OBJECTIVE: To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1. METHODS: The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry. RESULTS: The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×10 CONCLUSION Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.
Cell Line, Tumor ; Genetic Vectors ; Humans ; Interleukin-3 Receptor alpha Subunit ; K562 Cells ; Lentivirus/genetics* ; Leukemia, Lymphocytic, Chronic, B-Cell/genetics* ; Plasmids ; Transfection

OBJECTIVE: To investigate the expression of CD123 in patients with acute myeloid leukemia (AML) and its relationship between clinical features, concomitant fusion gene or gene mutation, efficacy and prognosis. METHODS: 365 patients with newly diagnosed AML (except M3) treated in the First Affiliated Hospital of Zhengzhou University were enrolled and retrospective analysis, and multi-parameter flow cytometry was performed to detect the expression of CD123 in myeloid leukemia cell population. CD123≥20% was defined as positive. Clinical features, concomitant fusion gene or gene mutation, efficacy and prognosis of CD123 RESULTS: The positive rate of CD123 in 365 newly diagnosed AML patients was 38.9%. Compared with the CD123 CONCLUSION CD123 positive indicates that AML patients have higher tumor burden and are more difficult to reach remission. It is an independent risk factor for OS and EFS in patients with normal karyotype and intermediate risk, which is important to evaluate the prognosis of patients with AML without specific prognostic marker.
Humans ; Interleukin-3 Receptor alpha Subunit ; Karyotype ; Leukemia, Myeloid, Acute/genetics* ; Mutation ; Patients ; Prognosis ; Retrospective Studies

Objective: To construct a new CD123- specific chimeric antigen receptor in order to provide a foundation for immunotherapy of CD123 positive leukemia. Methods: A hybridoma strain (6E11) capable of stably secreting CD123 antibody was obtained by a monoclonal screening technique, and the hybridoma cells were expanded and injected intraperitoneally to the pretreated Balb/c mice. Ascites was collected and purified to obtain the monoclonal antibody (mAb) . The affinity and specificity of 6E11 mAb were measured. The variable regions of the heavy and light chains of the 6E11 mAb were cloned by RT-PCR from the 6E11 mouse hybridoma. We generated a new CD123 specific chimeric antigen receptor with a scFv fragment derived from 6E11 antibody, designated as 6E11 CAR. T cells were transduced with lentiviral supernatant from 293T cells transfected with 6E11 CAR plasmid to generate 6E11 CAR-T cells. The specific cytotoxicity of 6E11 CAR-T against CD123(+) acute myeloid leukemia (AML) cell lines and primary AML cells in vitro were evaluated by co-culture experiments, degranulation experiments and cytokine releasing assay. Results: ① A hybridoma cell line 6E11 stably secreting anti-human CD123 antibody was developed and its variable region sequences were obtained. ② The 6E11 mAb has high affinity for CD123 protein (Kd value: 2.1 nmol/L) . The 6E11 mAb specifically recognizes CD123(+) cell line THP-1 cells and does not respond to CD123(-) cell line Jurkat cells. ③ 6E11 CAR-T cells were successfully generated with a CAR expression rate higher than 60%. ④ 6E11 CAR-T cells could specifically kill CD123(+) MV4-11 cell line but had no killing effect on the CD123(-) K562 cell line. Compared with vector-T cells, 6E11 CAR-T cells have higher killing rate to MV4-11 cells[ (98.60±1.20) %vs (20.28±6.74) %, P<0.001]. ⑤ MV4-11 cells activated 6E11 CAR-T cells significantly but not Vector-T cells[ (26.33±3.30) %vs (1.17±0.06) %, P<0.001]. ⑥ 6E11 CAR-T cells released more cytokines than vector-T cells when co-cultured with MV4-11[IL-2: (92.90±1.51) pg/ml vs (6.05±3.41) pg/ml, P<0.001; TNF-α: (1 407.20±91.95) pg/ml vs (7.86±0.85) pg/ml, P<0.001; IFN-γ: (5 614.60±170.17) pg/ml vs (8.42±2.70) pg/ml, P<0.001]. The IFN-γ, IL-2 and TNF-α in the 6E11 CAR-T group were similar to those in the Vector-T group when co-cultured with K562. ⑦ 6E11 CAR-T cells could be activated by bone marrow mononuclear cells (BMMNC) derived from CD123(+) AML patients and effectively kill these BMMNC cells from CD123(+) AML patients. Conclusion: 6E11 hybridoma cell line can stably secrete highly specific monoclonal antibodies against human CD123, which can be used to detect the expression of human CD123. It can also be used to target human CD123 protein in tumor immunotherapy. CD123 CAR-T cells with 6E11 Ig variable region sequence have specific anti-leukemic activity in vitro, which may provide a new option for further clinical research of AML.
Animals ; Cell Line, Tumor ; Humans ; Interleukin-3 Receptor alpha Subunit ; Leukemia, Myeloid, Acute ; Mice ; Receptors, Chimeric Antigen ; Single-Chain Antibodies

OBJECTIVE: To investigate the expression of CD44, CD87 and CD123 in acute leukemia and its correlation with cellular immune markers. METHODS: A total of 166 patients with acute leukemia (AL) admitted from May 2014 to February 2017 were enrolled in AL groups. Among these patients, 100 patients suffered from acute myeloid leukemia, 50 patients suffered from acute lymphoid leukemia, and 16 patients showed B/medullary phenotype. At the same time 50 patients with non-acute leukemia were enrolled in the control group. 5 ml of fasting venous blood collected from the patients in each group, and the percentage of CD44, CD87 and CD123 cells was determined by three-color flow cytometry. Symptomatic chemotherapy was given to the patients with confirmed acute leukemia, and the remission was evaluated after 2 treatmen courses. The Complete remission (CR) was recorded and the percentage of CD44, CD87 and CD123 cells under different curative efficacy were recorded. The correlation of the prognosis patients with CD44, CD87 and CD123 was analyzed by SPSS Pearson correlation analysis software. RESULTS: The positive rates of CD44, CD87 and CD123 in AL group were all higher than those in the control group (P＜0. 05). The positive rates of CD44 and CD123 in acute myeloid leukemia group were higher than those in acute lymphoblastic leukemia group and B/myeloid phenotype group (P＜0. 05). The positive rate of CD44 in acute lymphoid leukemia group was higher than that in B/medullary double phenotype group (P＜0.05). The treatment in the patients of AL group was successfully completed. 132 patients reachel to CR and 34 patients to PR+NR after 2 courses. The positive rates of CD44, CD87 and CD123 in CR patients were lower than those in PR+NR patients (P＜0.05). The results of SPSS Pearson correlation analysis showed that the prognosis of patients with acute leukemia negatively correlated with CD44 and CD87 (P＜0.05). CONCLUSION The expression of CD44, CD87 and CD123 in different phenotype of acute leukemia are different, which correlateds with prognosis. The determination of CD44, CD87 and CD123 can be used to evaluate the prognosis of patients for the reference of clinical treatment.
Humans ; Hyaluronan Receptors ; immunology ; Immunity, Cellular ; Interleukin-3 Receptor alpha Subunit ; immunology ; Leukemia, Myeloid, Acute ; Prognosis ; Receptors, Urokinase Plasminogen Activator ; immunology

To assess the value of immunohistochemical analysis for expressions of C3d, C4d, IgG, IgG4, and CD123 in the diagnosis of autoimmune skin diseases.
 Methods: We investigated the expressions of C3d, C4d, IgG, IgG4, and CD123 in paraffin-embedded, formalin-fixed tissues from 27 lupus erythematosus cases, including 8 discoid lupus erythematosus (DLE) cases, 4 subacute cutaneous lupus erythematosus (SCLE) cases, and 15 systemic lupus erythematosus (SLE) cases. Tissues from 15 dermatomyositis (DM) cases, 15 bullous pemphigoid (BP) cases, and 15 pemphigus cases were examined by immunohistochemical analysis. The differences in expression rates of C3d, C4d, IgG, IgG4, and CD123 between immunohistochemical staining and direct immunofluorescence were compared in the diagnosis of these diseases.
 Results: In the lupus erythematosus group, the positive rates of C3d and C4d deposited along the dermoepidermal junction were 85.2% and 51.9%, respectively. In the dermatomyositis group, the positive rates of C3d and C4d deposited along the dermoepidermal junction were 40% and 0, respectively. The expressions of C3d and C4d in lupus erythematosus tissues were significantly higher than those in DM tissues (P<0.05). The expression of CD123 protein in skin lesions of the lupus group was significantly higher than that in the DM group (P<0.05). In the BP group, the positive rates of C3d and C4d deposited along the dermoepidermal junction were 100% and 86.7%, respectively. In the pemphigus group, the positive rates of C3d and C4d deposited in the intercellular space of keratinocytes were 100% and 60%, respectively. The expressions of IgG and IgG4 in pemphigus tissues were higher than those in BP tissues (P<0.05). And the ratios of IgG4 to IgG in the pemphigus group was significantly higher than that in the BP group (P<0.05).
 Conclusion: The assays of C3d and C4d define an important diagnostic adjunct in evaluation of lupus erythematosus, BP and pemphigus. In some cases, it may even replace the direct immunofluorescence as a diagnostic adjunct. The expression of CD123 possesses certain clinical significance for the differential diagnosis of lupus erythematosus, and IgG4 and IgG expressions have adjunctive diagnostic significance for pemphigus.
Autoimmune Diseases ; Complement C3d ; Complement System Proteins ; Humans ; Immunoglobulin G ; Interleukin-3 Receptor alpha Subunit ; Lupus Erythematosus, Systemic ; Pemphigus

Objective: To investigate the characteristic and prognostic significance of leukemia stem cells associated antigens expressions including CD34, CD38, CD123, CD96 and TIM-3 in t (8;21) AML. Methods: Bone marrow samples of 47 t (8;21) AML patients were collected at diagnosis from October 2015 to April 2018 in Peking University Peoples' Hospital, then flow cytometry method was performed to detect the expression frequencies of CD34, CD38, CD123, CD96 and TIM-3 to analyze the relationship between leukemia stem cells associated antigens expressions and relapse. Results: Of 47 t (8;21) AML patients tested, the median percentages of CD34(+)CD38(-), CD34(+) CD38(-)CD123(+), CD34(+)CD38(-) CD96(+) and CD34(+) CD38(-) TIM-3(+) cells among nucleated cells were 2.37%, 0.24%, 0.27% and 0.06%, respectively. All the frequencies of CD34(+)CD38(-), CD34(+)CD38(-)CD123(+), CD34(+)CD38(-)CD96(+) and CD34(+) CD38(-)TIM-3(+) cells had no impact on the achievement of CR after the first course of induction. All higher frequencies of CD34(+)CD38(-), CD34(+)CD38(-)CD123(+), CD34(+)CD38(-)CD96(+) cells were related to higher 2-year CIR rate. Whereas, the frequency of CD34(+) CD38(-) TIM-3(+) cells had no impact on CIR rate. Both high frequency of CD34(+) CD38(-) cells and the high level of minimal residual diseases (patients with <3-log reduction in the RUNX1-RUNX1T1 transcript level after the second consolidation therapy) were independent poor prognostic factors of CIR[P=0.025, HR=6.9 (95%CI 1.3-37.4) ; P=0.031, HR=11.1 (95%CI 1.2-99.2) ]. Conclusion: Different leukemia stem cells associated antigens had distinct prognostic significance in t (8;21) AML. High frequencies of CD34(+) CD38(-), CD34(+) CD38(-) CD123(+) and CD34(+)CD38(-)CD96(+) cells at diagnosis predicted relapse in patients with t (8;21) AML.
ADP-ribosyl Cyclase 1 ; Antigens, CD ; Flow Cytometry ; Humans ; Interleukin-3 Receptor alpha Subunit ; Leukemia, Myeloid, Acute ; Neoplastic Stem Cells ; Prognosis ; Stem Cells

OBJECTIVE: To analyze the expression characteristics of leukemia stem cell (LSC) antigen in acute myeloid leukemia (AML) and to explore the correation of LSC-specific antigens with the subtypes, cytogenetics and clinical efficacy of AML. METHODS: A total of 61 newly diagnosed patients with AML (except M3) hospltalized in Department of Hematology of our hopital were selected from January 2013 to March 2016. The immun phenotypes and expression of Tim-3, CD96 and CD123 on leucamia cells were detected by direct immunofluorescenct flow cytometry. 61 patients were divided into positive expression and megative expression groups according to expression of Tim-3, CD96 and CD123; the correlation of LSC antigen expression level with high WBC count, chromosome and therapeutic efficacy was analyzed. RESULTS: Among 61 newly diagnosed patients with AML (except M3), the expression rate of Tim-3, CD96 and CD123 was 52.45%, 44.26% and 55.73% respectively. The expression rates of Tim-3, CD96 and CD123 between the AML subtypes and total patients was not stetistically different (P>0.05). The high WBC count occurred more easily in AML (except MS) patients with positive expression of Tim-3, CD96 and CD123, but compared with AML patients with negative espression, the difference was not statstically significant (P>0.05). The proportion of chromosone karyotype with poor prognosis detected in patients with positive expression of Tim-3 and CD96 was higher than that in patients with negative expreesion (P<0.05); while the preoprtion of chromosome karyotype with poor prognosis detected in patients with positive and negative expression of CD123 was not significantly different (P>0.05). After 2 courses of chemotherapy, the complete remission (CR) rate in patients with positive expression of Tim-3, CD96 and CD123 was significantly lower than that in patients with negative expression of Tim-3, CD96 and CD123 (P<0.05), the comparison of OS time in patients with positive and negative expression of Tim-3 and CD96 showed the statistical difference (P>0.05), while the difference of OS time in patients with positive and negative expression of CD123 was not significant (P>0.05). CONCLUSION The expression levels of Tim-3, CD96 and CD123 in newly diagnosed AML (except M3) sybtype patients are not significantly different form those in total patients. The high WBC count ocours more easily in patients with positive expression of Tim-3, CD96 and CD123. After 2 course of chemotherapy, the CR rate in patients with positive expression of Tim-3, CD96 and CD123 was significantly lower than that in patients with negative expression. The proportion of chromsome karyotype with poor prognosis detected in patients with positive expression of Tim-3 and CD96 is high, moreover, OS time in patients with positive expression of Tim-3 and CD96 is shorter than that in patients with negative expression.
Antigens, CD ; Flow Cytometry ; Humans ; Interleukin-3 Receptor alpha Subunit ; Leukemia, Myeloid, Acute ; Prognosis ; Stem Cells

OBJECTIVETo investigate the presence of leukemia stem cells (LSC) in acute myeloid leukemia (AML) and find out the relative position of leukemia cells in the figures of flow cytometry, and to analyze the relationship between minimal residual diseases (MRD) and the level of LSC, so as to explore the correlation of LSC changes with the curative effect and the prognosis during chemical therapy.

METHODSA total of 85 samples were collected from 50 AML (except M3) patients, including 50 samples from the newly diagnosed patients, 7 samples of AML patients with non-remission and 28 samples of AML patients with complete remission. All samples were used for detection of LSC from immune phenotype of CD34/CD38/CD123 by flow cytometry. The detection of immune phenotypic of leukemia cells was performed in the newly diagnosed patients. The detection of leukemia- associated immune phenotypes (LAIP) was implemented in the non-newly diagnosed patients.

RESULTSThe LSC was identified in the CD34/ CD38/ CD123 in AML and consistent with the relative position of the leukemia cell in flow cytometry figures. Statistical analysis showed significant difference in LSC content between the newly diagnosed AML group and the post-chemotherapy complete remission group(P<0.01),but did not between the newly diagnosed AML group and the post-chemotherapy non-remission group(P>0.05).There was significant positive correlation between the LSC content and MRD level in 28 AML patients with complete remission (r=0.680,P<0.01).

CONCLUSIONLSC exist in AML and the relative position are consistent with the leukemia cells in flow cytometry figures, the size characteristics and weak expression of CD45 are also similar to leukemia cells. The proportion of LSC decreases after chemotherapy. Detecting and tracking the LSC changes in bone marrow and combination with detecting minimal resident disease(MRD) may contribute to evaluate the theraputic efficacy and prognosis of leukemia patients.

BACKGROUND: The indirect basophil activation test using flow cytometry is a promising tool for autoimmune urticaria diagnosis. We aimed to identify better donor basophils (from atopic vs. non-atopic donors and interleukin-3 primed vs. unprimed basophils) and improve basophil identification and activation markers (eotaxin CC chemokine receptor-3 [CCR3] vs. CD123 and CD63 vs. CD203c). METHODS: Donor basophils were obtained from non-atopic and atopic group O donors. Positive control sera were artificially prepared to simulate autoimmune urticaria patients' sera. Patient sera were obtained from nine children with chronic urticaria. Assay sensitivity was compared among each variation by using positive control sera (n=21), applying cutoff values defined from negative control sera (n=20). RESULTS: For basophil identification, a combination of CCR3 and CD123 markers revealed a higher correlation with automated complete blood count (r=0.530) compared with that observed using CD123 (r=0.498) or CCR3 alone (r=0.195). Three activation markers on the atopic donor basophils attained 100% assay sensitivity: CD203c on unprimed basophils, CD63+CD203+ or CD63 alone on primed basophils; however, these markers on the non-atopic donor basophils attained lower assay sensitivity. CONCLUSIONS: For basophil identification markers, a combination of CD123 and CCR3 is recommended, while CD123 alone may be used as an alternative. Donor basophils should be obtained from an atopic donor. For basophil activation markers, either CD203c alone on unprimed basophils or CD203c and CD63 on primed basophils are recommended, while CD63 alone on primed basophils may be used as an alternative.
Autoimmune Diseases/blood/*diagnosis/immunology ; Basophils/*immunology/metabolism ; Biomarkers/blood ; Child ; Flow Cytometry ; Humans ; Interleukin-3 Receptor alpha Subunit/blood ; Male ; Receptors, CCR3/blood ; Urticaria/blood/*diagnosis/immunology

No abstract available.
Basophils/cytology/*metabolism ; Flow Cytometry ; HLA-DR Antigens/metabolism ; Humans ; Interleukin-3 Receptor alpha Subunit/metabolism ; Leukocyte Count ; Phosphoric Diester Hydrolases/metabolism ; Pyrophosphatases/metabolism ; Receptors, CCR3/metabolism ; Urticaria/*diagnosis/immunology/metabolism