High-throughput Sequencing Technology Detects The Effect Of Human Cytomegalovirus IE2 On Mouse T Cell Receptor Diversity

Object Human cytomegalovirus(HCMV)is the largest human herpesvirus that is usually acquired early in life and establishes a lifelong latent infection after the initial infection.Once a latent infection is established,the virus cannot be cleared from the host and can cause serious illness in immune-deficient individuals.IE2 is an important protein encoded by HCMV and plays a very important role in the replication and pathogenesis of viruses.As the most important immune cells,T lymphocytes have the ability to recognize the antigenic peptides via specific T cell receptor(TCR)on cell surface to activate the host's adaptive immune response.The diversity of T cell receptors determines the host's immune response and the prognosis of the several disease.Currently,HCMV has developed a variety of immune escape strategies,which enables the virus latently infected in the host.The purpose of this experiment was to investigate whether the T cell recognition receptor library and its diversity of the host have changed during HCMV latent infection.Due to the strict species specificity of HCMV,we have established the UL122 genetically modified mice which express IE2 protein stably and continuously.The establishment of this animal model broke the species specificity of HCMV,so we can studied the effect of IE2 protein on the TCR library of mouse immune system by in vivo experiments.Methods 1.The first generation of IE2 mice were obtained by microinjecting p AV.Ex1d-CMV-IE2-IRES/e GFP vector which contains the c DNA fragment of ie2 gene into fertilized eggs and embryo of C57/BL6 mouse.The expression of IE2 in transgenic mice was identified by PCR technique.After breeding the transgenic mice,we divided the next generation of mice into a positive group and a negative group by PCR.2.The expression of IE2 in the immune organs(spleen and lymph nodes)of the two groups was detected by immunohistochemistry,and the number of CD4+ T cells and CD8+ T cells in transgenic mice was detected by flow cytometry.3.High-throughput sequencing technology was used to detect and analyze the differences in V? and J? gene segments and diversity of T cell receptor which on surface of T lymphocyte in IE2 positive and negative mice.Results 1.The p AV.Ex Bi-CMV-IE2-IRES-e GFP expression vector was successfully constructed and four positive transgenic mice were obtained by microinjection and other techniques.The new generation of transgenic mice were obtained by crossing with wild type C57/BL6 mice.IE2 positive and negative mice were identified by extracting rat tail DNA and PCR for subsequent experiments.2.Immunohistochemistry results showed that IE2 protein displayed strong nuclear staining in the spleen and lymph nodes of IE2-positive transgenic mice,which proved that IE2 protein was widely expressed in host immune organs.There was no expression of IE2 protein in the spleen and lymph nodes of IE2-negative group.3.Flow cytometry results showed that the proportion of CD4+ and CD8+T cells in peripheral blood of transgenic mice was changed.Among them,the CD4+T cells of IE2-positive transgenic mouse remained at a lower level than the negative group,while the CD8+T lymphocytes maintained a higher level than the negative group.4.The results of high-throughput sequencing technology showed that the distribution ratio of TCR? chain was significantly lower in the positive group than in the negative group.There was no significant difference in the distribution of V?,J? gene and V-J combination between the two groups,but the proportion of unique CDR3 aa clonotypes in the positive group was lower.At the same time,the Gini index,the Simpson diversity index,the standardized Shannon diversity index and the Rank-abundance all showed that the positive transgenic mice had lower TCR diversity than the negative group.Conclusions 1.The IE2 transgenic mouse model which express IE86 protein stably and continuously was successfully constructed.2.The expression of IE2 protein affects the host immune organs and the development and distribution of peripheral blood T lymphocytes.3.Expression of IE2 causes changes in the host TCR library and reduces host TCR diversity.4.The low diversity of TCR library in transgenic model mice reveals that the expression of IE2 may lead to changes in cellular immunity and may be related to the body's antitumor immunity and viral escape.