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Editor | Xi's high-affinity neutralizing antibody is an important weapon for the body to resist infection by pathogenic microorganisms, and it is the basis for most vaccines to function.
High-frequency mutations and forward screening of variable regions of B cell receptors (BCRs) necessary for antibody affinity maturation occur in a tissue structure called GC within the spleen and lymph nodes.
GC is divided into two areas with different functions-dark area and light area.
B cells repeatedly migrate between different regions, and through the Darwinian evolutionary process, the forward screening of high-affinity BCR mutations is realized, and plasma cells that produce high-affinity antibodies are finally screened out.
Due to the lack of effective molecular markers in the field to distinguish and enrich GC B cells with high-affinity mutations from a polyclonal immune response, which intracellular molecular pathways are currently supporting the forward screening of high-affinity GC B cells Important issues have not been systematically studied.
On May 24, 2021, the Xu Heping group of West Lake University published online research results entitled Coupled analysis of transcriptome and BCR mutations reveals role of OXPHOS in affinity maturation in Nature Immunology.
Based on the integrated analysis of antibody mutation patterns and transcriptome information, combined with genetic experiments, it revealed the key role of oxidative phosphorylation (OXPHOS) pathway in the positive screening of germinal center (Germinal Center, GC) B cell affinity.
Based on the classic NP-KLH immune mouse model, Xu Heping’s research group innovatively correlated antibody mutation patterns with transcriptome expression levels at the single-cell level to break through this research problem (Figure 1).
NP-KLH is a T cell dependent antigen.
It is known that when the germinal center response induced by it in a mouse model reaches its peak, GC B cells that specifically bind to hapten NP mainly express antibody heavy chain variable region genes Fragment VH186.
2; In addition, after the 33rd tryptophan of the amino acid sequence encoded by this gene was mutated to leucine, the affinity of BCR and NP increased to 10 times, while other random mutations were not significantly Change affinity.
The above two important immune response characteristics allow researchers to distinguish the binding affinity of NP based on the mutation of the BCR sequence.
On this basis, the use of 5'-end single-cell transcriptome analysis technology can simultaneously obtain the BCR sequence mutation pattern and transcriptome information of each B cell, so that the molecular mechanisms associated with affinity maturation can be fully explored and analyzed.
Figure 1: Integrating BCR mutation information and transcriptome data reveals the key molecular mechanism of antibody affinity maturation.
Based on transcriptome data and known gene expression characteristics, the authors first confirmed the spatial regions and cell cycles of different types of GC B cells.
status.
In order to explore the changes in the expression of cell gene modules more comprehensively, the authors introduced topic model algorithms in machine learning.
Compared with the traditional method of comparing the differentially expressed genes between cells of different groups, the topic model algorithm can not only find group-specific gene characteristics, but also find gene modules across groups, and achieve a more refined depiction of the cell state.
For example, in the past, some studies believed that the oxygen content in the clear area of the germinal center was low, and thus believed that GC B cells could only perform glycolysis.
The thematic model analysis in this work revealed and compared the metabolic characteristics of GC B cells in different regions.
It was found that cells in the bright area expressed higher levels of glycolysis pathway-related genes.
On the contrary, cells in the dark area expressed more OXPHOS (oxidation).
Phosphorylation) and fatty acid oxidation pathway genes indicate that GC B cells in different regions have different metabolic activities; to a certain extent, this shows that the previous conclusion that GC B cells only perform glycolysis is incorrect.
In the preparation for submission of this study, a newly published work through in vitro detection of primary GC B cell metabolic activity also shows that GC B cells mainly carry out OXPHOS rather than glycolysis.
More importantly, by combining the BCR mutation pattern information, the authors found that NP-specific GC B cell populations with high-affinity mutations (the VH186.
2 gene of the W33L mutation) have higher OXPHOS pathway gene expression.
It implies that this pathway may play an important role in the process of affinity maturation.
In addition, in the more complex polyclonal immune response model induced by OVA protein antigen, the authors not only further confirmed the positive correlation between OXPHOS and affinity forward screening, but also demonstrated that the research method developed in this work can be used for Analyze more complex features of B cell immune response.
In collaboration with Professor Harinder Singh of the University of Pittsburgh, the authors used genetic means to verify the role of the OXPHOS pathway in the body in the process of positive affinity screening.
The Cox10 gene encodes heme IX farnesyl transferase, which is an important part of the cytochrome c oxidase complex on the mitochondrial electron transport chain, and its deletion will lead to impaired OXPHOS activity.
Compared with control mice, the proportion of GC B cells, proliferation and antibody affinity of Cox10-deficient mice among GC B cells decreased significantly, proving that GC B cells rely on OXPHOS to achieve positive affinity screening.
Finally, by trying a variety of small molecules that have been reported to promote the activity of OXPHOS, the authors found that a small molecule called oltipraz can promote the forward screening of GC B cells and the maturation of antibody affinity in vivo.
This is the first report so far.
Small chemical molecules with similar functions.
The results of this study not only prove that OXPHOS promotes forward screening and affinity maturation by promoting the rapid division of high-affinity GC B cells, but also provides new ideas and targets for small molecules to intervene in the process of antibody affinity maturation and vaccine design.
In addition, using the analytical methods and procedures developed by the project, this research work has also successfully analyzed the correlation between the BCR response mutation pattern of complex antigens and the expression of molecular pathways, proving that this method can be widely used to analyze autoimmunity and transplant rejection immunity.
The diversity of BCRs and molecular response characteristics in other complex diseases. Researcher Xu Heping from West Lake University and Professor Harinder Singh from the University of Pittsburgh are the co-corresponding authors of the paper.
Doctoral students Chen Tianyu and Wang Yan from West Lake University and the postdoc Godhev K Manakkat Vijay from the University of Pittsburgh are the co-first authors of this paper.
In addition, research assistant Fu Shujie is the project's advance Made an important contribution.
Attached with the introduction and recruitment information of Xu Heping's research groupThe Systemic Immunology Laboratory of West Lake University is dedicated to exploring the key molecular mechanisms and networks that regulate the humoral immune response and mucosal inflammation.
Based on the organic integration of genetic animal models, clinical samples, and cutting-edge bioinformatics, it focuses on the study of the interaction mechanism of the immune and nervous system and other tissue-specific microenvironments in the inflammatory response and antibody response.
In recent years, many academic papers have been published in mainstream journals such as Nature (2 articles), Nature Immunology (2 articles) and Immunity (1 article).
With the support of the National Overseas High-level Talents Project, the National Natural Science Foundation of China's Joint Fund Key Projects, and the Ministry of Science and Technology Key R&D Program, various projects in the laboratory are progressing smoothly.
There is an urgent need for young talents to join the team to grow and develop together! There are many postdoctoral, doctoral and research assistant positions in the laboratory, welcome to contact us! Resume delivery (if interested, please send your resume and other materials to): https://jinshuju.
net/f/ZqXwZt or scan the QR code to send the original resume link: 021-00936-y Platemaker: Instructions for reprinting on the eleventh [Non-original article] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights and offenders must be investigated.
High-frequency mutations and forward screening of variable regions of B cell receptors (BCRs) necessary for antibody affinity maturation occur in a tissue structure called GC within the spleen and lymph nodes.
GC is divided into two areas with different functions-dark area and light area.
B cells repeatedly migrate between different regions, and through the Darwinian evolutionary process, the forward screening of high-affinity BCR mutations is realized, and plasma cells that produce high-affinity antibodies are finally screened out.
Due to the lack of effective molecular markers in the field to distinguish and enrich GC B cells with high-affinity mutations from a polyclonal immune response, which intracellular molecular pathways are currently supporting the forward screening of high-affinity GC B cells Important issues have not been systematically studied.
On May 24, 2021, the Xu Heping group of West Lake University published online research results entitled Coupled analysis of transcriptome and BCR mutations reveals role of OXPHOS in affinity maturation in Nature Immunology.
Based on the integrated analysis of antibody mutation patterns and transcriptome information, combined with genetic experiments, it revealed the key role of oxidative phosphorylation (OXPHOS) pathway in the positive screening of germinal center (Germinal Center, GC) B cell affinity.
Based on the classic NP-KLH immune mouse model, Xu Heping’s research group innovatively correlated antibody mutation patterns with transcriptome expression levels at the single-cell level to break through this research problem (Figure 1).
NP-KLH is a T cell dependent antigen.
It is known that when the germinal center response induced by it in a mouse model reaches its peak, GC B cells that specifically bind to hapten NP mainly express antibody heavy chain variable region genes Fragment VH186.
2; In addition, after the 33rd tryptophan of the amino acid sequence encoded by this gene was mutated to leucine, the affinity of BCR and NP increased to 10 times, while other random mutations were not significantly Change affinity.
The above two important immune response characteristics allow researchers to distinguish the binding affinity of NP based on the mutation of the BCR sequence.
On this basis, the use of 5'-end single-cell transcriptome analysis technology can simultaneously obtain the BCR sequence mutation pattern and transcriptome information of each B cell, so that the molecular mechanisms associated with affinity maturation can be fully explored and analyzed.
Figure 1: Integrating BCR mutation information and transcriptome data reveals the key molecular mechanism of antibody affinity maturation.
Based on transcriptome data and known gene expression characteristics, the authors first confirmed the spatial regions and cell cycles of different types of GC B cells.
status.
In order to explore the changes in the expression of cell gene modules more comprehensively, the authors introduced topic model algorithms in machine learning.
Compared with the traditional method of comparing the differentially expressed genes between cells of different groups, the topic model algorithm can not only find group-specific gene characteristics, but also find gene modules across groups, and achieve a more refined depiction of the cell state.
For example, in the past, some studies believed that the oxygen content in the clear area of the germinal center was low, and thus believed that GC B cells could only perform glycolysis.
The thematic model analysis in this work revealed and compared the metabolic characteristics of GC B cells in different regions.
It was found that cells in the bright area expressed higher levels of glycolysis pathway-related genes.
On the contrary, cells in the dark area expressed more OXPHOS (oxidation).
Phosphorylation) and fatty acid oxidation pathway genes indicate that GC B cells in different regions have different metabolic activities; to a certain extent, this shows that the previous conclusion that GC B cells only perform glycolysis is incorrect.
In the preparation for submission of this study, a newly published work through in vitro detection of primary GC B cell metabolic activity also shows that GC B cells mainly carry out OXPHOS rather than glycolysis.
More importantly, by combining the BCR mutation pattern information, the authors found that NP-specific GC B cell populations with high-affinity mutations (the VH186.
2 gene of the W33L mutation) have higher OXPHOS pathway gene expression.
It implies that this pathway may play an important role in the process of affinity maturation.
In addition, in the more complex polyclonal immune response model induced by OVA protein antigen, the authors not only further confirmed the positive correlation between OXPHOS and affinity forward screening, but also demonstrated that the research method developed in this work can be used for Analyze more complex features of B cell immune response.
In collaboration with Professor Harinder Singh of the University of Pittsburgh, the authors used genetic means to verify the role of the OXPHOS pathway in the body in the process of positive affinity screening.
The Cox10 gene encodes heme IX farnesyl transferase, which is an important part of the cytochrome c oxidase complex on the mitochondrial electron transport chain, and its deletion will lead to impaired OXPHOS activity.
Compared with control mice, the proportion of GC B cells, proliferation and antibody affinity of Cox10-deficient mice among GC B cells decreased significantly, proving that GC B cells rely on OXPHOS to achieve positive affinity screening.
Finally, by trying a variety of small molecules that have been reported to promote the activity of OXPHOS, the authors found that a small molecule called oltipraz can promote the forward screening of GC B cells and the maturation of antibody affinity in vivo.
This is the first report so far.
Small chemical molecules with similar functions.
The results of this study not only prove that OXPHOS promotes forward screening and affinity maturation by promoting the rapid division of high-affinity GC B cells, but also provides new ideas and targets for small molecules to intervene in the process of antibody affinity maturation and vaccine design.
In addition, using the analytical methods and procedures developed by the project, this research work has also successfully analyzed the correlation between the BCR response mutation pattern of complex antigens and the expression of molecular pathways, proving that this method can be widely used to analyze autoimmunity and transplant rejection immunity.
The diversity of BCRs and molecular response characteristics in other complex diseases. Researcher Xu Heping from West Lake University and Professor Harinder Singh from the University of Pittsburgh are the co-corresponding authors of the paper.
Doctoral students Chen Tianyu and Wang Yan from West Lake University and the postdoc Godhev K Manakkat Vijay from the University of Pittsburgh are the co-first authors of this paper.
In addition, research assistant Fu Shujie is the project's advance Made an important contribution.
Attached with the introduction and recruitment information of Xu Heping's research groupThe Systemic Immunology Laboratory of West Lake University is dedicated to exploring the key molecular mechanisms and networks that regulate the humoral immune response and mucosal inflammation.
Based on the organic integration of genetic animal models, clinical samples, and cutting-edge bioinformatics, it focuses on the study of the interaction mechanism of the immune and nervous system and other tissue-specific microenvironments in the inflammatory response and antibody response.
In recent years, many academic papers have been published in mainstream journals such as Nature (2 articles), Nature Immunology (2 articles) and Immunity (1 article).
With the support of the National Overseas High-level Talents Project, the National Natural Science Foundation of China's Joint Fund Key Projects, and the Ministry of Science and Technology Key R&D Program, various projects in the laboratory are progressing smoothly.
There is an urgent need for young talents to join the team to grow and develop together! There are many postdoctoral, doctoral and research assistant positions in the laboratory, welcome to contact us! Resume delivery (if interested, please send your resume and other materials to): https://jinshuju.
net/f/ZqXwZt or scan the QR code to send the original resume link: 021-00936-y Platemaker: Instructions for reprinting on the eleventh [Non-original article] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights and offenders must be investigated.
