Michael J. H. Ratcliffe

The chicken B‐cell receptor complex and its role in avian B‐cell development

Summary: The bursa of Fabricius is critical to normal B‐lymphocyte development in birds. During embryonic life, B‐cell precursors migrate to the bursal rudiment and those which have undergone productive V(D)J recombination colonize lymphoid follicles and undergo immunoglobulin V gene diversification by gene conversion. The chicken surface IgM complex appears structurally and functionally equivalent to its mammalian counterpart, with homologs to CD79a and CD79b. Expression of a truncated Ig chain is sufficient to drive the early stages of B‐cell development in the embryo bursa. Bursal cells expressing the truncated receptor complex proliferate in bursal follicles, and those which contain V gene rearrangements undergo V gene diversification by gene conversion. The bursa is a gut‐associated organ and antigen is focused to bursal lymphoid follicles after hatch. While expression of the truncated chain is sufficient to support B‐cell development in the embryo, B cells expressing this receptor are rapidly eliminated after hatch. We suggest the possibility that B‐cell development in the bursa after hatch is driven by encounter with antigen leading to redistribution of B cells within the lymphoid follicle, B‐cell proliferation and V gene repertoire development by gene conversion.

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

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.

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

Antibody diversification in the bursa of Fabricius occurs by gene conversion: pseudogene‐derived sequences replace homologous sequences in rearranged immunoglobulin genes. Bursal cells expressing a truncated immunoglobulin μ heavy chain, introduced by retroviral gene transfer, bypass normal requirements for endogenous surface immunoglobulin expression. Immunoglobulin light chain rearrangements in such cells undergo gene conversion under conditions where the products are not selected based on their ability to encode a functional protein. The efficiency with which gene conversion maintains a productive reading frame exceeds 97% under such non‐selective conditions. By analysis of donor pseudogene usage we demonstrate that bursal cell development is not driven by a restricted set of antigenic specificities. We further demonstrate that gene conversion can restore a productive reading frame to out‐of‐frame VJL junctions, providing a rationale for the elimination of cells containing non‐productive VJL rearrangements prior to the onset of gene conversion in normal bursal cell development.

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

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.

The avian B-cell receptor complex: distinct roles of Igα and Igβ in B-cell development

Summary:  The bursa of Fabricius has evolved in birds as a gut‐associated site of B‐cell lymphopoiesis that is segregated from the development of other hematopoietic lineages. Despite differences in the developmental progression of chicken as compared to murine B‐cell lymphopoiesis, cell‐surface immunoglobulin (sIg) expression has been conserved in birds as an essential checkpoint in B‐cell development. B‐cell precursors that express an sIg complex that includes the evolutionarily conserved Igα/β heterodimer colonize lymphoid follicles in the bursa, whereas B‐cell precursors that fail to express sIg due to non‐productive V(D)J recombination are eliminated. Productive retroviral gene transfer has allowed us to introduce chimeric receptor constructs into developing B‐cell precursors in vivo. Chimeric proteins comprising the extracellular and transmembrane regions of murine CD8α fused to the cytoplasmic domain of chicken Igα efficiently supported B‐cell development in precursors that lacked endogenous sIg expression. By contrast, expression of an equivalent chimeric receptor containing the cytoplasmic domain of Igβ actively inhibited B‐cell development. Consequently, the cytoplasmic domains of Igα and Igβ play functionally distinct roles in chicken B‐cell development.

B cell development in gut associated lymphoid tissues

B lymphocyte development can occur in a variety of anatomical sites. While typically considered to be a process that occurs in the bone marrow throughout life, it is becoming clear that gut associates sites of B cell development are critically important in many species of veterinary importance. Among these sites, the bursa of Fabricius in chickens and the ileal Peyers patches of sheep are among the best studied. In these organs, it has become clear that many of the properties associated with B cell development in rodent and primate bone marrow do not apply. Thus while bone marrow B cell development typically involves an ongoing maturation of mature B cells from immature B lineage precursors that lack the expression of a surface immunoglobulin complex, gut associated lymphoid tissues (GALTs) may be colonized by a single wave of precursor cells during embryo development. Nonetheless, molecular analysis of the requirements for B lymphocyte development in GALTs reveals some striking parallels with requirements identified for B cell development in bone marrow. This article will discuss differences between B cell development in the bone marrow and GALTs and recent evidence emerging that yields insights into how these processes are regulated.

Cell surface immunoglobulin receptors in B cell development.

Expression of surface immunoglobulin (sIg) related receptors has been conserved in phylogenetically distinct species as a critical checkpoint in B cell development. The sIg receptor comprises extracellular IgM heavy and light chains, with the potential for ligand binding, complexed to the Igalpha/Igbeta heterodimer that is responsible for signal transduction through sIg. Experimental systems, from both avian and murine models of B cell development, have been designed to identify the function of individual receptor components in B cell development. In this review, we assess the regulatory functions of different components of the sIg receptor complex during early development in experimental systems from evolutionarily distinct species.

4 – B CELLS, THE BURSA OF FABRICIUS AND THE GENERATION OF ANTIBODY REPERTOIRES

Birds have a unique organ for primary B lymphopoiesis, the bursa of Fabricius. Thus, avian B cell development can be classified into pre-bursal, bursal and post-bursal stages. During embryonic development, pre-bursal B cell precursors migrate in a single wave into the bursal mesenchyme; cells which express cell surface immunoglobulin as a consequence of productive gene rearrangement subsequently colonize the follicle anlagen. B cells then proliferate rapidly in bursal follicles and diversify their receptor repertoire by means of gene conversion. After hatch, the formerly homogenous follicles separate into two compartments: an outer cortex and a central medulla. Starting around the time of hatch, B cells emigrate from the bursa, predominantly from the follicle cortex, to colonize peripheral lymphoid tissues. In the periphery, naive B cells encounter antigen, undergo a germinal center reaction and differentiate into antibody-secreting plasma cells. Many aspects of these processes are regulated by cytokines. While the function of BAFF and CD40L on peripheral B cells is largely conserved between birds and mammals, both of these TNF family members play unique roles in the bursa of Fabricius.

Biological roles of the major capsid proteins and relationships between the two existing serotypes of infectious bursal disease virus

SummaryNeutralizing monoclonal antibodies (n-MAbs) were produced against infectious bursal disease virus (IBDV) of serotypes 1 and 2. The n-MAbs recognizing the major antigenic proteins VP2 and VP3, were characterized using different strains of IBDV representing the existing two serotypes and a variant subtype of serotype 1. The biological properties of these viral antigens as defined by the MAbs in vitro, were studied utilizing post-adsorption virus neutralization tests and fluorescence-activated cell sorter analysis. The MAbs directed against the immunodominant epitopes on VP2 were capable of enhanced virus neutralization but did not inhibit the virus attachment to susceptible cells. These MAbs were able to neutralize the virus by interfering with an event subsequent to virus adsorption, possibly inhibiting virus penetration or uncoating. On the contrary, a MAb that immunoprecipitated the other capsid protein VP3 was able to prevent virus attachment although it possessed lower neutralization titers. Cross-immunoprecipitations of various virus strains by these MAbs and antisera revealed interrelationships between the two serotypes of IBDV.

BLNK Binds Active H-Ras to Promote B Cell Receptor-mediated Capping and ERK Activation

Cross-linked B cell receptor (BCR) aggregates on the cell surface, then assembles into the “cap” where Ras is co-localized, and transduces various intracellular signals including Ras-ERK activation. BCR signals induce proliferation, differentiation, or apoptosis of B cells depending on their maturational stage. The adaptor protein BLNK binds various signaling proteins and Igα, a signaling subunit of the BCR complex, and plays an important role in the BCR signal transduction. BLNK was shown to be required for activation of ERK, but not of Ras, after BCR cross-linking, raising a question how BLNK facilitates ERK activation. Here we demonstrate that BLNK binds the active form of H-Ras, and their binding is facilitated by BCR cross-linking. We have identified a 10-amino acid Ras-binding domain within BLNK that is necessary for restoration of BCR-mediated ERK activation in BLNK-deficient B cells and for anti-apoptotic signaling. The Ras-binding domain fused with a CD8α-Igα chimeric receptor could induce prolonged ERK phosphorylation, transcriptional activation of Elk1, as well as the capping of the receptor in BLNK-deficient B cells. These results indicate that BLNK recruits active H-Ras to the BCR complex, which is essential for sustained surface expression of BCR in the form of the cap and for the signal leading to functional ERK activation.