Kyoko Hayakawa

Identification of the Earliest B Lineage Stage in Mouse Bone Marrow

We have identified a very early stage of B lineage cells in the CD45R (B220)+CD24 (HSA) pre-pro-B fraction of mouse bone marrow delineated by expression of AA4.1, a molecule found on stem cells and early B lineage cells. These cells are B lineage precursors based on their capacity to generate B lineage cells rapidly in stromal-dependent culture and their expression of high levels of germline IgH transcripts in the absence of Rag-1/2. Half of these AA4.1+ precursors express low levels of CD4, characteristic of lymphoid progenitors, but few if any have up-regulated CD19, a molecule expressed very early in the B lineage. Furthermore, expression of genes encoding pre-B and B cell receptor components (mb-1, B29, and lambda 5) and transcription factors necessary for B lineage differentiation (BSAP, E12, E47, and Id) provide further support for designating these cells as the earliest B cell precursors.

CD5 B Cells, a Fetal B Cell Lineage

Publisher Summary This chapter presents a short background of CD5 expression on B cells and focuses on the issue of the relationship of CD5 B cells to B cell development, proposing a model that views this subset as the progeny of a fetal B cell differentiation pathway. Several features of this population that distinguish it from other B cells are reviewed, including a possible relationship between the normal population and the numerous CD5 + B cell neoplasias. The chapter discusses another feature that has evoked much interest, an apparent bias toward expressing autoreactive specificities along with skewed V gene usage and focuses on work with the putative homologous human B cell subset, highlighting similarities and current work. Expression of CD5 on B cell lines has been confirmed both by immunoprecipitation and by Northern blot analysis. The same CD5 gene is expressed on normal T and CD5 B cells in mice has been further confirmed by the homologous gene targeting method. A model for CD5 + B cell generation that regards fetal and adult B cell differentiation as significantly different is presented in the chapter.

Defective presentation of the CD1d1-restricted natural Va14Ja18 NKT lymphocyte antigen caused by β-d-glucosylceramide synthase deficiency

Va14Ja18 natural T (NKT) cells play an immunoregulatory role, which is controlled by a self glycolipid(s) presented by CD1d. Although the synthetic antigen α-D-galactosylceramide (α-D-GalCer) stimulates all Va14Ja18 NKT cells, α-anomeric D-glycosylceramides are currently unknown in mammals. We have used β-D-GalCer-deficient mice and β-D-glucosylceramide (β-D-GlcCer)-deficient cells to define the chemical nature of a natural NKT cell antigen. β-D-GalCer-deficient mice exhibit normal NKT cell development and function, and cells from these animals potently stimulate NKT hybridomas. In striking contrast, the same hybridomas fail to react to CD1d1 expressed by a β-D-GlcCer-deficient cell line. Importantly, human β-D-GlcCer synthase cDNA transfer, and hence the biosynthesis of β-D-GlcCer, restores the recognition of mutant cells expressing CD1d1 by the Va14Ja18 NKT hybridomas. Additionally, suppression of β-D-GlcCer synthesis inhibits antigen presentation to Va14Ja18 NKT cells. The possibility that β-D-GlcCer itself is the natural NKT cell antigen was excluded because it was unable to activate NKT hybridomas in a cell-free antigen-presentation assay. These findings suggest that β-D-GlcCer may play an important role in generating and/or loading a natural Va14Ja18 NKT antigen.

B-cell commitment, development and selection.

Summary: Here we review three areas in B‐cell development in the mouse, with a focus on relevance to B‐1/CD5+ B cells. Multiparameter flow cytometry has allowed the dissection of intermediate stages of developing B cells, both in fetal liver and bone marrow. In the first area, we present recent work that has delineated a fraction of pre‐pro‐B cells, committed to the B lineage, but lacking any immunoglobulin rearrangements. Next, the role of the pre‐B‐cell receptor in B‐cell repertoire selection has become clear in the past few years, but we present work suggesting that the action of this process during fetal life is different, resulting in selection of a very distinct repertoire compared with adult. Finally, we describe a new VH3609 antithymocyte Ig transgenic mouse model system that has provided the first definitive evidence for the role of self‐antigen in development and maintenance of natural autoreactive B cells.

Distinctive developmental origins and specificities of murine CD5+B cells

CD5+ B cells constitute a small fraction of cells in the spleen of adult mice that exhibit numerous features serving to distinguish them from the bulk of IgD++CD5−“conventional” B cells. In this review we focus on two major questions relating to this population: 1) the relationship of CD5+ B cells to other B cells; and 2) the distinctive enrichment of particular autoreactive specificities in this subset. The nature of their origins is clarified by a thorough analysis of intermediate stages of early B‐cell development in both fetal and adult tissues. The reactivity to bromeliad‐treated mouse red blood cells serves as a prototype system for the investigation of biased specificities in CD5+ B cells. These lines of investigation lead us to propose that CD5+ B cells in the adult are the remnant of a distinct fetal B‐cell differentiation pathway wherein selection of cells from this fetal/neonatal population into the adult long‐lived pool results in the over‐expression of certain germline‐encoded autoreactivities.

Recycling CD1d1 Molecules Present Endogenous Antigens Processed in an Endocytic Compartment to NKT Cells

Mouse CD1d1 molecules present endogenous glycolipids to NKT cells. Although glycolipid presentation requires CD1d1 transport through the endocytic pathway, the processing requirements for such endogenous Ag presentation by CD1d1 molecules are undefined. We examined CD1d1 Ag presentation to NKT cells by disrupting endocytic trafficking and function in cells expressing normal and mutated CD1d1 expressed by recombinant vaccinia viruses. Consistent with previous studies, we found that preventing CD1d1 localization to endosomes by altering its cytoplasmic targeting sequences abrogated recognition by Vα14Jα281+ NKT cells without affecting recognition by Vα14− NKT cells. Increasing the pH of acidic compartments by incubating cells with chloroquine or bafilomycin A1 blocked CD1d1 recognition by Vα14+ (but not Vα14−) NKT cells without reducing levels of cell surface CD1d1. Similar results were obtained with primaquine, which interferes with the recycling of cell surface glycoproteins. These results suggest that the loading of a subset of glycolipid ligands onto CD1d1 molecules entails the delivery of cell surface CD1d1 molecules and an acidic environment in the endocytic pathway.

Positive Selection of Anti–Thy-1 Autoreactive B-1 Cells and Natural Serum Autoantibody Production Independent from Bone Marrow B Cell Development

A natural serum autoantibody specific for the Thy-1 glycoprotein (anti–Thy-1 autoantibody [ATA]) is produced by B-1 cells that are positively selected by self-antigen. Here, using ATAμκ transgenic mice we show that cells with this B cell receptor are negatively selected during bone marrow (BM) development. In a Thy-1 null environment, BM ATA B cells progress to a normal follicular stage in spleen. However, in a self-antigen–positive environment, development is arrested at an immature stage in the spleen, concomitant with induction of CD5. Such cells are tolerant and short-lived, different from B-1. Nonetheless, ATA-positive selection was evident by self-antigen–dependent high serum ATA production, comprising ∼90% of serum immunoglobulin M in ATAμκ mice. Splenectomy did not eliminate ATA production and transfer of tolerant splenic B cells did not induce it. These findings demonstrate that B-1 positive selection, resulting in the production of natural serum ATA, arises independently from the major pathway of BM B cell development and selection.

Inhibition of glycolipid shedding rescues recognition of a CD1+ T cell lymphoma by natural killer T (NKT) cells.

Neoplastic transformation of cells is accompanied by an aberration of cell surface glycolipid composition. These tumor-associated, altered glycosphingolipids are often shed into the tumor cell microenvironment and mediate immunosuppressive activity. The nature and form of glycolipids shed by a variety of tumor cell lines and the mechanism(s) of shedding have been well characterized. The murine T cell lymphoma line, L5178Y-R, is known to shed a tumor-associated glycolipid, gangliotriaosylceramide, into the culture medium. We analyzed the effect of glycolipids from L5178Y-R on antigen presentation by murine CD1d1 molecules. CD1d1 molecules present glycolipid antigens to a specialized class of T cells called natural killer T (NKT) cells that mainly express a T cell receptor α chain (Vα14Jα281) associated with Vβ chains of limited diversity. In the current report, we found that L5178Y-R cells express CD1 on their cell surface yet are unable to stimulate CD1d1-specific NKT cells. We hypothesized that the glycolipid(s) shed by L5178Y-R inhibited antigen presentation by CD1d1. Pretreatment of CD1d1+ cells with conditioned medium from L5178Y-R inhibited CD1-specific stimulation of canonical (Vα14+) but not noncanonical (Vα5+) NKT cells. Exogenous addition of lipids extracted from L5178Y-R cells as well as purified gangliotriaosylceramide mimicked this effect. Inhibition of glycolipid shedding in L5178Y-R cells with d-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol resulted in the rescue of CD1d1 recognition by canonical (but not noncanonical) NKT cells. These results suggest that one means by which certain tumor cells can evade the hosts innate antitumor immune response is by shedding glycolipids that inhibit CD1-mediated antigen presentation to NKT cells.

Selection during development of VH11+ B cells: a model for natural autoantibody‐producing CD5+ B cells

Summary:  Natural autoantibodies constitute a large portion of serum immunoglobulin M (IgM) and bridge the adaptive and innate immune systems, serving as a rapid response to common pathogens. Many arise from a distinctive subset of B cells, termed B‐1, that express CD5. Here, we describe our studies with a representative CD5+ B‐cell‐derived natural autoantibody, the VH11Vκ9 B‐cell receptor (BCR) that binds a determinant on senescent erythrocytes. This specificity represents 5–10% of the CD5+ B‐cell subset, with a large portion accounted for by two novel BCRs, VH11Vκ9 and VH12Vκ4. We have found that the development of B‐lineage cells with a VH11 rearrangement is surprisingly restricted at several crucial bottlenecks: (i) one of the most common VH11 rearrangements generates a heavy‐chain protein that only inefficiently assembles a pre‐BCR, key for recombinase‐activating gene downregulation/allelic exclusion and pre‐B‐clonal expansion; (ii) cells containing VH11‐µ chains lacking N‐addition are favored for progression to the B‐cell stage, eliminating most bone marrow VH11 rearrangements; and (iii) only a subset of Vκ‐light chains combine with VH11 heavy chain to foster progression to the mature B‐cell stage. Together, these constrain VH11 generation to fetal development and may favor production of B cells with the prototype VH11Vκ9 BCR.

TCRβ Chain Influences But Does Not Solely Control Autoreactivity of Vα14J281T Cells

CD1d-dependent accumulation of αβ T cells bearing a canonical Vα14Jα281 α-chain (Vα14+ T cells) is thought to model positive selection of lipid-specific T cells, based on their ability to recognize CD1d-presented self glycolipid(s). However, it has been difficult to demonstrate self ligand specificity in this system, as most Vα14+ T cells do not exhibit significant autoreactivity despite high reactivity to α-galactosylceramide presented by CD1d (α-GalCer/CD1d). To assess the role of TCRβ chain in determining the α-GalCer/CD1d vs autoreactive specificity of Vα14+ T cells, we conducted TCRα or TCRβ chain transduction experiments. In this study we demonstrate, by combining different TCRβ chains with the Vα14 α-chain in retrovirally transduced T cell lines, that the Vα14 α-chain plays a primary role, necessary but not sufficient for imparting α-GalCer/CD1d recognition. β-Chain usage alone is not the sole factor that controls the extent of autoreactivity in Vα14+ T cells, since transduction of TCRαβ chains from a high CD1d autoreactive Vα14+ T cell line conferred the α-GalCer/CD1d specificity without induction of autoreactivity. Thus, heterogeneity of Vα14+ T cell reactivity is due to both β-chain diversity and control mechanism(s) beyond primary TCR structure.