Transitional B Cells in Early Human B Cell Development – Time to Revisit the Paradigm?

Abstract

The B cell repertoire is generated in the adult bone marrow by an ordered series of gene rearrangement processes that result in massive diversity of immunoglobulin (Ig) genes and consequently an equally large number of potential specificities for antigen. As the process is essentially random, the cells exhibiting excess reactivity with self-antigens are generated and need to be removed from the repertoire before the cells are fully mature. Some of the cells are deleted, and some will undergo receptor editing to see if changing the light chain can rescue an autoreactive antibody. As a consequence, the binding properties of the B cell receptor are changed as development progresses through pre-B ≫ immature ≫ transitional ≫ naïve phenotypes. Using long-read, high-throughput, sequencing we have produced a unique set of sequences from these four cell types in human bone marrow and matched peripheral blood, and our results describe the effects of tolerance selection on the B cell repertoire at the Ig gene level. Most strong effects of selection are seen within the heavy chain repertoire and can be seen both in gene usage and in CDRH3 characteristics. Age-related changes are small, and only the size of the CDRH3 shows constant and significant change in these data. The paucity of significant changes in either kappa or lambda light chain repertoires implies that either the heavy chain has more influence over autoreactivity than light chain and/or that switching between kappa and lambda light chains, as opposed to switching within the light chain loci, may effect a more successful autoreactive rescue by receptor editing. Our results show that the transitional cell population contains cells other than those that are part of the pre-B ≫ immature ≫ transitional ≫ naïve development pathway, since the population often shows a repertoire that is outside the trajectory of gene loss/gain between pre-B and naïve stages.

Immunoglobulin gene analysis as a tool for investigating human immune responses

“…83 We have also shown repertoire differences as B cells progress through bone marrow development and central tolerance. 84 These studies all serve to reinforce the view that repertoire studies should be conducted on sorted cells, be class and subclassspecific and the subjects should be age matched as well as possible.…”

“…We have used a levenstein distance, as opposed to a hamming distance, to build hierarchical clustering dendrograms in order to reduce error introduced by sequence indel errors. 84 This is important where HTS sequencing platforms are prone to homopolymer tract errors as CDR-H3 regions often have larger homopolymer tracts. We use an empirically determined cut off value to split the sequences into clonal groups which errs on the side of inclusivity.…”

“…149 During early B cell development of IgM, between preB cells to Naïve B cells, there is also a significant decrease in CDR-H3 size. 84,150 The size of CDR3 is determined partially by IGH gene use, and partially by factors involved in gene rearrangement at the junctions-particularly the activity of Terminal deoxynucleotidyl transferase (TdT) adding random nucleotides to the junction. There may be interindividual variation in TdT activity since, in young adults, the distribution of CDR3 size at baseline and day 28…”

“…151 Similarly, the level of N nucleotide addition in early B cell development is consistent between heavy, kappa and lambda chains within individuals, but differs between individuals. 152 Given the apparent importance of CDR3 size to an antibody response 82,149 and to central tolerance 84,150 these interindividual differences may warrant closer inspection in studies on immune disease, vaccination and infection as they may be biomarkers of response or autoimmunity.…”

“…147 We found that there is some selection against the use of cysteines in central tolerance; the percentage of sequences without any cysteines increases from 85% to 91% between preB and naïve B cells. Although it is difficult to infer an antibody's specificity based on its amino acid sequence, it has been observed that the CDR-H3 regions of antibodies in the bone marrow are on average longer, and more hydrophobic than those in the peripheral blood 84,1151,152 , indicating that these CDR-H3 characteristics are selected against during central tolerance. The charge at the binding site is also critical, the prevalence of positively charged arginines in the CDR3 has been associated with binding to (negatively charged) DNA in some antibodies and in SLE 153,154 and to phospholipid antigens.…”