DYSREGULATED MIR34A/DIACYLGLYCEROL KINASE ? INTERACTION ENHANCES T-CELL ACTIVATION IN ACQUIRED APLASTIC ANEMIA
(Abstract release date: 05/19/16)
EHA Library. Sun Y. 06/11/16; 135228; S472
Ms. Yuanxin Sun
Contributions
Contributions
Abstract
Abstract: S472
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:30 - 11:45
Location: Room H5
Background
As a paradigm of bone marrow failure syndrome, acquired aplastic anemia (AA) was thought to be a specific autoimmune disease for the aberrant T-cell immune homeostasis. MicroRNAs (miRNAs) down-regulate gene expression by binding to target messenger RNAs and play important roles in various immune processes. We screened for differentially expressed miRNAs in T-lymphocytes of sever AA (SAA) patients by miRNA genechip assays and found miR34a overexpressed in SAA. MiR34a has been demonstrated to directly and functionally target diacylglycerol kinase (DGK) ζ to promote T-cell response. There was no data referred to the role of miR34a or DGKζ in AA.
Aims
To investigate the role of miR34a and DGKζ in T-cell activation in AA.
Methods
Human bone marrow mononuclear cells (BMMCs) were isolated from 25 SAA patients, 16 moderate AA (MAA) patients and 20 healthy volunteers. Affymetrix miRNA genechip assays on CD3+ T-cells purified from BMMCs of three SAA patients and three healthy controls were undertaken. Then the relative expression of miR34a and DGKζ in BMMCs from the 41 AA patients and 20 healthy controls was quantified by real time polymerase chain reaction. The BMMCs from AA patients were transfected with miR34a inhibitor sponge in lentivirus and the level of T-cell activation marker CD69 on the transfected cells was analyzed by flow cytometry. The lymphocytes isolated from lymph nodes (LN) of wild-type (WT) and miR34a-knockout (KO) mice were labeled with CFSE and left unstimulated or stimulated with McAbs to CD3 and CD28. The cell division and the CD69 and CD25 expression was analyzed by flow cytometry. A murine model of bone marrow failure was created in which LN cells from WT or miR34a-KO mice (C57/B6 background) were transferred into CB6F1 mouse recipients preirradiated with 5 Gy total body irradiation. After euthanasia on Day 12, CD4+,CD8+ and Lin-Sca1+CD117+CD34- (LSKCD34-) BM cells were analyzed by flow cytometry.
Results
The microarray analysis demonstrated that miR34a expression increased 2.1-fold in T-cells from SAA patients compared with that in healthy controls. The significantly higher expression of miR34a was confirmed by PCR in BMMCs from the 41 AA patients compared with that from the 20 normal controls. The miR34a expression showed a negative correlation with peripheral blood neutrophil or reticulocyte counts in AA patients, and was higher in SAA than in MAA groups. The mRNA level of DGKζ in AA patients was much lower than that in controls, and was negatively correlated with the level of miR34a. The protein level of CD69 on T-cells transfected with miR34a inhibitor lentivirus was lower than that on cells transfected with control lentivirus. In murine models, upon stimulation with McAbs to CD3 and CD28, the CD4+ and CD8+ LN cells from miR34a-KO mice expressed much lower levels of CD69 and CD25 than those from WT mice, and proliferated less vigorously than the cells from WT mice. Consistently, the DGKζ expression in LN cells from miR34a-KO mice decreased to a less extent after stimulation than that in cells from WT mice. In vivo, the mouse recipients transferred with miR34a-KO lymphocytes demonstrated lower mortality on Day12 after irradiation, fewer CD8+ T-cells in BM and increased BM LSKCD34- cells compaired to those transferred with WT cells.
Conclusion
The data demonstrated that miR34a/DGKζ was dysregulated in AA, which resulted in enhanced T-cell activation and bone marrow failure. Our study indicated that miR34a/DGKζ played a critical role in T-cell immunity in AA and might be a new therapeutic target for AA.
Session topic: Bone marrow failure syndromes incl. PNH - Biology
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:30 - 11:45
Location: Room H5
Background
As a paradigm of bone marrow failure syndrome, acquired aplastic anemia (AA) was thought to be a specific autoimmune disease for the aberrant T-cell immune homeostasis. MicroRNAs (miRNAs) down-regulate gene expression by binding to target messenger RNAs and play important roles in various immune processes. We screened for differentially expressed miRNAs in T-lymphocytes of sever AA (SAA) patients by miRNA genechip assays and found miR34a overexpressed in SAA. MiR34a has been demonstrated to directly and functionally target diacylglycerol kinase (DGK) ζ to promote T-cell response. There was no data referred to the role of miR34a or DGKζ in AA.
Aims
To investigate the role of miR34a and DGKζ in T-cell activation in AA.
Methods
Human bone marrow mononuclear cells (BMMCs) were isolated from 25 SAA patients, 16 moderate AA (MAA) patients and 20 healthy volunteers. Affymetrix miRNA genechip assays on CD3+ T-cells purified from BMMCs of three SAA patients and three healthy controls were undertaken. Then the relative expression of miR34a and DGKζ in BMMCs from the 41 AA patients and 20 healthy controls was quantified by real time polymerase chain reaction. The BMMCs from AA patients were transfected with miR34a inhibitor sponge in lentivirus and the level of T-cell activation marker CD69 on the transfected cells was analyzed by flow cytometry. The lymphocytes isolated from lymph nodes (LN) of wild-type (WT) and miR34a-knockout (KO) mice were labeled with CFSE and left unstimulated or stimulated with McAbs to CD3 and CD28. The cell division and the CD69 and CD25 expression was analyzed by flow cytometry. A murine model of bone marrow failure was created in which LN cells from WT or miR34a-KO mice (C57/B6 background) were transferred into CB6F1 mouse recipients preirradiated with 5 Gy total body irradiation. After euthanasia on Day 12, CD4+,CD8+ and Lin-Sca1+CD117+CD34- (LSKCD34-) BM cells were analyzed by flow cytometry.
Results
The microarray analysis demonstrated that miR34a expression increased 2.1-fold in T-cells from SAA patients compared with that in healthy controls. The significantly higher expression of miR34a was confirmed by PCR in BMMCs from the 41 AA patients compared with that from the 20 normal controls. The miR34a expression showed a negative correlation with peripheral blood neutrophil or reticulocyte counts in AA patients, and was higher in SAA than in MAA groups. The mRNA level of DGKζ in AA patients was much lower than that in controls, and was negatively correlated with the level of miR34a. The protein level of CD69 on T-cells transfected with miR34a inhibitor lentivirus was lower than that on cells transfected with control lentivirus. In murine models, upon stimulation with McAbs to CD3 and CD28, the CD4+ and CD8+ LN cells from miR34a-KO mice expressed much lower levels of CD69 and CD25 than those from WT mice, and proliferated less vigorously than the cells from WT mice. Consistently, the DGKζ expression in LN cells from miR34a-KO mice decreased to a less extent after stimulation than that in cells from WT mice. In vivo, the mouse recipients transferred with miR34a-KO lymphocytes demonstrated lower mortality on Day12 after irradiation, fewer CD8+ T-cells in BM and increased BM LSKCD34- cells compaired to those transferred with WT cells.
Conclusion
The data demonstrated that miR34a/DGKζ was dysregulated in AA, which resulted in enhanced T-cell activation and bone marrow failure. Our study indicated that miR34a/DGKζ played a critical role in T-cell immunity in AA and might be a new therapeutic target for AA.
Session topic: Bone marrow failure syndromes incl. PNH - Biology
Abstract: S472
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:30 - 11:45
Location: Room H5
Background
As a paradigm of bone marrow failure syndrome, acquired aplastic anemia (AA) was thought to be a specific autoimmune disease for the aberrant T-cell immune homeostasis. MicroRNAs (miRNAs) down-regulate gene expression by binding to target messenger RNAs and play important roles in various immune processes. We screened for differentially expressed miRNAs in T-lymphocytes of sever AA (SAA) patients by miRNA genechip assays and found miR34a overexpressed in SAA. MiR34a has been demonstrated to directly and functionally target diacylglycerol kinase (DGK) ζ to promote T-cell response. There was no data referred to the role of miR34a or DGKζ in AA.
Aims
To investigate the role of miR34a and DGKζ in T-cell activation in AA.
Methods
Human bone marrow mononuclear cells (BMMCs) were isolated from 25 SAA patients, 16 moderate AA (MAA) patients and 20 healthy volunteers. Affymetrix miRNA genechip assays on CD3+ T-cells purified from BMMCs of three SAA patients and three healthy controls were undertaken. Then the relative expression of miR34a and DGKζ in BMMCs from the 41 AA patients and 20 healthy controls was quantified by real time polymerase chain reaction. The BMMCs from AA patients were transfected with miR34a inhibitor sponge in lentivirus and the level of T-cell activation marker CD69 on the transfected cells was analyzed by flow cytometry. The lymphocytes isolated from lymph nodes (LN) of wild-type (WT) and miR34a-knockout (KO) mice were labeled with CFSE and left unstimulated or stimulated with McAbs to CD3 and CD28. The cell division and the CD69 and CD25 expression was analyzed by flow cytometry. A murine model of bone marrow failure was created in which LN cells from WT or miR34a-KO mice (C57/B6 background) were transferred into CB6F1 mouse recipients preirradiated with 5 Gy total body irradiation. After euthanasia on Day 12, CD4+,CD8+ and Lin-Sca1+CD117+CD34- (LSKCD34-) BM cells were analyzed by flow cytometry.
Results
The microarray analysis demonstrated that miR34a expression increased 2.1-fold in T-cells from SAA patients compared with that in healthy controls. The significantly higher expression of miR34a was confirmed by PCR in BMMCs from the 41 AA patients compared with that from the 20 normal controls. The miR34a expression showed a negative correlation with peripheral blood neutrophil or reticulocyte counts in AA patients, and was higher in SAA than in MAA groups. The mRNA level of DGKζ in AA patients was much lower than that in controls, and was negatively correlated with the level of miR34a. The protein level of CD69 on T-cells transfected with miR34a inhibitor lentivirus was lower than that on cells transfected with control lentivirus. In murine models, upon stimulation with McAbs to CD3 and CD28, the CD4+ and CD8+ LN cells from miR34a-KO mice expressed much lower levels of CD69 and CD25 than those from WT mice, and proliferated less vigorously than the cells from WT mice. Consistently, the DGKζ expression in LN cells from miR34a-KO mice decreased to a less extent after stimulation than that in cells from WT mice. In vivo, the mouse recipients transferred with miR34a-KO lymphocytes demonstrated lower mortality on Day12 after irradiation, fewer CD8+ T-cells in BM and increased BM LSKCD34- cells compaired to those transferred with WT cells.
Conclusion
The data demonstrated that miR34a/DGKζ was dysregulated in AA, which resulted in enhanced T-cell activation and bone marrow failure. Our study indicated that miR34a/DGKζ played a critical role in T-cell immunity in AA and might be a new therapeutic target for AA.
Session topic: Bone marrow failure syndromes incl. PNH - Biology
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:30 - 11:45
Location: Room H5
Background
As a paradigm of bone marrow failure syndrome, acquired aplastic anemia (AA) was thought to be a specific autoimmune disease for the aberrant T-cell immune homeostasis. MicroRNAs (miRNAs) down-regulate gene expression by binding to target messenger RNAs and play important roles in various immune processes. We screened for differentially expressed miRNAs in T-lymphocytes of sever AA (SAA) patients by miRNA genechip assays and found miR34a overexpressed in SAA. MiR34a has been demonstrated to directly and functionally target diacylglycerol kinase (DGK) ζ to promote T-cell response. There was no data referred to the role of miR34a or DGKζ in AA.
Aims
To investigate the role of miR34a and DGKζ in T-cell activation in AA.
Methods
Human bone marrow mononuclear cells (BMMCs) were isolated from 25 SAA patients, 16 moderate AA (MAA) patients and 20 healthy volunteers. Affymetrix miRNA genechip assays on CD3+ T-cells purified from BMMCs of three SAA patients and three healthy controls were undertaken. Then the relative expression of miR34a and DGKζ in BMMCs from the 41 AA patients and 20 healthy controls was quantified by real time polymerase chain reaction. The BMMCs from AA patients were transfected with miR34a inhibitor sponge in lentivirus and the level of T-cell activation marker CD69 on the transfected cells was analyzed by flow cytometry. The lymphocytes isolated from lymph nodes (LN) of wild-type (WT) and miR34a-knockout (KO) mice were labeled with CFSE and left unstimulated or stimulated with McAbs to CD3 and CD28. The cell division and the CD69 and CD25 expression was analyzed by flow cytometry. A murine model of bone marrow failure was created in which LN cells from WT or miR34a-KO mice (C57/B6 background) were transferred into CB6F1 mouse recipients preirradiated with 5 Gy total body irradiation. After euthanasia on Day 12, CD4+,CD8+ and Lin-Sca1+CD117+CD34- (LSKCD34-) BM cells were analyzed by flow cytometry.
Results
The microarray analysis demonstrated that miR34a expression increased 2.1-fold in T-cells from SAA patients compared with that in healthy controls. The significantly higher expression of miR34a was confirmed by PCR in BMMCs from the 41 AA patients compared with that from the 20 normal controls. The miR34a expression showed a negative correlation with peripheral blood neutrophil or reticulocyte counts in AA patients, and was higher in SAA than in MAA groups. The mRNA level of DGKζ in AA patients was much lower than that in controls, and was negatively correlated with the level of miR34a. The protein level of CD69 on T-cells transfected with miR34a inhibitor lentivirus was lower than that on cells transfected with control lentivirus. In murine models, upon stimulation with McAbs to CD3 and CD28, the CD4+ and CD8+ LN cells from miR34a-KO mice expressed much lower levels of CD69 and CD25 than those from WT mice, and proliferated less vigorously than the cells from WT mice. Consistently, the DGKζ expression in LN cells from miR34a-KO mice decreased to a less extent after stimulation than that in cells from WT mice. In vivo, the mouse recipients transferred with miR34a-KO lymphocytes demonstrated lower mortality on Day12 after irradiation, fewer CD8+ T-cells in BM and increased BM LSKCD34- cells compaired to those transferred with WT cells.
Conclusion
The data demonstrated that miR34a/DGKζ was dysregulated in AA, which resulted in enhanced T-cell activation and bone marrow failure. Our study indicated that miR34a/DGKζ played a critical role in T-cell immunity in AA and might be a new therapeutic target for AA.
Session topic: Bone marrow failure syndromes incl. PNH - Biology
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