Line ◽  
T Cell Activation ◽  
Cell Activation ◽  
Model System ◽  
Kinase C

ABSTRACT Multiple isoforms of the protein tyrosine phosphatase CD45 are expressed on the surface of human T cells. Interestingly, the expression of these isoforms has been shown to vary significantly upon T-cell activation. In this report, we describe a novel cell line-based model system in which we can mimic the activation-induced alternative splicing of CD45 observed in primary T cells. Of the many proximal signaling events induced by T-cell stimulation, we show that activation of protein kinase C and activation of Ras are important for the switch toward the exclusion of CD45 variable exons, whereas events related to Ca2+ flux are not. In addition, the ability of cycloheximide to block the activation-induced alternative splicing of CD45 suggests a requirement for de novo protein synthesis. We further demonstrate that sequences which have previously been implicated in the tissue-specific regulation of CD45 variable exons are likewise necessary and sufficient for activation-induced splicing. These results provide an initial understanding of the requirements for CD45 alternative splicing upon T-cell activation, and they confirm the importance of this novel cell line in facilitating a more detailed analysis of the activation-induced regulation of CD45 than has been previously possible.

Primary T Cell Immunodeficiencies and Autoimmunity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-21-SCI-21
Author(s):  
Alain Fischer
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Autosomal Recessive Inheritance ◽  
Cell Receptor ◽  
Autoantibody Production ◽  
Cell Counts

Abstract Abstract SCI-21 There are a variety of primary T cell immunodeficiencies that can impair T cell differentiation or T cell activation. The latter include CD38δ deficiency, ZAP70 deficiency, Ca++ influx deficiency (ORAI1 and Stim1 deficiencies), and ITK deficiency as well. A new T cell activation deficiency as observed in a patient with defective T cell receptor triggered T cell activation and low CD4 T cell counts will be reported. A remarkable and quasi constant feature shared by all those T cell activation defects is the occurrence of autoimmune diseases, mostly related to autoantibodies, and inflammation such as colitis or panniculitis. Several mechanisms can account for these findings that include defective regulatory T cell development or function, defective negative selection impaired intrinsic feedback mechanism as well as non TCR-mediated T cell activation ultimately leading to proinflammatory cytokines release and autoantibody production by B cells. Another new form of primary T cell immunodeficiency with autosomal recessive inheritance observed in four patients from two families will be described. It is characterized by defective survival of naïve T cells. There again, autoimmunity appeared to be a significant component of the phenotype. Collectively, these results indicate that further insight into the role of key molecules in T cell activation/survival is provided by the analysis of new primary immunodeficiency phenotypes. In addition, the occurrence of autoimmunity in these settings stresses on one hand the role of T cells in the control of reactivity to self and, on the other hand, should be considered in the therapeutic strategy of these conditions. Disclosures: No relevant conflicts of interest to declare.

Imatinib Mesylate Impairs the Function of Human Regulatory T Cells (Tregs) and Reverses the Inhibition of Immune Responses to Imatinib-Resistant CML Cells by Tregs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4728-4728
Author(s):  
Masahiro Ogasawara ◽  
Toshihiro Matsukawa ◽  
Yuki Katsura ◽  
Kanako Shima ◽  
Minoru Kanaya ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Cell Line ◽  
Imatinib Mesylate ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Rapid Development ◽  
Inhibitory Effect ◽  
Peripheral Tolerance ◽  
Imatinib Resistant

Abstract Abstract 4728 Imatinib mesylate (imatinib) has been widely used clinically for the treatment of CML and Ph-positive ALL patients with tremendous success. However, the effect is limited in advanced stage by rapid development of resistance to imatinib. Tregs play a pivotal role in peripheral tolerance and their impairment results in autoimmune disease and GVHD. Previous studies have revealed that imatinib inhibits the proliferation of several types of immune cells. However, immunomodulatory function of imatinib in CML remains to be elucidated. In the present study, we investigated whether imatinib exerts an immunosuppressive effect on naturally occurring Tregs in T cell responses to CML, especially imatinib-resistant CML. Peripheral blood mononuclear cells were obtained from 5 healthy volunteers with informed consent. CD4+CD25+ and CD4+CD25- T cells were isolated by magnetic cell sorting using human CD4+CD25+ T regulatory cell isolation kit (Miltenyi Biotec). Both CD4+CD25+ and CD4+CD25- T cells, pre-stimulated with anti-CD3 and anti-CD28 monoclonal antibody-coated beads for 2 days, were cultured with increasing doses of imatinib for 3 days and survival of the cells was evaluated by a WST1 tetrazolium assay. Viability decreased at higher concentrations (>10μM) of imatinib. There was no difference in the sensitivity to imatinib between CD4+CD25+ and CD4+CD25- T cells. To examine whether imatinib exerts an inhibitory effect on the proliferation of Tregs, freshly isolated CD4+CD25+ and CD4+CD25- T cells were cultured with varying concentrations of imatinib together with CD3 and CD28 stimulation for 3 days. The proliferation of these cells was inhibited by higher concentrations of imatinib in a similar fashion. Expression of FoxP3 in CD4+CD25+ Tregs was inhibited with 10μM imatinib by apporoximately 70% as shown in Figure 1. Because Treg has been shown to suppress immunity against cancer, a similar inhibitory effect is expected to occur in CML. This prompts us to examine immune responses to CML. For this purpose, we utilized a cell line (TM) established by us from a patient with CML in blastic crisis and another imatinib-resistant cell line (TM-G) generated by culturing TM with increasing concentrations of imatinib. CFSE-labeled CD4+CD25- and CD8+ responder cells were cultured for 5 days with irradiated autologous CD3- antigen presenting cells which were loaded with cell-lysates from either TM or TM-G in the presence or absence of Tregs which were pre-incubated with increasing concentrations of imatinib for 2 days. Proliferation of both CD4+CD25- and CD8+ responder cells were inhibited by Tregs which were not exposed to imatinib. On the other hand, Tregs which were exposed to imatinib had reduced inhibitory effects on the proliferation of responder cells depending on the concentration of imatinib as shown in Figure 2. These results demonstrate the immunomodulatory functions of imtinib in CML and imply that the use of this drug is a potent inhibitor of Tregs in cancer immunotherapy. Disclosures: No relevant conflicts of interest to declare.

CRLF2 and JAK2 Mutations: Occurrence and Function In Pre-B ALL Cell Lines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5109-5109 ◽  
Author(s):  
Hilmar Quentmeier ◽  
Sonja Eberth ◽  
Roderick AF MacLeod ◽  
Stefan Nagel ◽  
Julia Romani ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Lymphoblastic Leukemia ◽  
Janus Kinase ◽  
Model Systems ◽  
Thymidine Uptake ◽  
Downstream Target

Abstract Abstract 5109 Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells promotes early B-cell development and activates dendritic cells. It has recently been reported that a subset of B-cell precursor acute lymphoblastic leukemia (pre-B ALL) overexpresses the TSLP receptor CRLF2. CRLF2 overexpression is linked to translocations between sex chromosomes – localizing CRLF2 – and the immunoglobulin heavy chain locus on chromosome 14, or to an interstitial deletion on the gonosomes. Both events, translocation and deletion juxtapose CRLF2 to a different promoter (IgH or P2RY8). Performing quantitative real-time PCR we tested pre-B ALL and acute myeloid leukemia (AML) cell lines for overexpression of CRLF2. AML cell lines were included in the screening because we knew from an earlier TSLP project that the AML cell line MUTZ-3 is TSLP-responsive, and thus positive for the cytokine receptor. Three of 63 (5%) pre-B ALL cell lines tested (INC, MHH-CALL4, MUTZ-5) overexpressed CRLF2 mRNA. CRLF2-high cell lines carry a t(14;Y). With respect to the 58 AML cell lines tested: some expressed CRLF2 mRNA, but none of them rivalled the aforementioned pre-B cell lines. Pre-B ALL cell lines show the association between chromosomal CRLF2 aberrations and JAK2 pseudokinase domain mutations that has been described for primary pre-B ALL cells: cell lines MHH-CALL4 (JAK2I682F) and MUTZ-5 (JAK2R683G) and – newly described - also the CRLF2-high pre-B ALL cell line INC express a mutated version of Janus kinase 2 (JAK2R683G). We established a PCR based assay system that allowed for the rapid detection of the JAK2R683G mutation: none of the CRLF2-low or –negative pre-B ALL cell lines exhibited this mutation. All three CRLF2-high/JAK2mu cell lines showed high phosphorylation levels of the JAK2 downstream target STAT5. Inhibition of the JAK kinase led to dephosphorylation of STAT5. However, repression of 3H-thymidine uptake and induction of apoptosis by inhibition of the JAK2/STAT5 pathway was weaker in the JAK2mu pre-B ALL cell lines than in the JAK2V617F positive essential thrombocythemia-derived cell line SET-2. Provided that these results reflect the situation in primary cells, mutated JAK2 seems to be of lesser importance for growth and survival of pre-B ALL cells than for cells from myeloproliferative neoplasms. The CRLF2-high/JAK2mu cell lines INC, MHH-CALL4 and MUTZ-5 are promising model systems for the study of the roles of high-level CRLF2 expression and of JAK2 mutations in pre-B ALL. Disclosures: No relevant conflicts of interest to declare.

Haploidentical Stem Cell Transplantation After Negative Depletion Of T Cells Expressing The αβ Chain Of The T-Cell Receptor (TCR) For Adults With Hematological Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4609-4609 ◽  
Author(s):  
Lucia Prezioso ◽  
Sabrina Bonomini ◽  
Chiara Lambertini ◽  
Chiara Schifano ◽  
Elena Rossetti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Conflicts Of Interest ◽  
Chronic Gvhd ◽  
Cell Depletion ◽  
T Cell Depletion ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Αβ T Cells

Introduction For many years, T cell depletion (TCD) of hematopoietic stem cells (HSCs) has been based on either positive or negative selection of mobilised peripheral blood cells (PBPCs). After CD34+ cell selection, the T cell repertoire is very narrow since the number of T lymphocytes in the graft has to be particularly low to prevent GvHD and ATG in the conditioning exerts an additional in vivo T cell depletion. Thus the immune recovery is slow and patients tend to remain susceptible to opportunistic infections for several months after HSCT. To hasten and improve post-transplant immune reconstitution broad repertoire various strategies of adoptive donor T cell immunotherapy (e.g. engineering with a suicide gene; depleting alloreactivity by means of photodynamic purging or through the use of freshly purified regulatory T cells) have been investigated over the past years. More recently, selective elimination of αβ+ T cells has been performed to achieve a 4,5–5 log TCD and to retain in the graft NK, dendritic cells, monocytes and γδT lymphocytes. Under this approach, a rapid immunological reconstitution and very promising outcome have been reported in pediatric patients. With the aims of confirming these results even in adults, we have recently launched this programme and here we report our preliminar clinical data. Methods Thirteen patients, median age 40 years (range 19-65), with AML (n=9), ALL (n=2), HL (n=1) or Rhabdomyosarcoma (n=1) entered the study. All but two patients, who were in first remission, were in advanced-stage disease at transplant with five patients in chemoresistant relapse. Conditioning consisted of ATG 1,5 mg/kg from day -13 to day -10, Treosulfan 12gr/sqm from -9 to –7, Fludarabine 30mg/sqm from -6 to -2 and Thiotepa 5mg/Kg on days -5 and -4. Ten μg/kg G-CSF was used to mobilize PBPCs from one-haplotype mismatched donors (4 mothers, 4 brothers, 2 sisters, 1 son, 1 daughter and 1 cousin). Mobilized mononuclear cells were incubated with a biotinylated anti-TcRαβ antibody and subsequently with an antibiotin antibody conjugated to magnetic microbeads (Miltenyi Biotec, Germany). Under a strong magnetic field, TcRαβ T lymphocytes were retained, whereas all nonmagnetized cells were recovered. Short sirolimus (1mg/day x3 weeks) was used as additional GVHD prophylaxis in 3 cases whose grafts contained more than 2x105/kg αβ+Tcells. Results Grafts contained a median of 12,3x106/kg CD34+ cells(range7-19), 6 x106 CD3+Tcells/kg (range 2,3-13)with 10,4x104/kg αβ+T cells (range 1,38-62) and 5,8x106 γδ+Tcells/kg (range2,1-12,6), 6x104B cells/kg (range 0,2–32) and 34x108 CD56+NKcells/kg (range10-91). All but one patient, who required a second graft from the same donor to boost hematopoietic reconstitution, achieved a full donor sustained engraftment. Median time to reach 500 neutrophils and 50,000 platelets was 13 (range 9-18) and 11 days (range 9-13), respectively. Four patients had skin grade I/II aGVHD. No patients has so far developed chronic GvHD. Median CD4+ cell counts at 30, 60, 90 and 120 days since the transplant were 33, 122, 190 and 251 n/mL, respectively. CMV reactivation occurred in only 2 cases (in one, CMV serology was unfavourable: CMV-negative donor/CMV-positive recipient). Overall, 3 patients have so far died (2 non-hematologic causes and 1 early relapse). Ten survive disease-free at a median follow-up of 104 days (range 30-178). Conclusions The infusion of αβ/CD19-depleted grafts was safe and effective also in adult setting, resulting into rapid donor hematopoietic engraftment and early expansion of donor-derived γδT lymphocytes, without life-threatening infectious complications. Disclosures: No relevant conflicts of interest to declare.

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