Implication of STAT-5 Recruitment in the Expression of IRF-8 and in the Impairment of Human Plasmacytoid Dendritic Cells Differentiation Induced by BCR-ABL and GM-CSF

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3196-3196
Author(s):  
Anne Barra ◽  
Lucie Brault ◽  
Ali G Turhan ◽  
Lydia Roy ◽  
François Guilhot ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Critical Role ◽  
Chronic Phase ◽  
Plasmacytoid Dendritic Cells ◽  
Myelogenous Leukemia ◽  
Molecular Response ◽  
Gm Csf ◽  
Cd34 Cells

Abstract It is currently established that the immune system plays a critical role in the control of chronic myelogenous leukemia (CML). Several cell populations including T cells, dendritic cells (Dc) and NK cells have been shown to exhibit potential anti-leukemic activities. We have previously shown that plasmacytoid dendritic cells (pDc) differentiation and IFN-a production was impaired along the chronic phase (CP) of CML and that the Tyrosine- Kinase (TK) activity of BCR-ABL was responsible for this impairment. Moreover, patients in complete cytogenetic remission (CCyR) after imatinib mesylate (IM) or IFN-a therapy present a partial restoration of the pDc compartment and IFN-a production. Some recent results (Esashi et al., Immunity, 2008) indicate that, in mice, STAT-5 but not STAT-3, block the expression of IRF-8, a transcription factor required for the differentiation of pDc and that the GM-CSF-induced STAT5 activation interferes with pDc differentiation. As STAT3 and STAT5 proteins are important targets of BCR-ABL TK activity, we postulate that their recruitment could be responsible for the CML pDc impairment and could be involved in the persistent pDc dysfunction in CR patients. We decided to investigate the effect of BCR-ABL or GM-CSF on STAT-3, STAT-5 and IRF8 status ex vivo, on CMN of patients who have achieved sustained CCyR and major molecular response (MMR) after IFN-a therapy and in whom treatment has been discontinued (n=8) compared to CMN of healthy subjects (HS) (n=5). The same investigations were also performed in vitro with the model of pDc differentiation of CD34+ haematopoietic progenitors from CP patients and HSs, using the combination of Stem Cell Factor, Flt3-Ligand and Thrombopoietin,. Flow cytometry analysis showed that the homeostasis of the IFN-a production was altered in CCyR patients : pDc (determined by the coexpression of CD303, CD123 and CD4), represent 34.6 ± 14.9 % of the IFN-a producing cells after stimulation by Influenza virus and intracellular detection of IFN-a, whereas in healthy donors (HD) IFN-a producing cells were principally pDc (72.1 ± 20.5 %of IFN-a+ cells, p<0.05 in comparison with the CCyR patients group). The IRF-8 expression analysed by flow cytometry was also deregulated as low levels of IRF8 were detected in these cells when compared with pDc of HD (based on mean fluorescence analysis of the IRF-8 staining). Moreover, higher levels of P-STAT5 were observed in CMN and pDc from CCyR patients (65.1 ± 18 % of pDc was P-STAT5 +) than from HD (0.75 ± 0.66 % of pDc are P-STAT5 +, p<0.01 in comparison with the CR patients group). The involvement of Human STAT5 in the repression of pDc differentiation was confirmed in our in vitro model. We showed that GM-CSF blocked the pDc differentiation of normal CD34+ cells and enhanced the P-STAT5 levels. We showed higher levels of P-STAT5 along differentiation of CD34+ cells of CML patients in CP, in correlation with the lack of differentiation and maturation of pDc. Altogether, our results suggest that in Human STAT5 is a target for BCR-ABL and GM-CSF and is responsible for the defect of pDc differentiation and IFN-a production. After CCyR, the pDc defect is only partially restored, principally because a STAT5 recruitment persists, possibly induced by soluble factors produced by the residual disease.

The Induction of Dendritic Cells with IFN-alpha and GM-CSF from Bone Marrow Mononuclear Cells from Patients with Chronic Myeloid Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4873-4873
Author(s):  
Shuier Zhen ◽  
Jie Jin ◽  
Xiangmin Tong
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Bone Marrow Mononuclear Cells ◽  
Gm Csf

Abstract Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease arising from the clonal expansion of a stem cell with the typical Philadelphia (Ph) chromosome cytogenetic abnormality. IFN-a has been proven to be effective for patients in the chronic phase of myelogenous leukemia (CML), yet the mechanisms of the antitumor action of these cytokines are still a matter of debate. Dendritc cells (DCs) are potent antigen-presenting cells that prime effective T-cell response aginst tumour antigens. Recent studies have shown that IFN-a can exert a variety of effects on dendritic cells (DCs), which may play an important role in the induction of an antitumor immunity. Human DCs can be generated in vitro from peripheral blood(PB) monocytes or from CD34+ haematopoietic precursor cells in culture medium containing human granulocyte macrophage-colony stimulating factor (GM-CSF), IL-4 and some other cytokines. Previous studies have shown a new effective protocol for the generation of human DCs from unseparated BM aspirate cells with excellent functional capacity of antigen uptake and of stimulating naive and memory T cell responses superior to that of DCs from peripheral blood(PB) monocytes. We, therefore, explored whether treatment with IFN-a may influence the CML bone marrow mononuclear cells(BMMNCs) derived DCs in vitro. Treatment BMMNCs of 12 patients with CML in chronic phase with IFN-a+rhGM-CSF(IFN-a-DC) generated DCs with more mature phenotype properties expressing higher of CD80,CD86,HLA-DR,CD83 compared to the CML- BMMNCs treated with rhGM-CSF+IL-4(IL-4-DC). And in parallel with phenotypes, IFN-a-DC also showed more effective than IL-4-DC in eliciting an allogeneic mixed lymphocyte reaction by MTT assay. FISH confirmed the DCs of both groups were leukemic origin. These findings demonstrate that IFN-a promotes the differentiation/maturation of DCs derived from BMMNCs of patients with CML in vitro, these studies also broaden the clinical scope of IFN-a as a promising agent in the immunotherapy of CML.

Fas-Mediated Modulation of Bcr/Abl in Chronic Myelogenous Leukemia Results in Differential Effects on Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 981-989 ◽  
Author(s):  
Carmine Selleri ◽  
Jaroslaw P. Maciejewski ◽  
Fabrizio Pane ◽  
Luigia Luciano ◽  
Anna Maria Raiola ◽  
...  
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Chronic Phase ◽  
Poor Response ◽  
Inhibitory Response ◽  
Myelogenous Leukemia ◽  
Poor Responders ◽  
Cd34 Cells ◽  
In Cells

Abstract Fas-R is expressed constitutively in CD34+ cells of patients with chronic myelogenous leukemia (CML); Fas-R triggering results in decreased proliferation rate due to apoptosis of clonogenic cells. We have already shown that α-interferon (IFN-α) enhances Fas-R expression on CML progenitor cells, thus increasing their sensitivity to Fas-R agonists. Although it appears that IFN-α can prime CML cells for the effects of Fas, the response to IFN-α in vivo is not a constant feature in CML patients. We studied the mechanisms of Fas-mediated apoptosis in 11 patients suffering from CML in chronic phase and tried to see whether there was a correlation between in vitro inducibility of apoptosis in CD34+ CML cells after Fas-R triggering and the clinical response to IFN-α. After priming with IFN-α, Fas triggering resulted in in vitro suppression of hematopoietic cell growth in seven of eight patients who had optimal hematologic response to IFN-α; in the same conditions, no inhibitory response to Fas-R agonist was observed in cells from three of three patients who proved to be poor responders to IFN-α. In responders to IFN-α, Fas-R agonist induced dose-dependent apoptosis of CD34+ cells; this effect was associated with a decrease in the bcr/abl protein level. In cells derived from patients with a poor response to IFN-α, the rate of apoptosis in culture remained unchanged in the presence of Fas-R agonist and nobcr/abl downmodulation was observed. Finally, we measuredbcr/abl mRNA by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and found that decreased bcr/ablprotein after Fas triggering was not associated with decreased amounts of specific mRNA, a finding which is consistent with a posttranscriptional regulation of the bcr/abl protein expression. It appears that Fas-mediated downmodulation of p210bcr/abl restores susceptibility to apoptosis of CML cells; in addition, in vitro studies on CML cells may predict response to IFN-α treatment. © 1998 by The American Society of Hematology.

Inhibition of mTOR suppresses IFNα production and the STING pathway in monocytes from systemic lupus erythematosus patients

Rheumatology ◽  
2020 ◽  
Vol 59 (10) ◽  
pp. 2992-3002 ◽  
Author(s):  
Goh Murayama ◽  
Asako Chiba ◽  
Taiga Kuga ◽  
Ayako Makiyama ◽  
Ken Yamaji ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Lupus Erythematosus ◽  
Mononuclear Cells ◽  
Intracellular Cytokine Staining ◽  
Plasmacytoid Dendritic Cells ◽  
Mtor Pathway ◽  
Pathway Activation ◽  
Sting Pathway

Abstract Objective Increased IFNα is important in the pathogenesis of SLE. Plasmacytoid dendritic cells are considered the main producer of IFNα upon Toll-like receptor pathway activation. However, which cells produce IFNα following stimulation with cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) in SLE remains unknown. We investigated the IFNα producing capacity of myeloid cells under cGAS-STING pathway stimulation. Methods IFNα levels in peripheral blood mononuclear cells from SLE patients and healthy controls stimulated with 2′3′c-GAMP, a stimulator of cGAS-STING, were measured by intracellular cytokine staining and flow cytometry. STING expression and its co-localization with TBK1 were examined by flow cytometry or confocal microscopy. The effects of in vitro exposure to IFNα on IFNα production and STING expression, and in vitro rapamycin treatment on IFNα production and STING, pTBK1 and IRF3 expression were examined. Results IFNα was produced by monocytes, conventional dendritic cells and plasmacytoid dendritic cells upon cGAS-STING pathway activation. The frequency of IFNα-producing monocytes positively correlated with SLE disease activity. STING expression and its co-localization with TBK1 were increased in lupus monocytes. Prior exposure to IFNα enhanced the IFNα-producing capacity of monocytes. Inhibition of the mechanistic target of the rapamycin (mTOR) pathway suppressed IFNα production from monocytes and downregulated enhanced STING expression and its downstream molecules. Conclusion Enhanced IFNα from lupus monocytes induced by augmented STING pathway activation is associated with SLE pathogenesis. Suppression of the mTOR pathway downregulated the enhanced STING expression and the subsequent IFNα production by monocytes.

Defective Production of Myeloid Dendritic Cells Results from Skewing of Chronic Myelogenous Leukemia Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4700-4700
Author(s):  
Martin Guimond ◽  
Radia Sidi Boumedine ◽  
Claude Perreault ◽  
Denis-Claude Roy
Keyword(s):  
Dendritic Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Myelogenous Leukemia ◽  
Immune Recognition ◽  
Erythroid Progenitors ◽  
Myeloid Dendritic Cells ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Clonogenic Cells

Abstract The robustness of the immunostimulatory response generated by dendritic cells (DCs) has positioned these cells as central and determining cellular mediators of T cell activation. Their production in sufficient number from hematopoietic precursors is crucial in order to drive immune responses. To determine the capacity of CML clonogenic cells to generate dendritic cells expressing endogenous CML-specific epitopes, we have evaluated the generation of DCs from CD34+ progenitor cells and CD14+ monocytes isolated from patients with CML (n=28). We found that the kinetics of production of DCs, in GM-CSF 800U/ml, TNF-a 50U/ml and IL-4 10U/ml, from CD34+ progenitors of CML patients were significantly delayed in comparison to healthy controls (p<0.05). Even after 20 day-cultures, DC numbers remained decreased (P<0.01). A proliferative defect of CML blasts cannot explain this finding since CD34+ cells divided normally according to total cell culture count and CFSE staining. Flow cytometry analysis of the few DCs produced from CML CD34+ cells showed the same phenotypic characteristics of expression of MHC (class I and II), costimulatory (CD80, CD86) and adhesion molecules (CD11a, CD11b and CD58), as DCs generated from control CD34+ cells, except for late downregulation of CD11a. Normal maturation of DCs from CML CD14+ cells and a sustained proportion of the bcr-abl+ DCs before and after DC expansion excluded a 9:22 translocation-induced differentiation defect. However, retarded down-modulation of the CD34 antigen at the surface of CML blasts, independently of IL-4 receptor synthesis, rather suggested decreased susceptibility of CML progenitors to IL-4 and GM-CSF-mediated DC growth conditions. Indeed, CML progenitors, plated in semi-solid media, showed an important skewing toward more mature progenitors (BFU-E and late CFU-GM) with a significant decrease in immature CFU-GM (> 500 cells) (CML = 29 vs normal = 95 colonies, P<0.001) and CFU-Mix (CML = 0.6 vs normal = 5.6 colonies, P=0.0006). Moreover, the number of DCs obtained after 10 and 15 day-cultures showed a strong correlation with the number of immature CFU-GM and CFU-Mix colonies (R=0.95). Thus, our study identifies a selective impairment in DC production from CD34+ progenitors of CML patients corresponding to a skewing of the progenitor cell compartment toward mature myeloid and/or erythroid progenitors. The lack of DCs derived from leukemia progenitors may explain the immunologic escape of CML clonogenic cells and provides clues to enhance their immune recognition.

Fas-Mediated Modulation of Bcr/Abl in Chronic Myelogenous Leukemia Results in Differential Effects on Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 981-989 ◽  
Author(s):  
Carmine Selleri ◽  
Jaroslaw P. Maciejewski ◽  
Fabrizio Pane ◽  
Luigia Luciano ◽  
Anna Maria Raiola ◽  
...  
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Chronic Phase ◽  
Poor Response ◽  
Inhibitory Response ◽  
Myelogenous Leukemia ◽  
Poor Responders ◽  
Cd34 Cells ◽  
In Cells

Fas-R is expressed constitutively in CD34+ cells of patients with chronic myelogenous leukemia (CML); Fas-R triggering results in decreased proliferation rate due to apoptosis of clonogenic cells. We have already shown that α-interferon (IFN-α) enhances Fas-R expression on CML progenitor cells, thus increasing their sensitivity to Fas-R agonists. Although it appears that IFN-α can prime CML cells for the effects of Fas, the response to IFN-α in vivo is not a constant feature in CML patients. We studied the mechanisms of Fas-mediated apoptosis in 11 patients suffering from CML in chronic phase and tried to see whether there was a correlation between in vitro inducibility of apoptosis in CD34+ CML cells after Fas-R triggering and the clinical response to IFN-α. After priming with IFN-α, Fas triggering resulted in in vitro suppression of hematopoietic cell growth in seven of eight patients who had optimal hematologic response to IFN-α; in the same conditions, no inhibitory response to Fas-R agonist was observed in cells from three of three patients who proved to be poor responders to IFN-α. In responders to IFN-α, Fas-R agonist induced dose-dependent apoptosis of CD34+ cells; this effect was associated with a decrease in the bcr/abl protein level. In cells derived from patients with a poor response to IFN-α, the rate of apoptosis in culture remained unchanged in the presence of Fas-R agonist and nobcr/abl downmodulation was observed. Finally, we measuredbcr/abl mRNA by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and found that decreased bcr/ablprotein after Fas triggering was not associated with decreased amounts of specific mRNA, a finding which is consistent with a posttranscriptional regulation of the bcr/abl protein expression. It appears that Fas-mediated downmodulation of p210bcr/abl restores susceptibility to apoptosis of CML cells; in addition, in vitro studies on CML cells may predict response to IFN-α treatment. © 1998 by The American Society of Hematology.

Production of GM-CSF by CML Cells Can Modulate the Anti-Proliferative and Pro-Apoptotic Effects of Imatinib on CML CD34+ Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2865-2865 ◽  
Author(s):  
Alan B. Lyons ◽  
Timothy P. Hughes ◽  
Pongtep Viboonjuntra
Keyword(s):  
Single Agent ◽  
Chronic Phase ◽  
Cell Number ◽  
Culture Conditions ◽  
Proliferation Index ◽  
Cytokine Signaling ◽  
Gm Csf ◽  
Proliferative Effect ◽  
Cd34 Cells

Abstract CML cells gain a survival advantage over normal cells due to dysregulated tyrosine kinase activity of BCR/ABL protein, which phosphorylates a number of proteins, potentially activating multiple signal transduction pathways. Imatinib specifically targets BCR/ABL protein and results in anti-proliferation and apoptosis. Even in the absence of mutation, the leukaemic clone may not be eradicated by imatinib, suggesting that leukaemic stem cells may not be absolutely reliant on BCR/ABL activity for survival. Reports on GM-CSF production by CML cells suggest a possible autocrine role, therefore we examined if GM-CSF could protect CML CD34+ cells from imatinib. CD34+ cells from peripheral blood of patients with CML in chronic phase (n=9) and from normal bone marrow donors (n=5), were labeled with CFSE (Carboxy-Fluorescein diacetate Succinimidyl Ester) to enable tracking of cell division. Normal and CML samples were cultured with and without GM-CSF (300pg/mL) to assess response to this cytokine as a single agent. CML CD34+ cells were cultured for 3 days in serum deprived medium with imatinib only, GM-CSF (300 pg/mL) + imatinib, GM-CSF (300pg/mL) + E21R (a GM-CSF analogue able to block cytokine binding) (10mg/mL), and GM-CSF (300pg/mL) + E21R (10mg/mL) + imatinib. In each condition, imatinib was titrated over the range of 0 to 10 microM. Cultures were analysed by flow cytometry to evaluate the proliferation index (PI), a ratio of final cultured cell to precursor cell number, where a PI of 1 represents no proliferation, and a PI of 2 corresponds to approximately three division cycles on average. Data are summarised in Table 1. Effect of GM-CSF and imatinib on proliferation Culture Proliferation Index Statistics using Student t test, symbols denote comparisons between culture conditions Normal Control 1.07+/−0.06 * NS Normal+GM-CSF 1.11+/−0.03 * # NS CML Control 1.43+/−0.18 ** p>0.01 #, & NS CML+GM-CSF 2.30+/−0.40 ** CML+10μM imatinib 1.18+/−0.09 *** NS CML+10μM imatinib+GM-CSF 1.54+/−0.19 @, & CML+10μM imatinib+GM-CSF+E21R 1.17+/−0.09 *** @ p>0.01 GM-CSF induced strong proliferation in all CML, but not normal samples. Imatinib reduced proliferation of CML CD34+ cells at 1 microM and above, and the addition of GM-CSF reduced this proliferative effect at concentrations of imatinib up to 10 microM. The protective effect of GM-CSF was clearly blocked using E21R. When the proliferation of CML CD34+ cells cultured within the total mononuclear fraction was compared to purified CD34+ cells, it was found that there was an approximate 40% enhancement of PI at each concentration of imatinib. This enhanced proliferation was inhibited by the addition of E21R, indicating GM-CSF may be produced by non-CD 34+ cells. ELISpot assay confirmed the production of GM-CSF by non-CD34+ CML cells (34±23%), but not CD34+ cells (0.06±0.02%). Fluorescent inhibitor of apoptosis (FLICA) assay showed that GM-CSF could reduce the proportion of cells with activated caspases induced by imatinib by over 40% during a 3 day in vitro culture, promoting increased cell survival. As the GM-CSF receptor and BCR/ABL may share common signaling pathways, during blockade of BCR/ABL activity by imatinib, GM-CSF can compensate to maintain cell viability and proliferation. These findings have implications for optimizing imatinib therapy by manipulating cytokine signaling.

Analysis of Molecular Data and the Emergence of Mutations for Chronic-Phase Chronic Myelogenous Leukemia (CML-CP) Patients Treated with Dasatinib After Imatinib Failure.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3282-3282
Author(s):  
Susan Branford ◽  
Andreas Hochhaus ◽  
Martin Mueller ◽  
Erkut Bahceci ◽  
Lynn Ploughman ◽  
...  
Keyword(s):  
Research Funding ◽  
Kinase Inhibitor ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Molecular Response ◽  
Second Line ◽  
Imatinib Resistance ◽  
Mutation Status ◽  
New Mutations

Abstract Abstract 3282 Poster Board III-1 The kinase inhibitor dasatinib has demonstrated efficacy in patients with CML-CP who fail imatinib due to resistance or intolerance. Imatinib failure is often associated with the acquisition of resistant mutations within BCR-ABL. Failure of dasatinib is associated with a limited spectrum of mutations, which are T315I/A, V299L, and F317L/I/V, suggesting these mutations are dasatinib resistant (DR). Only a fraction of patients with T315I respond to dasatinib, most patients with F317L have hematologic responses but few have cytogenetic responses, and V299L is uncommon in imatinib-resistant patients but is mostly seen after dasatinib failure. In vitro data suggests Q252H and E255K/V also confer a degree of dasatinib resistance. However, their clinical association with dasatinib resistance or inferior response is unclear. This retrospective analysis seeks to determine if the mutation status at the start of dasatinib impacts the initial molecular response, and to examine the type of mutations lost and gained during therapy. To assess the significance of intermediate sensitivity (DI) mutations (Q252H and E255K/V), BCR-ABL levels were measured against the International Scale (IS), and mutation status was assayed at initiation of dasatinib (baseline), at progression, or study termination or completion (progression/completion). Data were taken from 479 patients with CML-CP treated with dasatinib 70 mg twice daily, after imatinib failure for the phase II START-C or -R studies. Patients were grouped according to their baseline mutation status: DR, DI, dasatinib sensitive (DS, all other mutations not DR or DI), and no mutations. Patients with more than one mutation at baseline that qualified for more than one grouping were rare, and classified according to the most resistant mutation. By 6 months of dasatinib therapy, patients with DR mutations at baseline had the highest levels of BCR-ABL transcripts (Table 1). By comparison, those with DI, DS, or no mutations had a reduced proportion with high transcript levels. In addition, no patients with DR at baseline achieved a reduction of BCR-ABL by 6 months to IS ratio < 1, whereas significant reductions were identified in patients with DI, DS, or no mutations. A subset of 267 patients had mutation analysis performed at progression/completion. Of these, 115 had mutations at baseline and 152 had no mutations. The patients with baseline imatinib resistance were more likely than those with imatinib intolerance to acquire new mutations. Of the resistant patients with no mutations at baseline, 7% (9/124) developed new mutations and the majority (7/9) was DR. Among resistant patients with baseline mutations, 23% (26/112) had new mutations and the majority was DR (20/26). Although 36 patients gained new mutations (including 16 T315I/A, 8 F317L, and 6 V299L), many baseline mutations were no longer detectable at progression/completion. DS mutations were lost in 68/102 patients. In total, 76 patients had detectable mutations at progression/completion, with 51% (39/76) having DR mutations but only 5% (4/76) with DI mutations. Patients with baseline DR mutations were more likely to retain their DR mutations (11/13). Of 8 patients with DI mutations at baseline, only 3 retained their DI mutations and 3 gained DR mutations. Of 94 patients with only DS mutations at baseline, 36 (38%) retained their DS mutations, and 17 (18%) developed DR mutations. Patients with high levels of BCR-ABL at 3 months had the highest incidence of new DR mutations at progression/completion (18/145 evaluable patients; 12%) compared to patients with lower levels (5/79; 6%). In conclusion, the development of new mutations during second-line dasatinib is rare (36/267). Patients who harbored DI mutations at baseline were also rare, and an expanded prospective analysis of dasatinib efficacy in this cohort may be necessary to clarify their significance. However, from the current analysis there is no compelling clinical evidence to suggest that these patients have an inferior molecular response. Relative to DR mutations, the DI mutations (Q252H and E255K/V) were rarely present at progression/completion. Patients with baseline mutations and 6-month BCR-ABL transcript level after second-line dasatinib IS ratio ≤ 1.0 1 < IS ratio ’ 10 IS ratio > 10 DR 0% (0/13) 23% (3/13) 77% (10/13) DI 56% (5/9) 0% (0/9) 44% (4/9) DS 36% (46/128) 20% (25/128) 45% (57/128) No mutations 42% (92/221) 18% (40/221) 40% (89/221) Disclosures: Branford: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Hochhaus:Bristol-Myers Squibb: Research Funding; Novartis: Research Funding. Bahceci:Bristol-Myers Squibb: Employment. Ploughman:Bristol-Myers Squibb: Employment. Mukhopadhyay:Bristol-Myers Squibb: Employment. Hughes:Bristol-Myers Squibb: Advisor, Honoraria, Research Funding; Novartis: Advisor, Honoraria, Research Funding.

scholarly journals Plasmacytoid dendritic cells cross-prime naive CD8 T cells by transferring antigen to conventional dendritic cells through exosomes

2020 ◽  
Vol 117 (38) ◽  
pp. 23730-23741 ◽  
Author(s):  
Chunmei Fu ◽  
Peng Peng ◽  
Jakob Loschko ◽  
Li Feng ◽  
Phuong Pham ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cd8 T Cells ◽  
Critical Role ◽  
Plasmacytoid Dendritic Cells ◽  
Viral Immunity ◽  
Cell Immunity ◽  
Antigen Transfer

Although plasmacytoid dendritic cells (pDCs) have been shown to play a critical role in generating viral immunity and promoting tolerance to suppress antitumor immunity, whether and how pDCs cross-prime CD8 T cells in vivo remain controversial. Using a pDC-targeted vaccine model to deliver antigens specifically to pDCs, we have demonstrated that pDC-targeted vaccination led to strong cross-priming and durable CD8 T cell immunity. Surprisingly, cross-presenting pDCs required conventional DCs (cDCs) to achieve cross-priming in vivo by transferring antigens to cDCs. Taking advantage of an in vitro system where only pDCs had access to antigens, we further demonstrated that cross-presenting pDCs were unable to efficiently prime CD8 T cells by themselves, but conferred antigen-naive cDCs the capability of cross-priming CD8 T cells by transferring antigens to cDCs. Although both cDC1s and cDC2s exhibited similar efficiency in acquiring antigens from pDCs, cDC1s but not cDC2s were required for cross-priming upon pDC-targeted vaccination, suggesting that cDC1s played a critical role in pDC-mediated cross-priming independent of their function in antigen presentation. Antigen transfer from pDCs to cDCs was mediated by previously unreported pDC-derived exosomes (pDCexos), that were also produced by pDCs under various conditions. Importantly, all these pDCexos primed naive antigen-specific CD8 T cells only in the presence of bystander cDCs, similarly to cross-presenting pDCs, thus identifying pDCexo-mediated antigen transfer to cDCs as a mechanism for pDCs to achieve cross-priming. In summary, our data suggest that pDCs employ a unique mechanism of pDCexo-mediated antigen transfer to cDCs for cross-priming.

Experimental attempt to produce mRNA transfected dendritic cells derived from enriched CD34+ blood progenitor cells

Open Medicine ◽  
2008 ◽  
Vol 3 (1) ◽  
pp. 21-28
Author(s):  
Paula Lazarova ◽  
Gunnar Kvalheim ◽  
Liana Gercheva ◽  
Krassimir Metodiev
Keyword(s):  
Prostate Cancer ◽  
Dendritic Cells ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
High Dose ◽  
Serum Free ◽  
Gm Csf ◽  
Cd34 Cells

AbstractIt Peripheral blood progenitor enriched CD34+ cells (PBPC) are rather often used as stem cell background in cancer patients following high dose therapy. Keeping in mind that precursor dendritic cells (DCs) originate from haematopoietic progenitor cells, purified CD34+ cells might also serve as starting cells for ex-vivo production of DC. The aim of the present study is to develop a clinical grade procedure for ex-vivo production of DC derived from enriched CD34+ cells. Various concentrations of CD34+ cells were grown in gas-permeable Teflon bags with different serum-free and serum-containing media supplemented with GM-CSF, IL-4, TNF-a, SCF, Flt-3L and INF-a. Serum-free CellGroSCGM medium for 7 days followed by CellGroDC medium in 7 days gave equal results as serum-containing medium. Following incubation, the cultured cells containing immature DCs were concentrated and transfected with tumour mRNA from human prostate cancer cell lines employing a highly efficient electroporation procedure. Thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by ELISPOT assay using mock-transfected DCs as control. The results of our study show that frozen enriched CD34+ cells can be an alternative and efficient source for production of DCs for therapeutic purpose.

Natural Interferon Producing Cells Develop from Murine CD31+(high)/Ly6C- Marrow Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4169-4169
Author(s):  
Friederike Kreisel ◽  
Amanda Blasius ◽  
Marco Colonna ◽  
Marina Cella
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Critical Role ◽  
Surface Expression ◽  
Specific Marker ◽  
Murine Bone Marrow ◽  
Gm Csf ◽  
Ifn Alpha ◽  
Total Bone Marrow

Abstract Dendritic cells (DC) play a critical role in the initiation and regulation of immune responses. Based on immunophenotypic and functional charcateristics, murine CD11c+ DC can be classified into CD11c+, CD11b+, and B220− “conventional” DC and CD11c+, CD11b−/dim, B220+, and Gr-1+ interferon-producing cells (IPC) or plasmacytoid dendritic cells (pDC). The developmental origin of IPC remains controversial. In this study, we investigated the capacity of different murine bone marrow subpopulations to acquire immunophenotypic, morphologic and functional characteristics of IPC after in vitro culture with Flt-3L. Total bone marrow (BM) from 129/SvJ mice was sorted into six distinct BM subsets based on surface expression of the myeloid markers CD31 and Ly6C and subsequently cultured with Flt-3L for 8 days. Purified CD31high/Ly6C− BM cells were the only subset that consistently developed characteristics closely resembling that of IPC. Specifically, these cells expressed high levels of CD11c, low levels of CD11b, the lymphoid marker B220, and the IPC-specific marker 440C. Furthermore, these cells displayed the typical plasmacytoid morphology with eccentrically located nucleus showing mature lymphoid chromatin and lack of nucleoli. Functionally, these cells showed a high proliferation rate as demonstrated by the [3H] thymidine release assay and secreted detectable levels of IFN-alpha when stimulated with CPG ODN 2216. In contrast, all other sorted subsets failed to proliferate in the presence of Flt-3L. Similar results were obtained using BM from B6-RAG−/− mice. These findings indicate that, although both the CD31high/Ly6C− and CD31int/Ly6C− BM subsets contain early progenitor cells corresponding to “cobblestone area forming cells”, only the CD31high/Ly6C− is able to give rise to IPC when cultured with Flt-3L. Interestingly, CD31high/Ly6C− BM cells have been previously shown to generate also conventional DC if cultured with GM-CSF. Thus, IPC and DC may derive from common or closely related progenitors. Moreover, our results demonstrate that IPC development, in contrast to that of lymphocytes, does not require RAG proteins.

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