Andrew M. Knight

Additional microsatellite markers for mouse genome mapping

Mouse sequence information from the EMBL and GenBank databases, published sequences and genomic clones have been analyzed for simple repetitive elements or microsatellites. Each microsatellite has been amplified by the polymerase chain reaction (PCR) as a single locus marker. PCR primers were designed from unique sequence flanking each repeat. Size variation of PCR products less than 750 base pairs (bp) between mouse strains has been determined using ethidium bromide-stained acrylamide or agarose gels. A further 74 newly characterized microsatellites are presented in this paper, bringing to 185 the total we have analyzed. Of these, 157/185 (85%) have more than one allele, 143/178 (80%) vary in length between C57BL/6J and Mus spretus, and 82/168 (49%) vary between DBA/2J and C57BL/6J. Microsatellites provide informative single locus probes for linkage analysis in the construction of a genetic map of the mouse genome.

Co-segregation of a gene encoding a deletion ligand for Tcrb-V3+ T cells with Mtv-3

A gene encoding the endogenous superantigen Mlsc, which deletes Tcrb-V3+ T cells in the NOD inbred mouse strain, was found to co-segregate with Mtv-3 on chromosome 11. This identifies a fourth gene encoding a deletion ligand for Tcrb-V3+ T cells and extends recently published observations in support of the hypothesis that a number of endogenous superantigens are the products of Mtv proviruses.

Biochemical analysis of the mouse mammary tumor virus long terminal repeat product. Evidence for the molecular structure of an endogenous superantigen.

Recent reports have shown that both exogenous and endogenous mouse mammary tumor viruses (MMTV) can encode superantigens. Transfection and transgenic studies have identified the open reading frame (ORF) present in the 3′ long terminal repeat (LTR) as encoding superantigen function. In this study, we have used an in vitro translation system in an attempt to characterize the molecular nature of the protein encoded by the 3′ ORF of Mtv‐8. Using various constructs encoding full‐length and truncated versions of the ORF product, we report that the hydrophobic region close to the amino terminus of the 36‐kDa protein can function as a transmembrane domain. Protease digestion experiments also demonstrate that the protein has a type‐II transmembrane conformation with an extra‐cytoplasmic carboxy terminus. Since this hydrophobic region is conserved between all known MMTV, we speculate that LTR ORF, including those proposed to encode the minor lymphocyte stimulatory antigens, are also capable of encoding type‐II transmembrane glycoproteins. The polymorphism between MMTV LTR ORF products, which correlates with deletion phenotypes, is predominantly in the carboxy‐terminal extracellular region, consistent with a major role in interaction with the T cell receptor.

Distinct Sequences in the Cytoplasmic Domain of Complement Receptor 2 Are Involved in Antigen Internalization and Presentation

B cells express randomly rearranged surface Ig that forms part of a multiprotein complex known as the B cell receptor (BCR). Recognition of Ag via this receptor results in its capture, internalization, proteolysis and presentation to CD4+ T cells. The recognition of Ag by CD4+ T cells is critical for the selection of individual B cells, leading to the eventual secretion of a high affinity version of the BCR as an effective circulating Ab. B cells also express other receptors that recognize Ags associated with components of innate immunity. One of these receptors, CR2, binds Ags coated with activated complement components. Studies have shown that cross-linking CR2 and the BCR with complement-tagged Ags leads to enhanced Ag presentation by B cells. In addition, Ags targeted to B cell CR2 in the absence of BCR coligation are also efficiently presented to T cells. In this report, we identify several distinct sequences within the cytoplasmic domain of mouse CR2 (mCR2) that are essential for mCR2-mediated Ag presentation in both the presence and the absence of BCR cross-linking. The finding that distinct sequences in the cytoplasmic domain of mCR2 are essential for BCR-independent Ag presentation leads us to propose a novel role for CR2.

Regulated release of B cell‐derived exosomes: Do differences in exosome release provide insight into different APC function for B cells and DC?

The induction of CD4 TH cell activity is a crucial component of the mammalian acquired immune response. In order to activate TH cells, pathogen‐derived peptides are displayed on the plasma membrane of specialized cells termed APC. As well as unravelling common mechanisms in this process, considerable attention has been given to the distinct roles of the various cell types involved. In this issue of the European Journal of Immunology, a study examining the release by B cells of small vesicles termed exosomes is presented. This commentary reports that the control of exosome release from B cells appears to differ from that seen in DC. How these differences may relate to important features that differentiate the antigen presenting cell function of B cells and DC in vivo is also discussed.

Presentation of the candidate rheumatoid arthritis autoantigen aggrecan by antigen-specific B cells induces enhanced CD4+ T helper type 1 subset differentiation

Effective immune responses require antigen uptake by antigen‐presenting cells (APC), followed by controlled endocytic proteolysis resulting in the generation of antigen‐derived peptide fragments that associate with intracellular MHC class II molecules. The resultant peptide–MHC class II complexes then move to the APC surface where they activate CD4+ T cells. Dendritic cells (DC), macrophages and B cells act as efficient APC. In many settings, including the T helper type 1 (Th1) ‐dependent, proteoglycan‐induced arthritis model of rheumatoid arthritis, accumulating evidence demonstrates that antigen presentation by B cells is required for optimal CD4+ T cell activation. The reasons behind this however, remain unclear. In this study we have compared the activation of CD4+ T cells specific for the proteoglycan aggrecan following antigen presentation by DC, macrophages and B cells. We show that aggrecan‐specific B cells are equally efficient APC as DC and macrophages and use similar intracellular antigen‐processing pathways. Importantly, we also show that antigen presentation by aggrecan‐specific B cells to TCR transgenic CD4+ T cells results in enhanced CD4+ T cell interferon‐γ production and Th1 effector sub‐set differentiation compared with that seen with DC. We conclude that preferential CD4+ Th1 differentiation may define the requirement for B cell APC function in both proteoglycan‐induced arthritis and rheumatoid arthritis.

Antigen-specific B lymphocytes acquire proteoglycan aggrecan from cartilage extracellular matrix resulting in antigen presentation and CD4+ T-cell activation

The majority of studies examining antigen‐presenting cell (APC) function have focused on the capture and presentation of antigens released from pathogens or damaged cells. However, antigen‐specific B cells are also capable of efficiently extracting antigens that are either tethered to, or integrally part of the plasma membrane of various target cells. In this study we show that B cells are also highly efficient at extracting integral components of the extracellular matrix (ECM) for subsequent presentation. In particular we demonstrate that B cells specific for aggrecan, an integral component of cartilage ECM, acquire this rheumatoid arthritis candidate autoantigen in both a B‐cell‐receptor‐dependent and a contact‐dependent manner. We also demonstrate that the subsequent presentation of aggregan from ECM leads to CD4+ T‐cell activation and effector cell formation. Recent studies have identified B‐cell‐mediated antigen presentation as essential for the development of autoimmunity, but a unique role for B cells compared with other APC has yet to be defined. Our findings lead us to propose that the acquisition of ECM‐derived autoantigens represents a mechanism that defines the APC requirement for B cells in the development of autoimmunity.

Lowering the affinity between antigen and the B cell receptor can enhance antigen presentation

The B cell receptor (BCR) enables antigen‐specific B cells to bind, internalize and target antigens for processing into small peptide fragments. These epitopes are then expressed on the plasmamembrane in association with MHC class II molecules for recognition by CD4+ T cells. The affinity of the interaction between the BCR and antigen plays an important part in determining T cell epitope generation. In this report we provide evidence that the efficiency of antigen presentation by specific B cells does not need to be directly proportional to antigen/BCR affinity. We show that increased presentation can result from lowering the affinity of the antigen/BCR interaction. This finding suggests a novel mechanism by which B cells can recruit T cell help and obtain survival signals. Activation of these cells may have consequences for the generation of the B cell repertoire.

PCR-analyzed microsatellites of the mouse genome—additional polymorphisms among ten inbred mouse strains

Eighty sequences from the mouse genome database containing microsatellites (simple sequence repeats) have been analyzed for size variation among ten different inbred strains of mice; 62/80 (77.5%) showed polymorphism of at least three alleles. We have been able to detect all the polymorphims by agarose gel electrophoresis, often running the gels for up to 3 h. Between individual pairs of mouse strains to be used in chromosomal mapping studies in our laboratory, 35–60% polymorphism occurred. There are potentially enough microsatellites within the mouse and human genome to have a marker at every 1-cM distance. This simple approach will, therefore, continue to be useful in genome mapping studies, leading eventually to high-resolution maps of both the mouse and human genomes; this should allow for physical mapping and cloning of specific genes.

Modulation of murine complement receptor type 2 (CR2/CD21) ectodomain shedding by its cytoplasmic domain.

Ectodomain shedding is a mechanism that regulates numerous functions of cell surface proteins. The extracellular domain of the human complement receptor 2 (CR2/CD21) is released by proteolytic cleavage as a soluble protein through a variety of stimuli including the thiol antioxidants N-acetylcysteine (NAC) and glutathione (GSH), and the oxidant pervanadate (PV). In addition, PV mimics B cell antigen receptor (BCR) signaling. Here, we show that murine CD21 is shed upon those stimuli and that the cytoplasmic domain is an important modulator for CD21-shedding. B cells expressing a mutant CD21 cytoplasmic domain with only three amino acids (KHR) showed increased CD21-shedding and required lower stimuli concentrations. At lower PV concentrations, wildtype CD21 was up-regulated on the cell surface, whereas at higher PV concentrations the ectodomain was shed. These findings further indicate that GSH and NAC utilize different pathways than PV to activate CD21-shedding. Altogether, as pre-activated B cells express higher CD21 levels than resting mature B cells or fully activated and antigen-experienced B cells, we suggest CD21-shedding to be a mechanism to fine-tune B cell activation.