Research

B-1 cells were originally identified as CD5+ B cells. Making up for approximately 5% of total B cells in mouse, this small B cell subset is found to differ from conventional B cell, namely B-2 cells in many aspects. They arise earlier during ontogeny (fetal and neonatal stage) and maintain as a self-replenishing thereafter. They can be readily detected in a variety of tissues including spleen, peritoneal cavity (PerC), pleural cavity and intestines, and establish themselves as a non-redundant subset in immune system.

Functionally, B-1 cells are well known to be the major producers of antibodies that circulate in serum of normal animal in the absence of exogenous antigenic stimulation. These pre-exiting antibodies, called natural antibodies, are found to be critical for the survival of animal against pathogen invasion. Not only conferring the protection via secreting natural antibodies in a relatively passive manner, B-1 cells also actively participate in immune defense by initiating antibody response upon pathogen invasion. These responses can be induced by stimulation with innate-related molecules that are conserved among pathogens through engaging mitogenic receptor, e.g., Toll like receptors (TLRs) regardless of antigen specificities. In addition, earlier studies with response to phosphorylcholine (PC), a major epitope presented on a variety of S. pneumoniae strains, show that T15 idotype (T15id) PC-specific CD5+ B-1 clone is responsible for up to 90% of primary anti-PC response, arguing that CD5+ B-1 cells can be activated via BCR in antigen-specific manner. Nevertheless, B-1 mediated responses usually do not require T cell help and rarely undergo somatic mutation.

Despite the protective role, B-1 cells are also implicated in variety of diseases of B cell dysfunction, including leukemia and autoimmune disease. In essence, mechanisms that shape their repertoire, govern their generation and control their responses are critical for the survival of animal and are the fundamental issues for immunologist/biologists to understand.

My research with murine B-1 cells are brunched into two major directions: one is focused on their function, i.e., how they are mobilized to initiate the antibody response following pathogen invasion and how the response is regulated, as modeled by the Salmonella typhimurium LPS (Ty-LPS) and by a unique LPS from Francisella tularensis (Ft-LPS) that does not engage TLR; another direction aims to explore the molecular events that govern B-1 development during fetal and neonatal life, with particular attention towards elucidating the rules or pathways that distinguish B-1 generation occurred at these two stages. The recent findings (published and unpublished) and specific aims are summed up below. 

1. Ty-LPS stimulation triggered B-1 migration, cell division and plasma cell differentiation.

My studies with in vivo B-1 response to stimulation with Ty-LPS, a potent TLR4 ligand, surprisingly reveal a complexed B-1 cell migration and plasma cell differentiation events: an immediate response produced by resident splenic B-1a cells, which differentiate into plasma cells within 1-2 days without undergoing cell division and the immigration of B-1 cells from PerC to spleen, where they divide at least once before/during they differentiate to plasma cells. Based on these findings, studies are proposed to determine the Ty-LPS stimulated mechanisms that trigger the migration of B-1 cells from PerC to spleen and mechanisms that concomitantly trigger the splenic resident B-1 cells directly differentiate to plasma cells without undergoing cell division.

Relate publication:

Yang Yang, Tung, JW, Ghosn, EB, Herzenberg, LA and Herzenberg LA. “ Division and differentiation of natural antibody-producing cells in mouse spleen”. PNAS, 104: 4542-6, 2007

2. Antigen-specific B-1a antibody response and unique B-1 memory induced by immunization of mice with Ft-LPS, an unusual LPS that does not engage TLR2 and TLR4.

By collaboration with Dr. Stephanie Vogel lab (U. Maryland), who investigates the pathogenesis of F. tularensis infection and vaccine study in a mouse model of human tularemia, my studies with immunization of mice with Ft-LPS, which does not engage TLR, have found that Ft-LPS immunization activates B-1a cells with BCR recognizing Ft-LPS to initiate a robust T-independent antigen-specific response. Importantly, this antigen-specific antibody response provides long-term protection against lethal Ft live vaccine strain (LVS) infection.

Of note, Ft-LPS immunization also results in development of Ft-LPS specific B-1 cells that persist in PerC and manifest features resembling typical memory B cells. Boosting with the identical antigen used for priming, however, fails to generate memory response. Furthermore, a regulatory mechanism induced by Ft-LPS immunization is found to inhibit secondary in situ antibody response by these Ft-LPS specific memory B-1 cells. Preliminary data suggests that activation of Ft-LPS specific memory B-1 cells is regulated by mechanism accessible to intact organisms.

Since B-1 cells are rarely recognized to be capable of generating memory B cells and producing B cell memory, both of which are thought to be restricted, if not exclusively, to T-dependent antibody response, these findings surprisingly uncover a distinct type of memory B cells that are derived from B-1 cells and display with unique properties with respect to anatomic location and function quality. Ongoing work is focusing on discovering the feedback regulatory mechanism induced by Ft-LPS priming as well as mechanisms that enable intact F. tularensis to activate Ft-LPS+ B-1 memory cells.

Relate publication:

Cole, LE, Yang Yang (co-first author), Elkins, KL, Fernandez, ET, Qureshi N, Shlomchik, MJ, Herzenberg, LA, Herzenberg LA and Vogel SN. “Antigen-specific B-1a antibodies induced by Francisella tularensis LPS provide long-term protection against F.tularensis LVS challenge ”. PNAS, 106: 4343-8, 2009.

Yang Yang, Cole, LE, Ghosn, EB, Herzenberg, LA and Herzenberg LA. “ Novel property of B-1 memory”.  In preparation.

3. Distinguish B-1 lymphopoiesis and B-1 repertoire during fetal stage from their generation in neonatal life.

Earlier adoptive cell transfer studies demonstrate that B-1 cells are readily developed from progenitor in fetal/neonatal life but are limitedly reconstituted from adult bone marrow (BM). Terminal deoxynucleotidyl transferase (TdT),  the enzyme that adds non(N)-templated nucleotides at VDJ genes junctions during BCR recombination thus further diversify the BCR repertoire is not expressed in fetus, which marks the major difference for B cell development in fetus from B cell development in adult BM. Consistently, sequence studies with hybridoma cell lines with B-1 origin found that their repertoires are highly restricted and lack N-insertion between V-D and D-J junction. However, previous work in Herzenberg lab, in which FACS sorted single B-1 cell was sequenced to study B-1 repertoire in less-biased way, reveals a much more diversified pattern: only 1/3 B-1a cells lack N-insertion whereas about 2/3 B-1a cells have N-insertion at one or both junctions with the number of N-insertions being much fewer compared to B-2 cells. These data strongly argues that substantial number of B-1a cells is developed in the presence of functional TdT. Some of these B-1a cells with N-insertion are likely generated during neonate stage, when TdT gene starts to be expressed.

FACS analysis of neonate (1 week) spleen and BM reveals the active B cell lymphopoiesis at both tissues. However, analysis of neonate IL-7-deficient mice found that B cell development starts to show blockage after day 2. This preliminary data suggests that B cell development before or around birth is independent of IL-7 whereas IL-7 becomes indispensable part of B cell development program at majority of neonate stage. IL-7 is a growth factor secreted by stromal cells in hematopoietic tissues and IL-7 signaling plays a crucial role in B cell development in adult BM. Although B-1 cells are readily detectable in IL-7 deficient mice, but their total number is 5-10 fold lower compared to wide type mice. Furthermore, B-1 repertoire expressed in IL-7-deficient mice differs from that expressed in WT mice. Taken together, we propose that IL-7 is involved in B-1 generation during the neonate stage and dependence of IL-7 represents fundamental difference distinguishing B-1 development in fetus vs their generation in neonatal life. Research efforts will focus on discovering the molecular mechanisms that are responsible for two distinct developmental pathways.  

Other selected publication:

Yang, Y, Contag, CH, Felsher, D, Shachaf, CM, Herzenberg, LA and Tung, JW. The E47 transcription factor negatively regulates CD5 expression during thymocyte development. PNAS, 101(11): 3898-3902, 2004.

Baumgarth, N, Choi, Y. Y, Yang Y and Herzenberg, LA. B cell lineage contributions to antiviral host responses. Curr Top Microbiol Immunol. 319:41-61, 2008

 

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