Differentially regulated expression and function of CD22 in activated B-1 and B-2 lymphocytes

CD22 is a B cell-restricted transmembrane protein that apparently controls signal transduction thresholds initiated through the B cell Ag receptor (BCR) in response to Ag. However, it is still poorly understood how the expression of CD22 is regulated in B cells after their activation. Here we show that the expression levels of CD22 in conventional B-2 cells are markedly down-regulated after cross-linking of BCR with anti-IgM mAb but are up-regulated after stimulation with LPS, anti-CD40 mAb, or IL-4. In contrast, treatment with anti-IgM mAb barely modulated the expression levels of CD22 in CD5(+) B-1 cells, consistent with a weak Ca(2+) response in anti-IgM-treated CD5(+) B-1 cells. Moreover, in CD22-deficient mice, anti-IgM treatment did not trigger enhanced Ca(2+) influx in CD5(+) B-1 cells, unlike CD22-deficient splenic B-2 cells, suggesting a relatively limited role of CD22 in BCR signaling in B-1 cells. In contrast, CD22 levels were markedly down-regulated on wild-type B-1 cells in response to LPS or unmethylated CpG-containing oligodeoxynucleotides. These data indicate that the expression and function of CD22 are differentially regulated in B-1 and conventional B-2 cells, which are apparently implicated in innate and adaptive immunity, respectively.     

Cutting edge commentary: two B-1 or not to be one

B-1 cells differ from conventional B-2 cells both phenotypically and functionally. Two seemingly mutually exclusive hypotheses have been proposed to explain the origin of B-1 cells. The lineage hypothesis holds that certain B cell precursors are destined early on to become B-1 cells. The differentiation hypothesis holds that every B cell has the same potential to acquire B-1 characteristics. Reconsideration of previous studies of transgenic and knockout mice, plus recent results identifying differences between splenic and peritoneal B-1 cells, point to unexpected complexity in the pathway leading to B-1 status. A new paradigm is suggested, in which surface Ig signaling is required for B-1 cell production, but in which the signaling threshold and context that lead to B-1 cell development and/or expansion differ for particular B cell precursors. Surface Ig signaling may also produce receptor editing, apoptotic deletion, and tolerance induction; how these different outcomes are determined remains uncertain.     

Identification of a precursor to phosphatidyl choline-specific B-1 cells suggesting that B-1 cells differentiate from splenic conventional B cells in vivo: cyclosporin A blocks differentiation to B-1

The origin of B-1 cells is controversial. The initial paradigm posited that B-1 and B-2 cells derive from separate lineages. More recently it has been argued that B-1 cells derive from conventional B cells as a result of T-independent Ag activation. To understand B-1 cell differentiation, we have generated Ig transgenic (Tg) mice using the H and L chain genes (VH12 and Vkappa4) of anti-phosphatidyl choline (anti-PtC) B cells. In normal mice anti-PtC B cells segregate to B-1. Segregation is intact in VH12 (6-1) and VH12/Vkappa4 (double) Tg mice that develop large numbers of PtC-specific B cells. However, if B-1 cell differentiation is blocked, anti-PtC B cells in these Tg mice are B-2-like in phenotype, suggesting the existence of an Ag-driven differentiative pathway from B-2 to B-1. In this study, we show that double Tg mice have a population of anti-PtC B cells that have the phenotypic characteristics of both B-2 and B-1 cells and that have the potential to differentiate to B-1 (B-1a and B-1b). Cyclosporin A blocks this differentiation and induces a more B-2-like phenotype in these cells. These findings indicate that these cells are intermediate between B-2 and B-1, further evidence of a B-2 to B-1 differentiative pathway.     

Cutting edge: absence of expression of RAG1 in peritoneal B-1 cells detected by knocking into RAG1 locus with green fluorescent protein gene

It has been proposed that Ig gene rearrangement in the peritoneal cavity (Pc) B-1 cells might be involved in autoantibody generation. To study possible secondary B cell maturation, we prepared mice carrying a target integration of gfp gene into a rag1 locus (rag1/gfp mice). The GFP+ cells express rag1 mRNA and are undergoing Ig gene rearrangement. RAG1 expression was studied in Pc B-1 cells to detect cells during the stage of Ig gene rearrangement. In contrast to previous reports, Pc B-1 cells did not show RAG1 expression in adolescent or elderly mice. RAG1 expression was not induced in Pc B-1 cells in vivo after stimulation by oral or i.p. administration of LPS. Our results suggest that RAG1 expression in Pc B-1 cells is inhibited for a long period under normal condition and that this suppression is an essential state which maintains allelic exclusion of Ig genes.     

Interferon-alpha induction of STATs1, -3 DNA binding and growth arrest is independent of Lck and active mitogen-activated kinase in T cells

Type I interferons (IFNs) are a family of cytokines that have antiviral and antiproliferative effects. Data regarding the processes by which these cytokines transduce signals from the cell membrane to the nucleus are becoming increasingly complex. The most characterized pathway is via JAK-STAT signaling. Previous studies established a potential role for the Src-family kinase Lck in JAK-STAT signaling. Therefore, this study was designed to analyze the role of Lck in IFN-alpha signaling by using the Jurkat, JCam (an Lck-defective cell line derived from Jurkat), and JCam/Lck (JCam cells with Lck restored). The results show that IFN-alpha can induce MAPK activity, but only in cells containing Lck. Furthermore, STATs1 and -3 are effectively phosphorylated and activated to bind DNA in the absence of Lck expression in IFN-alpha-treated cells. Finally, the results demonstrate that IFN-alpha exerts an antiproliferative effect in all three cell lines. These data indicate that Lck and active MAPK do not affect IFN-alpha-induced growth arrest or induction of STAT1s1 and -3 DNA binding ability.     

RhoH regulates subcellular localization of ZAP-70 and Lck in T cell receptor signaling

RhoH is an hematopoietic-specific, GTPase-deficient Rho GTPase that plays a role in T development. We investigated the mechanisms of RhoH function in TCR signaling. We found that the association between Lck and CD3ζ was impaired in RhoH-deficient T cells, due to defective translocation of both Lck and ZAP-70 to the immunological synapse. RhoH with Lck and ZAP-70 localizes in the detergent-soluble membrane fraction where the complex is associated with CD3ζ phosphorylation. To determine if impaired translocation of ZAP-70 was a major determinant of defective T cell development, Rhoh(-/-) bone marrow cells were transduced with a chimeric myristoylation-tagged ZAP-70. Myr-ZAP-70 transduced cells partially reversed the in vivo defects of RhoH-associated thymic development and TCR signaling. Together, our results suggest that RhoH regulates TCR signaling via recruitment of ZAP-70 and Lck to CD3ζ in the immunological synapse. Thus, we define a new function for a RhoH GTPase as an adaptor molecule in TCR signaling pathway.     

The unique antigen receptor signaling phenotype of B-1 cells is influenced by locale but induced by antigen

Normal animals contain an autoreactive B lymphocyte subset, the B-1 subset, which is controlled by undefined mechanisms to prevent autoimmunity. Using a V(H)11V(kappa)9 Ig transgenic mouse, with a specificity prototypic of the subset, we have explored conditions responsible for the previously reported Ag hyporesponsiveness of these cells. We report that peritoneal V(H)11V(kappa)9 B cells exhibit typical B-1 behavior with high basal intracellular free Ca(2+) and negligible receptor-mediated calcium mobilization. However, splenic B cells from this mouse, while phenotypically similar to their peritoneal counterparts, including expression of CD5, mount robust B-2-like responses to Ag as measured by calcium influx and altered tyrosine phosphorylation responses. When these splenic cells are adoptively transferred to the peritoneal cavity and encounter their cognate self-Ag, they acquire a B-1 signaling phenotype. The ensuing hyporesponsiveness is characterized by increases in both basal intracellular calcium and resting tyrosyl phosphorylation levels and is highlighted by a marked abrogation of B cell receptor-mediated calcium mobilization. Thus, we show that self-Ag recognition in specific microenvironments such as the peritoneum, and we would propose other privileged sites, confers a unique form of anergy on activated B cells. This may explain how autoreactive B-1 cells can exist while autoimmunity is avoided.     

Cutting edge commentary: origins of B-1 cells

The origin of B-1a cells, a minority population of B cells that express CD5, are abundant in coelomic cavities, and often produce autoantibodies, has been the subject of study for many years. Accumulating evidence demonstrates that the hypothesis that only B cells arising in fetal or neonatal tissues have the potential to become B-1a cells cannot be true. Rather, B cell receptor-mediated signaling initiated by ligation of autoantigen has now been shown to be required for induction of the B-1a phenotype. Furthermore, cells with a functional B-1a phenotype can be induced from adult precursors by appropriate Ag. At the same time, microenvironment-specific events may determine the likelihood that a given B cell, either adult or fetal derived, enters this pathway. CD5 expression and possibly localization to the peritoneum appear to provide some protection to autoreactive cells otherwise slated for elimination.     

Interaction of human MUC1 and beta-catenin is regulated by Lck and ZAP-70 in activated Jurkat T cells

The MUC1 transmembrane glycoprotein is aberrantly expressed by diverse hematologic malignancies, including those of the T cell lineage. The MUC1 cytoplasmic domain (CD) interacts with beta-catenin; however, the role of MUC1 in T cells is not known. In the present work, MUC1 was studied as a potential downstream effector of the Lck and ZAP-70 tyrosine kinases that are essential for T cell activation. The results demonstrate that anti-CD3-induced or PMA+ionomycin-induced activation of Jurkat T cells is associated with increased binding of MUC1 and Lck. Lck phosphorylates MUC1-CD on Y-46 and, in turn, stimulates the binding of MUC1 to beta-catenin. The results further demonstrate that MUC1 interacts with ZAP-70. In contrast to Lck, ZAP-70 phosphorylates MUC1-CD predominantly on Y-20. However, like Lck, ZAP-70-mediated phosphorylation of MUC1 Y-20 stimulates binding of MUC1 and beta-catenin. These findings indicate that MUC1 functions as a substrate for Lck and ZAP-70 in activated Jurkat T cells and that MUC1 integrates T cell receptor signaling with the beta-catenin pathway.     

Spontaneously Ig-secreting B-1 cells violate the accepted paradigm for expression of differentiation-associated transcription factors

B-1 cells spontaneously secrete natural Ig that acts as a primary line of defense against infection. A major shortfall in our understanding of this key process centers on the molecular mechanisms regulating natural Ab secretion by B-1 cells. Herein, we demonstrate that secreting B-1 cells use some aspects of the recently recognized plasmacytic differentiation program but deviate from it in important ways. Specifically, we show that key repressors of the plasmacytic program, B cell leukemia/lymphoma-6 and paired box gene 5, are reduced in spontaneously secreting B-1 B cells, as in stimulated differentiated B-2 cells. Surprisingly, we find that key promoters of the plasmacytic program, B lymphocyte inducer of maturation program 1 and X-box binding protein 1, are not up-regulated in secreting B-1 cells, in contrast to secreting B-2 cells. These data demonstrate that B-1 cells operate under a differentiation program that is unique and differs from the paradigm associated with Ig-secreting B-2 cells.     

SHP-1 requires inhibitory co-receptors to down-modulate B cell antigen receptor-mediated phosphorylation of cellular substrates

Signaling through the B cell antigen receptor (BCR) is negatively regulated by the SH2 domain-containing protein-tyrosine phosphatase SHP-1, which requires association with tyrosine-phosphorylated proteins for activation. Upon BCR ligation, SHP-1 has been shown to associate with the BCR, the cytoplasmic protein-tyrosine kinases Lyn and Syk, and the inhibitory co-receptors CD22 and CD72. How SHP-1 is activated by BCR ligation and regulates BCR signaling is, however, not fully understood. Here we demonstrate that, in the BCR-expressing myeloma line J558L mu 3, CD72 expression reduces the BCR ligation-induced phosphorylation of the BCR component Ig alpha/Ig beta and its cytoplasmic effectors Syk and SLP-65. Substrate phosphorylation was restored by expression of dominant negative mutants of SHP-1, whereas the SHP-1 mutants failed to enhance phosphorylation of the cellular substrates in the absence of CD72. This indicates that SHP-1 is efficiently activated by CD72 but not by other pathways in J558L mu m3 cells and that inhibition of SHP-1 specifically activated by CD72 reverses CD72-induced dephosphorylation of cellular substrates in these cells. Taken together, BCR-induced SHP-1 activation is likely to require inhibitory co-receptors such as CD72, and SHP-1 appears to mediate the negative regulatory effect of CD72 on BCR signaling by dephosphorylating Ig alpha/Ig beta and its downstream signaling molecules Syk and SLP-65.     

Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.     

Specific dephosphorylation of the Lck tyrosine protein kinase at Tyr-394 by the SHP-1 protein-tyrosine phosphatase

The protein-tyrosine phosphatase SHP-1 has been shown to be a negative regulator of multiple signaling pathways in hematopoietic cells. In this study, we demonstrate that SHP-1 dephosphorylates the lymphoid-specific Src family kinase Lck at Tyr-394 when both are transiently co-expressed in nonlymphoid cells. We also demonstrate that a GST-SHP-1 fusion protein specifically dephosphorylates Lck at Tyr-394 in vitro. Because phosphorylation of Tyr-394 activates Lck, the fact that SHP-1 specifically dephosphorylates this site suggests that SHP-1 is a negative regulator of Lck. The failure of SHP-1 to inactivate Lck may contribute to some of the lymphoid abnormalities observed in motheaten mice.     

Evidence for a ligand-mediated positive selection signal in differentiation to a mature B cell

Positive selection is required for B cell differentiation, as indicated by the requirement for expression of the pre-B cell receptor (pre-BCR) and the BCR at the pre-B and immature B cell stages, respectively. Positive selection mediated by a tonic signal from these receptors is sufficient to drive B cell differentiation beyond the pre-B and immature B cell stages, but it is unclear whether additional positive selection signals are required for differentiation to a mature B-2 cell. We have identified a population of Ig transgenic B cells that differentiatively arrest at a transitional B cell stage in the spleen. They exhibit no evidence of Ag encounter or negative selection and can differentiate to mature B-2 cells in vivo upon weak BCR stimulation or adoptive transfer to irradiated hosts. These data are consistent with a requirement for a ligand-mediated BCR signal for differentiation to a mature B-2 cell.     

Superantigen-induced T cell:B cell conjugation is mediated by LFA-1 and requires signaling through Lck, but not ZAP-70.

The formation of a conjugate between a T cell and an APC requires the activation of integrins on the T cell surface and remodeling of cytoskeletal elements at the cell-cell contact site via inside-out signaling. The early events in this signaling pathway are not well understood, and may differ from the events involved in adhesion to immobilized ligands. We find that conjugate formation between Jurkat T cells and EBV-B cells presenting superantigen is mediated by LFA-1 and absolutely requires Lck. Mutations in the Lck kinase, Src homology 2 or 3 domains, or the myristoylation site all inhibit conjugation to background levels, and adhesion cannot be restored by the expression of Fyn. However, ZAP-70-deficient cells conjugate normally, indicating that Lck is required for LFA-1-dependent adhesion via other downstream pathways. Several drugs that inhibit T cell adhesion to ICAM-1 immobilized on plastic, including inhibitors of mitogen-activated protein/extracellular signal-related kinase kinase, phosphatidylinositol-3 kinase, and calpain, do not inhibit conjugation. Inhibitors of phospholipase C and protein kinase C block conjugation of both wild-type and ZAP-70-deficient cells, suggesting that a phospholipase C that does not depend on ZAP-70 for its activation is involved. These results are not restricted to Jurkat T cells; Ag-specific primary T cell blasts behave similarly. Although the way in which Lck signals to enhance LFA-1-dependent adhesion is not clear, we find that cells lacking functional Lck fail to recruit F-actin and LFA-1 to the T cell:APC contact site, whereas ZAP-70-deficient cells show a milder phenotype characterized by disorganized actin and LFA-1 at the contact site.     

Superantigen stimulation reveals the contribution of Lck to negative regulation of T cell activation

The conventional paradigm of T cell activation through the TCR states that Lck plays a critical activating role in this signaling process. However, the T cell response to bacterial superantigens does not require Lck. In this study we report that not only is Lck dispensable for T cell activation by superantigens, but it actively inhibits this signaling pathway. Disruption of Lck function, either by repression of Lck gene expression or by selective pharmacologic inhibitors of Lck, led to increased IL-2 production in response to superantigen stimulation. This negative regulatory effect of Lck on superantigen-induced T cell responses required the kinase activity of Lck and correlated with early TCR signaling, but was independent of immunological synapse formation and TCR internalization. Our data demonstrate that the multistage role of Lck in T cell signaling includes the activation of a negative regulatory pathway of T cell activation.