Lineage tree analysis of high throughput immunoglobulin sequencing clarifies B cell maturation pathways

Transitional (TR) B cells are immature B cells that have migrated from the bone marrow to peripheral lymphoid organs, but can still undergo selection against autoreactivity. TR cells that survive selection eventually develop into mature naïve B cells (CD27-IgD+, NA). Upon exposure to antigen, NA cells may become IgM memory (CD27+IgD+, MM) or "classical", classswitched memory (CD27+IgD-, SM) B cells. Although MM immunoglobulin (Ig) genes do not undergo class switching, they do undergo somatic hypermutation, albeit with lower frequency than SM. It has been postulated that MM B cells originate from T-independent immune responses, while SM cells originate from T-dependent responses. Alternatively, MM cells may be early emigrants from T-dependent germinal centers. Double negative B cells (CD27-IgD-, DN) have been said to be exhausted memory cells, but their precise origin is unclear. Therefore, a definitive elucidation of lineage relationships between these different B cell subsets is needed. In this study, we used lineage tree analysis of Ig heavy chain gene sequences from five human B cell subsets (TR, NA, MM, SM and DN) from three individuals, to study the relationships between these B cell populations and garner insights regarding their roles in immune responses. Our analyses confirmed that both MM and SM branches can include DN Ig sequences, sometimes identical to SM Ig sequences. MM trees were significantly shorter than SM trees. Even when they belonged to the same clone, MM branches were shorter, consistent with either an early exit of MM cells from germinal centers in a T-dependent response, before accumulating many mutations, or their generation by T-independent responses. Our finding of combined trees that included both MM and SM sequences suggests that at least some MM cells originate from the same clones as SM, rather than develop separately.