Efficient antibody diversification by gene conversion in vivo in the absence of selection for V(D)J-encoded determinants

Self Cite

During embryonic development, the avian bursa of Fabricius selects B cell precursors that have undergone productive V(D)J recombination for expansion in oligoclonal follicles. During this expansion, Ig diversity is generated by gene conversion. We have used retroviral gene transfer in vivo to introduce surface Ig molecules that lack V(D)J-encoded determinants into B cell precursors. This truncated μ heavy chain supports both B cell expansion within embryo bursal lymphoid follicles and gene conversion. We show that individual follicles can be colonized exclusively by cells expressing the truncated μ chain and lacking endogenous surface IgM, ruling out a requirement for V(D)J-encoded determinants in the establishment of bursal lymphoid follicles. In striking contrast to their normal development in the embryo, bursal cells expressing the truncated μ-chain exhibit reduced rates of cell division and increased levels of apoptosis after hatching. The level of apoptosis in individual follicles reflects the proportion of cells within the follicle that express the truncated μ-chain. In particular, high levels of apoptosis are associated with follicles containing exclusively cells expressing the truncated μ receptor. Thus, apoptotic elimination of such cells is not due to competition within the follicle by cells expressing endogenous surface IgM receptors. This provides the first direct demonstration that the regulation of B cell development in the avian bursa after hatching differs fundamentally from that seen in the embryo. The requirement for intact IgM expression when the bursa is exposed to exogenous Ag implicates a role for Ag in avian B cell development after hatching.

Section: Perinatal Deletion Of B Cells Expressing Surface Ig Moleculementioning

confidence: 95%

Section: Perinatal Deletion Of B Cells Expressing Surface Ig Moleculementioning

confidence: 99%

Perinatal Deletion of B Cells Expressing Surface Ig Molecules That Lack V(D)J-Encoded Determinants in the Bursa of Fabricius Is Not Due to Intrafollicular Competition

Sayegh

Ratcliffe

2000

Self Cite

“…PCR products were electrophoresed, and bands were quantitated by scanning densitometry. Complementarity-determining region 3 length of VJ L sequences (23) and gene conversion (32,33) were assessed, as before. VJ L sequences were cloned into PCR2.1 (Invitrogen, San Diego, CA) and sequenced (York University, North York, Ontario, Canada).…”

Section: Pcr Amplification and Cloning Of V Gene Sequencesmentioning

confidence: 99%

The Cytoplasmic Domain of Igα Is Necessary and Sufficient to Support Efficient Early B Cell Development

Pike

Iacampo

Friedmann

et al. 2004

The B cell receptor complex (BcR) is essential for normal B lymphocyte function, and surface BcR expression is a crucial checkpoint in B cell development. However, functional requirements for chains of the BcR during development remain controversial. We have used retroviral gene transfer to introduce components of the BcR into chicken B cell precursors during embryonic development. A chimeric heterodimer, in which the cytoplasmic domains of chicken Igα and Igβ are expressed by fusion with the extracellular and transmembrane domains of murine CD8α and CD8β, respectively, targeted the cytoplasmic domains of the BcR to the cell surface in the absence of extracellular BcR domains. Expression of this chimeric heterodimer supported all early stages of embryo B cell development: bursal colonization, clonal expansion, and induction of repertoire diversification by gene conversion. Expression of the cytoplasmic domain of Igα, in the absence of the cytoplasmic domain of Igβ, was not only necessary, but sufficient to support B cell development as efficiently as the endogenous BcR. In contrast, expression of the cytoplasmic domain of Igβ in the absence of the cytoplasmic domain of Igα failed to support B cell development. The ability of the cytoplasmic domain of Igα to support early B cell development required a functional Igα immunoreceptor tyrosine-based activation motif. These results support a model in which expression of surface IgM following productive V(D)J recombination in developing B cell precursors serves to chaperone the cytoplasmic domain of Igα to the B cell surface, thereby initiating subsequent stages of development.

“…B cell precursors that have undergone successful Ig gene rearrangement, leading to functional BCR expression, migrate across the basement membrane and expand to form the bursal buds from which follicles develop (12,13). Strong evidence supports a model in which BCR expression in the absence of ligation is necessary and sufficient to support transition through this developmental checkpoint (14)(15)(16).…”

mentioning

confidence: 87%

Ligation of Surface Ig by Gut-Derived Antigen Positively Selects Chicken Bursal and Peripheral B Cells

Davani

Pancer

Ratcliffe

2014

In many mammals and birds, B cell lymphopoiesis takes place in GALT, such as the avian bursa of Fabricius. Although BCR expression is sufficient for bursal colonization, the role of BCR ligation in the later stages of bursal B cell lymphopoiesis remains elusive. To address this directly, we introduced a surface Ig–related construct with defined Ag specificity containing the Ag-binding portion of a lamprey variable lymphocyte receptor specific for PE fused to a truncated chicken μ-chain (VLRPETμ) into developing chick embryos. VLRPETμ expression supports bursal follicle colonization, clonal expansion, and Ig V gene diversification. VLRPETμ-expressing B cells migrate to the periphery in the absence of the Ag starting from day 18 of embryogenesis. VLRPETμ-expressing B cells declined rapidly in the bursa and periphery in the absence of Ag after hatch; however, intrabursal injection of PE prolonged survival of VLRPETμ+ bursal and peripheral B cells. Intrabursal introduction of Ag increased emigration of short-lived LT2+ B cells. Peripheral VLRPETμ+ B cells were maintained following intrabursal PE application and contained both short-lived LT2+ and long-lived LT2− B cells. In the chicken bursa, the later stages of B cell development occur in the presence of gut-derived Ag; therefore, we conclude that Ag-mediated ligation of BCR in bursal B cells acts to positively select bursal B cells into both short-lived and long-lived peripheral B cell populations.