ACTIVATION INDUCED DEAMINASE SPLICE VARIANTS EXPRESSED IN B CELL LEUKEMIA AND LYMPHOMA DO NOT RETAIN THEIR CATALYTIC ACTIVITY
(Abstract release date: 05/19/16)
EHA Library. Zápražná K. 06/09/16; 132594; E1045
Dr. Kristína Zápražná
Contributions
Contributions
Abstract
Abstract: E1045
Type: Eposter Presentation
Background
Activation induced deaminase (AID) is essential for enhancing antibody affinity and effector functions in B cells during secondary immune response. It induces somatic hypermutation (SHM) and class switch recombination (CSR) in the immunoglobulin (Ig) genes. AID is a potent mutator -SHM frequency in the Ig genes is a million times higher than the frequency of spontaneous mutagenesis (10-3 -10-4 mutations/ cell/ generation). AID studies performed in mouse models have shown a correlation between AID activity and genomic instability. AID is aberrantly expressed in various B lymphoid malignancies and also in some solid tumors.
Aims
In this project, we set out to investigate the function of four alternatively spliced AID variants that differ from the full-length protein in the C terminal domain. These variants are present in an increased proportion in chronic lymphocytic leukemia (CLL), Ph-positive acute lymphoblastic leukemia (Ph-positive ALL) and mantle cell lymphoma (MCL).
Methods
Individual AID splice variants were cloned from peripheral blood CLL cells into an HA-tagged retroviral vector. Mature B cells purified from AID knockout spleens were stimulated with lipopolysaccharide and interleukin-4 and subsequently infected with retroviruses carrying individual AID splice variants. Cells were cultured ex vivo for a total of 4 or 5 days and then assessed for CSR to IgG1 by flow-cytometry or sorted based on high GFP expression marking the infected cells. The level of SHM in the Ig switch region Sm was assessed by ultra-deep next generation sequencing. The amount of double strand breaks in the Ig Sm was quantified by ligation-mediated PCR and hybridization with an Sm probe.
Results
While full-length AID induced CSR, SHM and double strand breaks in the Ig genes, alternatively spliced AID variants behaved as catalytically inactive in all performed functional assays. Immunoblotting with an anti-HA antibody revealed that AID splice variants had lower protein expression compared to full-length AID. It is a well-established fact in the AID field that in most situations, AID levels correlate with its activity. Therefore, lower expression of alternatively spliced AID variants might have contributed to their loss-of function in our model system.
Conclusion
Alternatively spliced AID transcripts were previously shown to be expressed in CLL, Ph-positive ALL and MCL. We hypothesized that alternative splicing might modulate their activity and consequently change AID deamination levels in the Ig genes or its targeting in the whole genome. Results of AID splice variant analysis done in AID knockout mouse B cells stimulated ex vivo indicated their loss of function. Therefore, the role of alternatively spliced AID expressed in B-lymphoid leukemia and lymphoma remains unclear, and their function is to be further analyzed using human cell lines derived from B-cell malignancies. This work was supported by project No.3SGA5792 financed from the SoMoPro II Programme that has acquired a financial grant from the People Programme (Marie Curie Action) of the Seventh Framework Programme of EU according to the REA Grant Agreement No. 291782 and was further co-financed by the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein.
Session topic: E-poster
Keyword(s): Alternative splicing, Lymphoid malignancy, Somatic hypermutation
Type: Eposter Presentation
Background
Activation induced deaminase (AID) is essential for enhancing antibody affinity and effector functions in B cells during secondary immune response. It induces somatic hypermutation (SHM) and class switch recombination (CSR) in the immunoglobulin (Ig) genes. AID is a potent mutator -SHM frequency in the Ig genes is a million times higher than the frequency of spontaneous mutagenesis (10-3 -10-4 mutations/ cell/ generation). AID studies performed in mouse models have shown a correlation between AID activity and genomic instability. AID is aberrantly expressed in various B lymphoid malignancies and also in some solid tumors.
Aims
In this project, we set out to investigate the function of four alternatively spliced AID variants that differ from the full-length protein in the C terminal domain. These variants are present in an increased proportion in chronic lymphocytic leukemia (CLL), Ph-positive acute lymphoblastic leukemia (Ph-positive ALL) and mantle cell lymphoma (MCL).
Methods
Individual AID splice variants were cloned from peripheral blood CLL cells into an HA-tagged retroviral vector. Mature B cells purified from AID knockout spleens were stimulated with lipopolysaccharide and interleukin-4 and subsequently infected with retroviruses carrying individual AID splice variants. Cells were cultured ex vivo for a total of 4 or 5 days and then assessed for CSR to IgG1 by flow-cytometry or sorted based on high GFP expression marking the infected cells. The level of SHM in the Ig switch region Sm was assessed by ultra-deep next generation sequencing. The amount of double strand breaks in the Ig Sm was quantified by ligation-mediated PCR and hybridization with an Sm probe.
Results
While full-length AID induced CSR, SHM and double strand breaks in the Ig genes, alternatively spliced AID variants behaved as catalytically inactive in all performed functional assays. Immunoblotting with an anti-HA antibody revealed that AID splice variants had lower protein expression compared to full-length AID. It is a well-established fact in the AID field that in most situations, AID levels correlate with its activity. Therefore, lower expression of alternatively spliced AID variants might have contributed to their loss-of function in our model system.
Conclusion
Alternatively spliced AID transcripts were previously shown to be expressed in CLL, Ph-positive ALL and MCL. We hypothesized that alternative splicing might modulate their activity and consequently change AID deamination levels in the Ig genes or its targeting in the whole genome. Results of AID splice variant analysis done in AID knockout mouse B cells stimulated ex vivo indicated their loss of function. Therefore, the role of alternatively spliced AID expressed in B-lymphoid leukemia and lymphoma remains unclear, and their function is to be further analyzed using human cell lines derived from B-cell malignancies. This work was supported by project No.3SGA5792 financed from the SoMoPro II Programme that has acquired a financial grant from the People Programme (Marie Curie Action) of the Seventh Framework Programme of EU according to the REA Grant Agreement No. 291782 and was further co-financed by the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein.
Session topic: E-poster
Keyword(s): Alternative splicing, Lymphoid malignancy, Somatic hypermutation
Abstract: E1045
Type: Eposter Presentation
Background
Activation induced deaminase (AID) is essential for enhancing antibody affinity and effector functions in B cells during secondary immune response. It induces somatic hypermutation (SHM) and class switch recombination (CSR) in the immunoglobulin (Ig) genes. AID is a potent mutator -SHM frequency in the Ig genes is a million times higher than the frequency of spontaneous mutagenesis (10-3 -10-4 mutations/ cell/ generation). AID studies performed in mouse models have shown a correlation between AID activity and genomic instability. AID is aberrantly expressed in various B lymphoid malignancies and also in some solid tumors.
Aims
In this project, we set out to investigate the function of four alternatively spliced AID variants that differ from the full-length protein in the C terminal domain. These variants are present in an increased proportion in chronic lymphocytic leukemia (CLL), Ph-positive acute lymphoblastic leukemia (Ph-positive ALL) and mantle cell lymphoma (MCL).
Methods
Individual AID splice variants were cloned from peripheral blood CLL cells into an HA-tagged retroviral vector. Mature B cells purified from AID knockout spleens were stimulated with lipopolysaccharide and interleukin-4 and subsequently infected with retroviruses carrying individual AID splice variants. Cells were cultured ex vivo for a total of 4 or 5 days and then assessed for CSR to IgG1 by flow-cytometry or sorted based on high GFP expression marking the infected cells. The level of SHM in the Ig switch region Sm was assessed by ultra-deep next generation sequencing. The amount of double strand breaks in the Ig Sm was quantified by ligation-mediated PCR and hybridization with an Sm probe.
Results
While full-length AID induced CSR, SHM and double strand breaks in the Ig genes, alternatively spliced AID variants behaved as catalytically inactive in all performed functional assays. Immunoblotting with an anti-HA antibody revealed that AID splice variants had lower protein expression compared to full-length AID. It is a well-established fact in the AID field that in most situations, AID levels correlate with its activity. Therefore, lower expression of alternatively spliced AID variants might have contributed to their loss-of function in our model system.
Conclusion
Alternatively spliced AID transcripts were previously shown to be expressed in CLL, Ph-positive ALL and MCL. We hypothesized that alternative splicing might modulate their activity and consequently change AID deamination levels in the Ig genes or its targeting in the whole genome. Results of AID splice variant analysis done in AID knockout mouse B cells stimulated ex vivo indicated their loss of function. Therefore, the role of alternatively spliced AID expressed in B-lymphoid leukemia and lymphoma remains unclear, and their function is to be further analyzed using human cell lines derived from B-cell malignancies. This work was supported by project No.3SGA5792 financed from the SoMoPro II Programme that has acquired a financial grant from the People Programme (Marie Curie Action) of the Seventh Framework Programme of EU according to the REA Grant Agreement No. 291782 and was further co-financed by the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein.
Session topic: E-poster
Keyword(s): Alternative splicing, Lymphoid malignancy, Somatic hypermutation
Type: Eposter Presentation
Background
Activation induced deaminase (AID) is essential for enhancing antibody affinity and effector functions in B cells during secondary immune response. It induces somatic hypermutation (SHM) and class switch recombination (CSR) in the immunoglobulin (Ig) genes. AID is a potent mutator -SHM frequency in the Ig genes is a million times higher than the frequency of spontaneous mutagenesis (10-3 -10-4 mutations/ cell/ generation). AID studies performed in mouse models have shown a correlation between AID activity and genomic instability. AID is aberrantly expressed in various B lymphoid malignancies and also in some solid tumors.
Aims
In this project, we set out to investigate the function of four alternatively spliced AID variants that differ from the full-length protein in the C terminal domain. These variants are present in an increased proportion in chronic lymphocytic leukemia (CLL), Ph-positive acute lymphoblastic leukemia (Ph-positive ALL) and mantle cell lymphoma (MCL).
Methods
Individual AID splice variants were cloned from peripheral blood CLL cells into an HA-tagged retroviral vector. Mature B cells purified from AID knockout spleens were stimulated with lipopolysaccharide and interleukin-4 and subsequently infected with retroviruses carrying individual AID splice variants. Cells were cultured ex vivo for a total of 4 or 5 days and then assessed for CSR to IgG1 by flow-cytometry or sorted based on high GFP expression marking the infected cells. The level of SHM in the Ig switch region Sm was assessed by ultra-deep next generation sequencing. The amount of double strand breaks in the Ig Sm was quantified by ligation-mediated PCR and hybridization with an Sm probe.
Results
While full-length AID induced CSR, SHM and double strand breaks in the Ig genes, alternatively spliced AID variants behaved as catalytically inactive in all performed functional assays. Immunoblotting with an anti-HA antibody revealed that AID splice variants had lower protein expression compared to full-length AID. It is a well-established fact in the AID field that in most situations, AID levels correlate with its activity. Therefore, lower expression of alternatively spliced AID variants might have contributed to their loss-of function in our model system.
Conclusion
Alternatively spliced AID transcripts were previously shown to be expressed in CLL, Ph-positive ALL and MCL. We hypothesized that alternative splicing might modulate their activity and consequently change AID deamination levels in the Ig genes or its targeting in the whole genome. Results of AID splice variant analysis done in AID knockout mouse B cells stimulated ex vivo indicated their loss of function. Therefore, the role of alternatively spliced AID expressed in B-lymphoid leukemia and lymphoma remains unclear, and their function is to be further analyzed using human cell lines derived from B-cell malignancies. This work was supported by project No.3SGA5792 financed from the SoMoPro II Programme that has acquired a financial grant from the People Programme (Marie Curie Action) of the Seventh Framework Programme of EU according to the REA Grant Agreement No. 291782 and was further co-financed by the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein.
Session topic: E-poster
Keyword(s): Alternative splicing, Lymphoid malignancy, Somatic hypermutation
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