Congenic nonobese diabetic mouse strains fail to confirm linkage of a marginal zone B lymphocyte phenotype to the Idd11 locus on chromosome 4.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
David M Tarlinton
Publications and source records attributed to David M Tarlinton.
CD45 links the B cell receptor with cell survival and is required for the persistence of germinal centers.
To segregate the many contributions that B cell receptor (BCR)-mediated signals make to immune responses, we have analyzed here B cells deficient in the 'pan-leukocyte' marker CD45. BCR ligation of Cd45-/- B cells failed to activate phosphatidylinositol-3-OH kinase, NF-kappaB, Erk1 or Erk2 kinases or to upregulate cell survival proteins and instead induced apoptosis. Immunization of Cd45-/- B cell chimeras induced germinal centers and antigen-specific immunoglobulin G1 antibody-forming cells early, but both cellular compartments decreased by day 14. Proliferation of Cd45-/- B cells induced by CD40 ligand in vitro was impaired as a result of abrogation by BCR ligation of the upregulation of prosurvival proteins. In contrast, enforced expression of the antiapoptotic factor Bcl-xL prevented the collapse of Cd45-/- B cell germinal centers. These results show mechanistic differences in B cell survival during germinal center initiation and propagation; CD40 signaling is sufficient for the former, whereas the latter requires signaling from the BCR.
Animals↗
Lyn-deficient mice develop severe, persistent asthma: Lyn is a critical negative regulator of Th2 immunity.
The etiology of asthma, a chronic inflammatory disorder of the airways, remains obscure, although T cells appear to be central disease mediators. Lyn tyrosine kinase has been implicated as both a facilitator and inhibitor of signaling pathways that play a role in allergic inflammation, although its role in asthma is unclear because Lyn is not expressed in T cells. We show in the present study that Lyn-/- mice develop a severe, persistent inflammatory asthma-like syndrome with lung eosinophilia, mast cell hyperdegranulation, intensified bronchospasm, hyper IgE, and Th2-polarizing dendritic cells. Dendritic cells from Lyn-/- mice have a more immature phenotype, exhibit defective inhibitory signaling pathways, produce less IL-12, and can transfer disease when adoptively transferred into wild-type recipients. Our results show that Lyn regulates the intensity and duration of multiple asthmatic traits and indicate that Lyn is an important negative regulator of Th2 immune responses.
Allergens↗
The development of functional B lymphocytes in conditional PU.1 knock-out mice.
An abundance of research has entrenched the view that the Ets domain containing transcription factor PU.1 is fundamental to the development and function of B lymphocytes. In this study, we have made use of a conditional PU.1 allele to test this notion. Complete deletion of PU.1 resulted in the loss of B cells and all other lineage-positive cells in the fetal liver and death between E18.5 and birth; however, specific deletion of PU.1 in the B lineage had no effect on B-cell development. Furthermore, deletion of PU.1 in B cells did not compromise their ability to establish and maintain an immune response. An increased level of apoptosis was observed in vitro upon B-cell receptor (BCR) cross-linking; however, this was partially rescued by interleukin-4 (IL-4). These findings suggest that PU.1 is not essential for the development of functional B lymphocytes beyond the pre-B stage.
Animals↗
A requirement for CD45 distinguishes Ly49D-mediated cytokine and chemokine production from killing in primary natural killer cells.
Engagement of receptors on the surface of natural killer (NK) cells initiates a biochemical cascade ultimately triggering cytokine production and cytotoxicity, although the interrelationship between these two outcomes is currently unclear. In this study we investigate the role of the cell surface phosphatase CD45 in NK cell development and intracellular signaling from activating receptors. Stimulation via the major histocompatibility complex I-binding receptor, Ly49D on CD45(-/-) primary NK cells resulted in the activation of phosphoinositide-3-kinase and normal cytotoxicity but failed to elicit a range of cytokines and chemokines. This blockage is associated with impaired phosphorylation of Syk, Vav1, JNK, and p38, which mimics data obtained using inhibitors of the src-family kinases (SFK). These data, supported by analogous findings after CD16 and NKG2D stimulation of CD45(-/-) primary NK cells, place CD45 upstream of SFK in NK cells after stimulation via immunoreceptor tyrosine-based activation motif-containing receptors. Thus we identify CD45 as a pivotal enzyme in eliciting a precise subset of NK cell responses.
Animals↗
Differential requirement for OBF-1 during antibody-secreting cell differentiation.
Resting B cells can be cultured to induce antibody-secreting cell (ASC) differentiation in vitro. A quantitative analysis of cell behavior during such a culture allows the influences of different stimuli and gene products to be measured. The application of this analytical system revealed that the OBF-1 transcriptional coactivator, whose loss impairs antibody production in vivo, has two effects on ASC development. Although OBF-1 represses early T cell-dependent (TD) differentiation, it is also critical for the completion of the final stages of ASC development. Under these conditions, the loss of OBF-1 blocks the genetic program of ASC differentiation so that Blimp-1/prdm1 induction fails, and bcl-6, Pax5, and AID are not repressed as in control ASC. Retroviral complementation confirmed that OBF-1 was the critical entity. Surprisingly, when cells were cultured in lipopolysaccharide to mimic T cell-independent conditions, OBF-1-null B cells differentiated normally to ASC. In the OBF-1(-/-) ASC generated under either culture regimen, antibody production was normal or only modestly reduced, revealing that Ig genes are not directly dependent on OBF-1 for their expression. The differential requirement for OBF-1 in TD ASC generation was confirmed in vivo. These studies define a new regulatory role for OBF-1 in determining the cell-autonomous capacity of B cells to undergo terminal differentiation in response to different immunological signals.
Animals↗
Early appearance of germinal center-derived memory B cells and plasma cells in blood after primary immunization.
Immunization with a T cell-dependent antigen elicits production of specific memory B cells and antibody-secreting cells (ASCs). The kinetic and developmental relationships between these populations and the phenotypic forms they and their precursors may take remain unclear. Therefore, we examined the early stages of a primary immune response, focusing on the appearance of antigen-specific B cells in blood. Within 1 wk, antigen-specific B cells appear in the blood with either a memory phenotype or as immunoglobulin (Ig)G1 ASCs expressing blimp-1. The memory cells have mutated V(H) genes; respond to the chemokine CXCL13 but not CXCL12, suggesting recirculation to secondary lymphoid organs; uniformly express B220; show limited differentiation potential unless stimulated by antigen; and develop independently of blimp-1 expression. The antigen-specific IgG1 ASCs in blood show affinity maturation paralleling that of bone marrow ASCs, raising the possibility that this compartment is established directly by blood-borne ASCs. We find no evidence for a blimp-1-expressing preplasma memory compartment, suggesting germinal center output is restricted to ASCs and B220(+) memory B cells, and this is sufficient to account for the process of affinity maturation.
Adoptive Transfer↗
Antigen delivery via two molecules on the CD8- dendritic cell subset induces humoral immunity in the absence of conventional "danger".
Targeting antigen to dendritic cells (DC) in vivo might be an effective method of modulating immune responses. Given the functional specializations among DC subsets, we investigated how targeting different receptors on different DC subsets may influence antibody (Ab) production. We show here that targeting FIRE (F4/80-like receptor) or CIRE (C-type lectin receptor), two molecules expressed on the surface of immature CD8- DC in the mouse, increases Ab production 100-1000-fold over a non-targeted control. This response was equivalent to that achieved with CpG adjuvant. In contrast, targeting CD205, which is primarily expressed on CD8+ DC, did not elicit an Ab response unless an adjuvant was added. Strong Ab responses in FcRgamma-/- mice, and with the use of F(ab')2 fragments, confirmed that FIRE and CIRE targeting was due to specific rather than FcR or complement binding. Our findings may reflect differences in the ability of CD8+ and CD8- DC subsets to stimulate immune responses in vivo. Although the consensus view is that Ag presentation on DC in their steady state leads to tolerance, the Ab enhancement from FIRE and CIRE targeting in the apparent absence of any "danger" or inflammatory signal would suggest that targeting certain DC molecules can supplant the need for external adjuvants for eliciting immune responses.
Animals↗
Lyn tyrosine kinase: accentuating the positive and the negative.
Lyn, one of several Src-family tyrosine kinases in immune cells, is noted for its ability to negatively regulate signaling pathways through phosphorylation of inhibitory receptors, enzymes, and adaptors. Somewhat paradoxically, it is also a key mediator in several pathways of B cell activation, such as CD19 and CD180. Whether Lyn functions to promote or inhibit immune cell activation depends on the stimulus and the developmental state, meaning that the consequences of Lyn activity are context dependent. The importance of regulating Lyn activity is exemplified by the pathological conditions that develop in both lyn-/- and lyn gain-of-function mice (lynup/up), including lethal antibody-mediated autoimmune diseases and myeloid neoplasia. Here, we review the outcomes of altered Lyn activity within the framework of B cell development and differentiation and the circumstances that appear to dictate the outcome.
Animals↗
Plasma cell ontogeny defined by quantitative changes in blimp-1 expression.
Plasma cells comprise a population of terminally differentiated B cells that are dependent on the transcriptional regulator B lymphocyte--induced maturation protein 1 (Blimp-1) for their development. We have introduced a gfp reporter into the Blimp-1 locus and shown that heterozygous mice express the green fluorescent protein in all antibody-secreting cells (ASCs) in vivo and in vitro. In vitro, these cells display considerable heterogeneity in surface phenotype, immunoglobulin secretion rate, and Blimp-1 expression levels. Importantly, analysis of in vivo ASCs induced by immunization reveals a developmental pathway in which increasing levels of Blimp-1 expression define developmental stages of plasma cell differentiation that have many phenotypic and molecular correlates. Thus, maturation from transient plasmablast to long-lived ASCs in bone marrow is predicated on quantitative increases in Blimp-1 expression.
Animals↗
Perturbed myelo/erythropoiesis in Lyn-deficient mice is similar to that in mice lacking the inhibitory phosphatases SHP-1 and SHIP-1.
The Lyn tyrosine kinase plays essential inhibitory signaling roles within hematopoietic cells by recruiting inhibitory phosphatases such as SH2-domain containing phosphatase-1 (SHP-1), SHP-2, and SH2-domain containing 5'-inositol phosphatase (SHIP-1) to the plasma membrane in response to specific stimuli. Lyn-deficient mice display a collection of hematopoietic defects, including autoimmune disease as a result of autoantibody production, and perturbations in myelopoiesis that ultimately lead to splenomegaly and myeloid neoplasia. In this study, we demonstrate that loss of Lyn results in a stem/progenitor cell-intrinsic defect leading to an age-dependent increase in myeloid, erythroid, and primitive hematopoietic progenitor numbers that is independent of autoimmune disease. Despite possessing increased numbers of erythroid progenitors, and a more robust expansion of these cells following phenylhydrazine challenge, Lyn-deficient mice are more severely affected by the chemotherapeutic drug 5-fluorouracil, revealing a greater proportion of cycling progenitors. We also show that mice lacking SHIP-1 have defects in the erythroid and myeloid compartments similar to those in mice lacking Lyn or SHP-1, suggesting an intimate relationship between Lyn, SHP-1, and SHIP-1 in regulating hematopoiesis.
Animals↗
CD45: direct and indirect government of immune regulation.
The protein tyrosine phosphatase (PTP) CD45 is abundantly expressed on all nucleated hematopoietic cells and is critical for classical antigen receptor signalling indicated by the arrested development of B and T cells in mice deficient for CD45. Despite its clear role in positive regulation of signalling through the activation of the Src family of tyrosine kinases, many reports have shown CD45 to also negatively regulate this process. Given such a dichotomy in CD45 function and a poor understanding of the mechanism underlying the phenotype of CD45(-/-) lymphocytes, we considered it timely to review the existing data and attempt to determine whether aspects of the CD45(-/-) phenotype result from excessive positive or negative kinase activity and the target molecules that may mediate such effects.
Animals↗
Targeting plasma cells in autoimmune diseases.
Antibodies specific for self-antigens mediate life-threatening pathology in several autoimmune diseases. Clearly the ability to target the plasma cells (PCs) producing the autoantibodies would be of great clinical benefit. Current immunosuppressive therapies are based on the premise that autoreactive PCs are short-lived and replenished from ongoing immune responses. However, recent results question this assumption and suggest that optimizing the treatment of severe autoimmune conditions will require a significant investment in elucidating the details of PC biology.
Autoimmune Diseases↗
Evidence from the generation of immunoglobulin G-secreting cells that stochastic mechanisms regulate lymphocyte differentiation.
Naive B lymphocytes undergo isotype switching and develop into immunoglobulin-secreting cells to generate the appropriate class and amount of antibody necessary for effective immunity. Although this seems complex, we report here that the generation of immunoglobulin G-secreting cells from naive precursors is highly predictable. The probabilities of isotype switching and development into secreting cells change with successive cell divisions and interleave independently. Cytokines alter the probability of each differentiation event, while leaving intact their independent assortment. As a result, cellular heterogeneity arises automatically as the cells divide. Stochastic division-linked regulation of heterogeneity challenges the conventional paradigms linking distinct phenotypes to unique combinations of signals and has the potential to simplify our concept of immune complexity considerably.
Animals↗
Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim inhibits BCR stimulation-induced apoptosis and deletion of autoreactive B cells.
During development, the stochastic process assembling the genes encoding antigen receptors invariably generates B and T lymphocytes that can recognize self-antigens. Several mechanisms have evolved to prevent the activation of these cells and the concomitant development of autoimmune disease. One such mechanism is the induction of apoptosis in developing or mature B cells by engagement of the B cell antigen receptor (BCR) in the absence of T cell help. Here we report that B lymphocytes lacking the pro-apoptotic Bcl-2 family member Bim are refractory to apoptosis induced by BCR ligation in vitro. The loss of Bim also inhibited deletion of autoreactive B cells in vivo in two transgenic systems of B cell tolerance. Bim loss prevented deletion of autoreactive B cells induced by soluble self-antigen and promoted accumulation of self-reactive B cells developing in the presence of membrane-bound self-antigen, although their numbers were considerably lower compared with antigen-free mice. Mechanistically, we determined that BCR ligation promoted interaction of Bim with Bcl-2, inhibiting its survival function. These findings demonstrate that Bim is a critical player in BCR-mediated apoptosis and in B lymphocyte deletion.
Animals↗
Generation and analysis of Siah2 mutant mice.
Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.
Animals↗
Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity.
Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lyn(up/up) mice). Lyn(up/up) mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lyn(up/up) B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cgamma2 in resting Lyn(up/up) B cells. Similarly, Lyn(up/up) B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lyn(up/up) mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.
Animals↗
The activation and subsequent regulatory roles of Lyn and CD19 after B cell receptor ligation are independent.
The cell surface glycoprotein CD19 and the Src-related protein tyrosine kinase Lyn are key mediators of, respectively, positive and negative signaling in B cells. Despite the apparent opposition of their regulatory functions, a recent model of the biochemical events after B cell receptor (BCR) ligation intimately links the activation of Lyn and CD19. We examined the biochemical consequences of BCR ligation in mouse B cells lacking either Lyn or CD19 for evidence of interaction or codependence. In contrast to published results, we found CD19 phosphorylation after BCR ligation to be unaffected by the absence of Lyn, yet dependent on Src family protein tyrosine kinases as it was inhibited fully by PP2, an Src family-specific inhibitor. Consistent with normal CD19 phosphorylation in lyn(-/-) B cells, the recruitment of phosphoinositide-3 kinase to CD19 and the ability of CD19 to enhance both intracellular calcium flux and extracellular signal-regulated kinase 1/2 activation after coligation with the BCRs were intact in the absence of Lyn. Similarly, unique functions of Lyn were found to be independent of CD19. CD19(-/-) B cells were normal for increased Lyn kinase activity after BCR ligation, inhibition of BCR-mediated calcium flux after CD22 coligation, and inhibition of extracellular signal-regulated kinase phosporylation after FcgammaRIIB coligation. Collectively, these data show that the unique functions of Lyn do not require CD19 and that the signal amplification mediated by CD19 is independent of Lyn. We conclude that the roles of Lyn and CD19 after BCR ligation are independent and opposing, one being primarily inhibitory and the other stimulatory.
Amino Acid Sequence↗