As the age of mice increases, the thymus and spleen T cells TCR CDR3 repertoire will change [22, 23], at the same time, the number and response capacity of central and peripheral B cells will change accordingly. The homogeneity and heterogeneity of the composition and characteristics of the central and peripheral B cells in younger/middle/old-aged mice is an important basis for the study of the responsive ability and mechanism of the aging immune system. In this experiment, 3-, 12-, and 20-month-old mice were used as subjects, and HTS was used to compare and analyze the homogeneity and heterogeneity of the productive, pseudogene, and out-of-frame sequences of the BCR H-CDR3 repertoire in bone marrow (central) and spleen (peripheral).
The study found that the white pulp of the spleen was more regular at 3 months of age, the density of lymphocytes was larger, and the shape of the shifting area is obvious. With the increase in the age of the mice, the structure of the white pulp became irregular at the ages of 12 months and 20 months. Some of them are relatively loose, with only a small number of lymphocytes, and the shape of the area became irregular. The white pulp structure is typically associated with antigenic stimulation; as the mouse age increased, the mice received more antigenic stimulation, resulting in an irregular white pulp structure in aged mice, which is consistent with previous literature reports[24].
The diversity of the BCR H-CDR3 repertoire is derived from the V(D)J gene rearrangement, insertion, deletion, and high-frequency mutations in somatic cells. As mice age, changes in BCR H-CDR3 repertoire diversity can be demonstrated by random combinations of light and heavy chains[25]. The frequency of the V, D, and J genes was related to the advantages of naive rearrangement, B cell self-tolerance selection, B cell clonal proliferation, immune response, etc. In theory, the frequency of the usage of the V, D, and J genes reflects the key features of the CDR3 recognition of specific antigens. This study found a high frequency of the usage of IGHV1-4, IGHV14-1, IGHV1-50, and IGHV1-64 in the productive and out-of-frame sequences of bone marrow B cells, spleen B cells and spleen memory B cells at different ages. In the pseudogene sequences, the frequency of IGHV1-67 and IGHV1-83 was significantly higher than that of other genes; IGHV1 had high-frequency usage at different ages and in different tissues, showing homogeneity, which is consistent with other researchers' reports[8]. This finding suggests that in the naive rearrangement, there is a significant advantage of some V gene in the naive rearrangement, resulting in its high frequency in the self-tolerance selection and peripheral response BCR repertoire. In the productive sequences, mouse bone marrow B cell IGHV1-54 and IGHV1-84; spleen B cell IGHV4-1, IGHV1-87, IGHV5-12, and IGHV9-1; and spleen memory B cell IGHV1-4, IGHV5-12-1, and IGHV5-9 usage significantly changed with age. Significant changes in gene usage may be related to B cell tolerance, clonal proliferation, and the frequency of the immune response and further indicate that the BCR H-CDR3 repertoire is partially heterogeneous at different ages and in different tissues. In the analysis of the frequency of IGHV gene family use, it was found that some IGHV gene families did not show regular changes with the increase of months (3 months, 12 months, and 20 months). This may require a more detailed analysis of the B cell subsets sorted in the bone marrow and spleen. The experiment needs to further clarify the characteristics of the naïve B cells and memory B cells (and different types of memory B and subsets) CDR3 repertoire in different parts of the bone marrow or spleen (Such as IGHV gene family utilization frequency) changes dynamically with the age of the month.
A pseudogene sequence is produced by the deletion of the initiation codon and the premature introduction of a termination codon[9]. A pseudogene sequence also participates in rearrangement. Because of the inefficiency of rearrangement, the pseudogene sequence better reflects the frequency characteristics of the V, D and J genes in the naive rearrangement, and, together with the out-of-frame rearrangement of functional genes, it can be used to compare and analyze the composition and specificity of the productive rearrangement of functional genes[26].
The pseudogene sequence showed significant differences in bone marrow B cell IGHV1-25, IGHV1-83, IGHD2-3 and IGHD2-4 with age and significant differences in spleen B cell IGHV1-32, IGHV1-67, IGHD3-1, IGHD3-3, and IGHD5-1 genes. Compared with the productive sequences, there were differences in V and D gene usage, suggesting that the pseudogene sequence better reflects naive rearrangement. A out-of-frame sequence is the result of non-productive rearrangementwe.When we analyzed the V genes usage, we found that the out-of-frame sequence and the Productive sequence have many similarities, mainly because the out-of-frame sequence sequence is a translational reading frame change caused by insertion and deletion of nucleotides during gene rearrangement and cannot be translated into functionality AA. Therefore, we believe that the difference in the out-of-frame sequence in the bone marrow tissue with age changes is mainly affected by gene rearrangement, or it may be the cause of individual differences. Therefore, we believe that the difference in the age of the out-of-frame sequence in the central and peripheral tissues is mainly affected by gene rearrangement, or it may be affected by the antigen response in the periphery.The frequency of the acquisition of the V, D, and J genes in mice was almost the same, and there were some differences with changes in mouse age, which is consistent with the study of the V, D, and J genes in the peripheral blood of newborns and adults[27].
The closer the cluster analysis is, the more similar the gene access is. The cluster analysis of V-J pairing in spleen B cells and memory B cells shows that the clustering distances of 12 months and 20 months are the closest, while the cluster distance of 3 months is more. Indicating that the 12-month-old and 20-month-old genes are more similar, suggesting that it may be similar to the longer-term in vitro antigen stimulation, and B cells produce corresponding responses and clonal proliferation.The clustering distance of pseudogene sequences was closer at 3 months and 12 months than at 20 months in the bone marrow and spleen B cells. In the out-of-frame sequences in bone marrow B cells and spleen memory B cells at the ages of 3 months and 12 months, the clustering distance was closer than that at 20 months. It is suggested that more V and J gene naïve rearrangements have higher advantages and matching. The specific sources and mechanisms of various V-J pairing B cell populations need to explore its regular changes in experiments with more mouse-month-old groups.
For the insertions and deletions of the BCR H-CDR repertoire, in the productive, pseudogene, and out-of-frame sequences, the bone marrow B cells had significant differences in 5'J trimming with age and may be mainly pro-B cells and pre-B cells.In the pro-B cell repertoire, the rearrangement is generally random, but there is a certain difference in the involvement of the J gene in functional and nonfunctional rearrangements. Therefore, in the next experiment, it is necessary to sort the pro-B cells and pre-B cells, naïve B cells and memory B cells in the bone marrow of mice of different months, and compare and analyze their characteristics in mice of different months. To explore the mechanism and significance of changes in central B cell rearrangement, selection and differentiation in the process of mice increasing with age. There were significant differences in spleen B cell and memory B cell N1 insertion, N2 insertion, P5'D insertion, and 5'D trimming, suggesting that the BCR H-CDR3 repertoire and B cell self-tolerance selection, immune response, and clonal proliferation are related. Due to the response of multiple autoantibodies and external antigens, the CDR3 repertoire participates in this process in addition to the naive rearrangement, high-frequency mutations of somatic cells and the secondary rearrangement of the BCR, resulting in N1 insertion, N2 insertion, P5'D insertion, and 5'D trimming differences. Therefore, in the peripheral immune organs, it is more complicated to explore the mechanism and significance of insertion and deletion of the CDR3 repertoire of different subsets of B cells in the process of changing with the age of mice. It needs to be carried out on the basis of mouse disease models of different months.
The diversity of the BCR H-CDR3 repertoire in different ages of mice showed that the diversity in bone marrow B cells and spleen memory B cells of 3-month-old mice was higher than that of 12- and 20-month old mice. In spleen B cells, the diversity of the 3-month-old mouse repertoire was significantly higher than that of the 12- and 20-month old mouse repertoire. The diversity of the BCR H-CDR3 repertoire is closely related to gene rearrangement and the immune response generated by external antigen stimulation. The diversity of the repertoire in the bone marrow is mainly due to the rearrangement/insertion/deletion of the V, D, and J genes[28]. As the mouse age increases, the B cell output from the bone marrow to the periphery decreases, and the diversity decreases[5]. The findings may have occurred in the bone marrow, and this reduction may also reflect changes in the subset[20, 25].
The response of spleen B cells to external antigen stimulation, as the body ages to increase the body's depletion of B cells, also leads to a decrease in B cell diversity, and B cell clonal proliferation changes with increasing age. Not only are the unique CDR3 sequence species reduced, but the clonal proliferation that occurs is also greater. This phenomenon also indicates that the clonal distribution caused by the steady-state proliferation and peripheral selection in the aging process is more unbalanced. Related studies indicate that the imbalance of clonal distribution can reflect the response ability and the peripheral selection of self-identification[29]. he changes in the diversity of the central and peripheral BCR H-CDR3 repertoire in mice at 3, 12, and 20 months of age suggest that they are due to both central and peripheral causes, providing further insight into immune system aging and results from diversity studies, basic data and new research techniques.
The AA composition of the BCR H-CDR3 region was found to be high-frequency tyrosine in the B cells of different ages and tissue-derived B cells, consistent with the literature[30]. The use of isoleucine (I) in the spleen B cell repertoire was significantly higher at 12 months than at 3 months and 20 months, and the use of asparagine (N) was significantly lower at 20 months than at 12 months. In the spleen memory B cell repertoire, the frequency of asparagine (N) was significantly lower at 3 months and 20 months than at 12 months. It is suggested that the difference in AA in the peripheral BCR repertoire of mice at different ages may be related to the immune response generated by specific antigen stimulation.
The length distribution analysis of CDR3 revealed that the length of the bone marrow B cells, spleen B cells and spleen memory B cells of mice at different ages showed a Gaussian distribution with 11-12 AA residues. This is consistent with the result previously reported for mouse CDR3, which was an average length of 11.5±1.9 AA residues[17]. Compared with 3 months of age, the CDR3 length distribution in the BCR H-CDR3 repertoire of bone marrow B cells, spleen B cells, and spleen memory B cells shifted to the left at 12 months and shifted to the right at 20 months, suggesting that with the increase in age, recognition antigen-producing responses, mutations, and clonal proliferation have a tendency to become longer and may be related to the antigens that are exposed[20]. Pickman Y., et al. also found healthy elderly people's BCR CDR3 length distributions can be distinguished from those of the young[31].
The overlap rate analysis of the B cell CDR3 repertoire is closely related to VDJ gene recombination selection, self-tolerance selection, and environmental immune response. This study found that the overlap ratio of the B cell CDR3 repertoire of mice of different months is higher (Fig. 6 and Tab. 2). The specific mechanism needs to be further explored, but through comparative analysis, it is found that the proportion of overlap in the bone marrow B cells and spleen B cells of mice at different ages was lower at 3 months and higher at 12 months and 20 months. The overlap of unique amino acids at different ages reflects not only clonal proliferation but also the presence of new, effective, unique sequences. Our results showed that the overlap rate of bone marrow and spleen B cells at 3 months was lower, and the overlap rate at 12 and 20 months was higher, which suggests that clonal proliferation increases with age. Aranburu A., et al. found age-associated B cells (ABCs) in autoimmune mice are comprised of autoreactive MBCs expressing highly restricted H-CDR3 repertoires[32]. In our study, the spleen memory B cells have no obvious regularity, which is mainly affected by the stimulation of the external antigen response.
Although this study found that the CDR3 repertoire of bone marrow and spleen B cells and spleen memory B cells in mice have multiple heterogeneity and other characteristics with the change of months, However, it is necessary to further clarify the relationship between naïve B cells (CD19+IgD+CD27-) and memory B cells (CD45R+CD27+) in different parts of the bone marrow or spleen,and even the memory B cells (CD45R+CD27+) in the bone marrow and spleen need to be further sorting by the method, conduct research on the CDR3 repertoire, such as comparing the CDR3 repertoire of T-bet- and T-bet+ memory B cells in the spleen germinal center or circulation with age[33]. Exploring the characteristics of B cell BCR in mice of different months of age can provide a basis for clinical disease mechanism research. For example, Zhang J., et al. found that B cell BCR activation is different in similar diseases of different ages, adult AML samples have significantly higher level of B cell activation and more secondary Ig class switch events than pediatric AML or non-tumor samples[34, 35].