Laboratory of B Cell Development and Differentiation

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Research in the Laboratory of B Cell Development and Differentiation is focused on the humoral immune response. The laboratory director, Garnett Kelsoe, is a member of Duke’s Autoimmunity Center of Excellence, the Haynes’ Collaboration for AIDS Vaccine Discovery, and CHAVI ID Scientific Leadership Group. In addition, the laboratory is closely allied with Duke’s Center for the Study of Aging and Human Development, the Comprehensive Cancer Center, and the Center for AIDS Research.  The laboratory staff, Dongmei Liao, Yi Li, Kristen Hopper, Xiaoe Liang, Michael Cunningham, Sergio Sanders, and Deborah Feyes, provide exceptional research and administrative expertise.

Kelsoe and his collaborators have long been interested in the population genetics and dynamics of lymphocyte populations and especially the generation and selection of B cells and their receptors. Research topics addressed by Kelsoe and his collaborators include, the rules of V(D)J recombination, identification and function of physiologic and cryptic recombination signals, germinal center responses and the affinity maturation of antibody, the role of complement in B-cell activation by foreign and self-antigens, humoral immunity to microbial pathogens, inflammation’s effects on hematopoiesis, and regulated AID expression during B-cell development.

Regulated expression of Aicda during B-cell development (Masayuki Kuraoka, Ph.D., Pilar Snowden, Alex Reynolds). These studies are carried out in association with the Duke Autoimmune Center for Excellence (T.F. Tedder and E.W. St. Clair) and the Department of Immunology (S. Unniraman).  Our research is aimed to understand better the regulation of B lymphocyte development, and especially repertoire diversification and immune tolerance.  Recently we demonstrated that the Aicda gene (activation-induced cytidine deaminase) is expressed in developmentally immature mouse and human B cells and found that the Aicda gene product, the AID mutator, plays a significant role in establishing self-tolerance in these B-cell compartments.  Remarkably, this tolerizing function of AID in early B-cells is observed in both mice and humans.  These observations identify a novel role for AID and a new pathway for central B-cell tolerance that may be an important role in autoimmune disease and responses to certain microbial pathogens, including HIV-1.

Induction versus selection in immunological tolerance (Masayuki Kuraoka, Ph.D.).  This work is carried out in collaboration with another member of the DHVI (Thomas B. Kepler).  During their development and maturation in the bone marrow, B cells that recognize self-antigens are removed by physiologic tolerance mechanisms.  Two of these, deletion by apoptosis and inactivation by anergy act at the level of cells, whereas the third tolerance pathway, receptor editing, is thought to induce genetic changes in autoreactive receptors. Determining, however, whether receptor editing is inductive or selective is not simple and the answer profoundly affects our understanding of how immunological tolerance operates. This research group has used novel molecular and mathematical approaches to distinguish inductive vs. selective tolerization of B cells. The results of these studies support models of tolerizing selection and are inconsistent with inductive models.

The role of self-tolerance in shaping humoral responses to HIV-1 (Guang Yang, Laurie McWilliams, M.D., Kuei‑Ying Su M.D., Takuya Nojima, Ph.D., Pilar Snowden, Masayuki Kuraoka, Ph.D.). This work is carried out in association with other members of the DHVI (L. Verkoczy, L. Liao, M.A. Moody, S.M. Alam and B.F. Haynes). Our studies test the hypothesis that certain broadly neutralizing antibodies (BnAb) to HIV-1 are purged from the mouse and human B-cell pools during development because of their cross-reactivity to self-antigens.  To this end, we have demonstrated the loss of potential BnAb precursors at well-known tolerance check-points in transgenic and normal mice.  We have identified candidate self-antigens that cross-react with HIV-1 epitopes and developed a novel culture system that supports B‑cell development and maturation in the absence of the tolerizing influence of bone marrow. These cultures allow the creation of chimeric mice that produce high levels of IgM and IgG autoantibody and respond significantly better to MPER peptide antigens than controls.

B-cell lymphopoiesis during human development (Laurie McWilliams, M.D., Kuei-ying Su, M.D., Takuya Nojima, Ph.D.). This research is carried out in association with another member of the DHVI (M.A. Moody and B.F. Haynes).  The human B lymphopoiesis research group characterizes human B-cell development in tissues that represent distinct stages of human development (fetal liver, cord blood, young and old adult bone marrow) in order to delineate the origin(s) of CD27+ B cells. Our work shows that the earliest CD27+ B cell populations develop in the fetal liver with CD27 expressed on membrane Ig- cells that are phenotypically identical to pre- and pro-B cells. This lineage is largely restricted to fetal development, as only very low frequencies of CD27+ pro-B and pre-B cells are present in adult bone marrow.  We hypothesize that these early CD27+ B cells represent precursors to human B1 cell lineage based on their surface expression of CD43, CD27and low frequencies of somatic hypermutation.

Regulation of hematopoiesis by inflammation (Derek W. Cain, Ph.D., Alex Reynolds, Pilar Snowden, Guang Yang). This research group has demonstrated that inflammatory signals alter hematopoietic output to promote myelopoiesis over lymphopoiesis.  This research team is now dissecting the extracellular cues and transcriptional pathways that mediate the “emergency” production of granulocytes during acute infection.  In addition, we recently uncovered a transcriptional pathway that directs the terminal differentiation of certain tissue-resident macrophages; mice deficient in this pathway lack mature macrophages in the peritoneal cavity, but the resident macrophage populations of spleen, lymph node, and liver are intact.  Remarkably, the presence of peritoneal macrophages inhibits the maturation of macrophage precursors, suggesting that tissue pools of macrophages are governed by the availability of growth/differentiation resources.  Future studies will include the identification of factors that regulate tissue macrophage “niches.”

Understanding the natural antigenicity of native and haptenated protein antigens (Pilar Snowden, Masayuki Kuraoka, Ph.D., Derek Cain, Ph.D.). Haptenated proteins have long served as model antigens in basic immunological research, but recent studies on the contribution of Toll-like receptor (TLR) signaling to antibody responses have concluded that the antigenic properties of native- and haptenated protein antigens are fundamentally different: strong responses to native proteins requires TLR signaling whereas responses to hapten-protein conjugates do not.  The suggestion that haptenation qualitatively changes humoral immune responses calls into question foundational principles of immunology.  We have, therefore, used standard methods to immunize C57BL/6 mice with native- or haptenated protein in alum and determined the kinetics and magnitude both protein- and hapten-specific serum antibody responses, the germinal center (GC) response, Ig VH gene segment hypermutation and affinity maturation, and humoral memory in cohorts of congenic animals sufficient or deficient in the TLR signaling adaptor, MyD88.  In our hands, both native- and haptenated proteins elicit comparable serum antibody, GC, and memory responses regardless of the presence or absence of MyD88 signaling.

Regulation of plasmacyte maintenance and selection in the bone marrow (Derek Cain, Ph.D., Alex Reynolds). B lymphocytes differentiate into memory B cells or antibody secreting plasmacytes (PCs) upon antigen encounter. PCs migrate to the bone marrow where they are retained in a microenvironment essential for their survival. This research group is investigating the molecular signals that determine the threshold number of PCs that can inhabit the bone marrow. Following cessation of the germinal center reaction, antigen-specific PCs continue to undergo affinity maturation in the bone marrow. The mechanisms responsible for this phenomenon are also being researched. The results of these studies will provide insight into the development and maintenance of long-lived high-affinity antibody secreting cells in the bone marrow; both those that produce protective antibody, and those that form self-reactive antibody.