Comparative Study On The Diversity Of T Cell Receptor α Chain Repertoire In Spleen And Intestine Of Zebrafish

T cells are the basis of the adaptive immune response of vertebrates.T cell receptor(TCR)is a characteristic molecule on the surface of all T cells and is expressed as a heterodimer,more than 95% are αβ type,and a few are γδ type,which mediates the cellular adaptive immune response of antigen.Each T cell expresses a unique TCR that specifically recognizes a single antigenic determinant,the adaptive immune system has a highly diverse TCR repertoire that ensures that T cells are more rapid,accurate,and effective in identifying and killing exogenous antigens.At the same time,it also directly reflects the immune response status of the body,that is,the diversity of antigen-specific TCR changes during the immune response.In this study,zebrafish was intraperitoneally injected with antigen to activate specific immunity,and the 5’RACE method was combined with the second generation high-throughput sequencing technology and platform to target the complementarity determining region(CDR3)of T cell receptors α chain(TCRA),revealing the T cell receptor alpha chain repertoire in the spleen and intestine of zebrafish.In order to gain a deeper understanding of the zebrafish TCRA repertoire diversity,we compared the TCRA repertoire between the normal and stimulated groups of the spleen,between the normal and stimulated groups of the intestine,between the normal groups of spleen and intestine,between the stimulated groups of the spleen and intestine.As a result,it was found that there were some differences in the use of different V/J genes and their combinations,CDR3 length distribution,richness and diversity,CDR3 sequence similarity and colinearity of the VJ gene and their combinations between samples.Comparative analysis of zebrafish spleen and intestine before and after stimulation found that the stimulation group produced more unique V genes than the normal group.After stimulation,the V and J genes and their combinations changed and the frequency of use increased and decreased to varying degrees.Some V and J genes are preferentially used for VJ pairing,and the phenomenon of VJ combination bias occurs,resulting in more new VJ combinations,but most of the frequencies are lower.It is speculated that these VJ combinations may play an important role in T cell receptors after antigen stimulation,that is,an antigen-specific binding immunoassay reaction process.This indicates that TCR produced a targeted rearrangement after antigen stimulation,and specific T cell clones appeared,and some T cells were selectively expressed.Some TCRAs are highly abundant due to antigen specificity.Amplification by a few unique clones and some public clones predominate.The TCRA richness and diversity in the stimulation group were higher than those in the normal group,indicating that the T cell response was significantly enhanced after antigen stimulation,and the TCRA CDR3 region of T cells was rearranged and showed high diversity.Comparative analysis of the spleen and intestine of the normal group and the spleen and intestine of the stimulation group revealed that the normal spleen had more unique V genes than the normal intestine;the richness and diversity of TCRA CDR3 in normal spleen is higher than that in normal intestine,and the diversity in stimulating spleen are also higher than that in stimulating intestine.This all indicates that the spleen as an important secondary lymphoid organ of zebrafish has more T lymphocytes than the intestine.In addition,the stimulated intestine has more unique V genes and more unique CDR3 sequences than the stimulated spleen.It is indicated that the diversity of TCRA repertoire in the intestine after stimulation was more obvious.Although there are no lymph nodes in the zebrafish intestine,the antigenic response of T cells can occur in the intestine,and the intestine may be the site of T cell production.It also shows that the zebrafish has a highly developed mucosal immune system,and the intestinal mucosal immune response plays an important role.In addition,the zebrafish TCRA repertoire has some similarities.There is a shared CDR3 region between each sample,and the V gene and VJ combination between samples have certain collinearity.This confirms that the actual expressed TCR repertoire is much smaller than the potential TCR repertoire.Antigen stimulation expands public clones in TCRA,and this shared CDR3 may play an important role in antigenic responses.In the similarity of samples,the similarity of V gene is higher,the similarity of VJ gene combination is lower,and there are some V gene and VJ combinations with different differences.This indicates that the V gene and the VJ gene increase TCRA diversity by various combinations of somatic cell rearrangements.At present,TCR studies are mostly concentrated on the individual level of human and mouse.In summary,This study used immunological repertoire sequencing to characterize and compare the TCRA repertoire diversity and changes in the spleen and intestine of the zebrafish at the tissue level.At present,there are no relevant reports at home and abroad.It provides new clues for the study of TCR diversity in fish and other species,helping to better understand the characteristics of the immune system and the etiology and progression of the disease,particularly the etiology and progression of unknown antigen triggers,it provides new ideas for disease prevention,diagnosis,treatment and prognosis monitoring.