T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse

The ability of antigen receptors to engage self-ligands with varying affinity is crucial for lymphocyte development. To further explore this concept, we generated transgenic mice expressing GFP from the immediate early gene Nr4a1 (Nur77) locus. GFP was up-regulated in lymphocytes by antigen receptor stimulation but not by inflammatory stimuli. In T cells, GFP was induced during positive selection, required major histocompatibility complex for maintenance, and directly correlated with the strength of T cell receptor (TCR) stimulus. Thus, our results define a novel tool for studying antigen receptor activation in vivo. Using this model, we show that regulatory T cells (T(reg) cells) and invariant NKT cells (iNKT cells) perceived stronger TCR signals than conventional T cells during development. However, although T(reg) cells continued to perceive strong TCR signals in the periphery, iNKT cells did not. Finally, we show that T(reg) cell progenitors compete for recognition of rare stimulatory TCR self-ligands.     

Receptor editing and marginal zone B cell development are regulated by the helix-loop-helix protein, E2A

Previous studies have indicated that the E2A gene products are required to initiate B lineage development. Here, we demonstrate that E2A(+/-) B cells that express an autoreactive B cell receptor fail to mature due in part to an inability to activate secondary immunoglobulin (Ig) light chain gene rearrangement. Both RAG1/2 gene expression and RS deletion are severely defective in E2A(+/-) mice. Additionally, we demonstrate that E2A(+/-) mice show an increase in the proportion of marginal zone B cells with a concomitant decrease in the proportion of follicular B cells. In contrast, Id3-deficient splenocytes show a decline in the proportion of marginal zone B cells. Based on these observations, we propose that E-protein activity regulates secondary Ig gene rearrangement at the immature B cell stage and contributes to cell fate determination of marginal zone B cells. Additionally, we propose a model in which E-proteins enforce the developmental checkpoint at the immature B cell stage.     

B cell activation. III. B cell plasma membrane depolarization and hyper- Ia antigen expression induced by receptor immunoglobulin cross-linking are coupled

We report investigation of the relationship between ligand-induced B cell plasma membrane depolarization and increased expression of membrane-associated, I-A subregion encoded (mI-A) antigens. Results demonstrate that equal frequencies of B cells are stimulated to undergo membrane depolarization and to increase mI-A expression in response to mitogen, anti-Ig, and thymus-independent (TI) or thymus-dependent (TD) antigens. Further, a cause-and-effect relationship between these two events is suggested by results that demonstrate that inhibition of anti- Fab--induced depolarization by valinomycin also inhibits the subsequent increase in mI-A antigen expression and "passive" (non-ligand-mediated) depolarization of murine B cells by K+ results in hyper-mI-A antigen expression. Based upon these results we hypothesize that antigen- mediated receptor cross-linking results in signal transduction via membrane depolarization, which is resultant in increased mI-A antigen synthesis and cell surface expression. This increase in mI-A antigen density may render the B cell more receptive to subsequent interaction with I-region-restricted helper T cells.     

RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement

Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603-607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837-847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-alpha, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.     

B cell antigen receptor specificity and surface density together determine B-1 versus B-2 cell development.

Mice expressing the immunoglobulin (Ig) heavy (H) chain variable (V) region from a rearranged V(H)12 gene inserted into the IgH locus generate predominantly B-1 cells, whereas expression of two other V(H) region transgenes (V(H)B1-8 and V(H)glD42) leads to the almost exclusive generation of conventional, or B-2, cells. To determine the developmental potential of B cells bearing two distinct B cell antigen receptors (BCRs), one favoring B-1 and the other favoring B-2 cell development, we crossed V(H)12 insertion mice with mice bearing either V(H)B1-8 or V(H)glD42. B cells coexpressing V(H)12 and one of the other V(H) genes are readily detected in the double IgH insertion mice, and are of the B-2 phenotype. In mice coexpressing V(H)12, V(H)B1-8 and a transgenic kappa chain able to pair with both H chains, double H chain-expressing B-2 cells, and B-1 cells that have lost V(H)B1-8 are generated, whereas V(H)B1-8 single producers are undetectable. These data suggest that B-1 but not B-2 cells are selected by antigenic stimuli in whose delivery BCR specificity and surface density are of critical importance.     

CTLA-4 is a second receptor for the B cell activation antigen B7

Functional interactions between T and B lymphocytes are necessary for optimal activation of an immune response. Recently, the T lymphocyte receptor CD28 was shown to bind the B7 counter-receptor on activated B lymphocytes, and subsequently to costimulate interleukin 2 production and T cell proliferation. CTLA-4 is a predicted membrane receptor from cytotoxic T cells that is homologous to CD28 and whose gene maps to the same chromosomal band as the gene for CD28. It is not known, however, if CD28 and CTLA-4 also share functional properties. To investigate functional properties of CTLA-4, we have produced a soluble genetic fusion between the extracellular domain of CTLA-4 and an immunoglobulin C gamma chain. Here, we show that the fusion protein encoded by this construct, CTLA4Ig, bound specifically to B7-transfected Chinese hamster ovary cells and to lymphoblastoid cells. CTLA4Ig also immunoprecipitated B7 from cell surface 125I-labeled extracts of these cells. The avidity of 125I-labeled B7Ig fusion protein for immobilized CTLA4Ig was estimated (Kd approximately 12 nM). Finally, we show that CTLA4Ig was a potent inhibitor of in vitro immune responses dependent upon cellular interactions between T and B lymphocytes. These findings provide direct evidence that, like its structural homologue CD28, CTLA-4 is able to bind the B7 counter-receptor on activated B cells. Lymphocyte interactions involving the B7 counter-receptor are functionally important for alloantigen responses in vitro.     

Stimulation of CD5 enhances signal transduction by the T cell antigen receptor.

After the addition of a CD3 monoclonal antibody to peripheral T cells that have been previously stimulated with phytohemagglutinin, inositol phosphates are produced at a rapid rate for 2 min and at a much slower rate thereafter. Stimulation of CD5 allows CD3-mediated production of inositol phosphates to be sustained at a brisk rate for greater than 20 min and augments the initial CD3-mediated increase in inositol trisphosphate and release of intracellular Ca2+. Thus, perturbation of CD5 by monoclonal antibody enhances the ability of the CD3-antigen receptor complex to couple to the inositol phospholipid pathway. This effect of CD5 is independent of any direct effect of the CD5 monoclonal antibody on the levels of inositol phosphates.     

Allogeneic T cell responses are regulated by a specific miRNA-mRNA network

Donor T cells that respond to host alloantigens following allogeneic bone marrow transplantation (BMT) induce graft-versus-host (GVH) responses, but their molecular landscape is not well understood. MicroRNAs (miRNAs) regulate gene (mRNA) expression and fine-tune the molecular responses of T cells. We stimulated naive T cells with either allogeneic or nonspecific stimuli and used argonaute cross-linked immunoprecipitation (CLIP) with subsequent ChIP microarray analyses to profile miR responses and their direct mRNA targets. We identified a unique expression pattern of miRs and mRNAs following the allostimulation of T cells and a high correlation between the expression of the identified miRs and a reduction of their mRNA targets. miRs and mRNAs that were predicted to be differentially regulated in allogeneic T cells compared with nonspecifically stimulated T cells were validated in vitro. These analyses identified wings apart-like homolog (Wapal) and synaptojanin 1 (Synj1) as potential regulators of allogeneic T cell responses. The expression of these molecular targets in vivo was confirmed in MHC-mismatched experimental BMT. Targeted silencing of either Wapal or Synj1 prevented the development of GVH response, confirming a role for these regulators in allogeneic T cell responses. Thus, this genome-wide analysis of miRNA-mRNA interactions identifies previously unrecognized molecular regulators of T cell responses.     

Isolation and characterization of a B lymphocyte mutant with altered signal transduction through its antigen receptor

A receptor surface Ig (sIg) signaling variant of WEHI-231 was constructed to investigate components and linkages between various signaling events associated with signal transduction through sIg. Unlike the wildtype, crosslinking of sIgM on VS2.12-cl.2 did not result in downregulation of proliferation. Similarly, receptor crosslinking was uncoupled from inositol phospholipid (PI) hydrolysis and upregulation of c-fos expression in the variant. The signaling defect in VS2.12-cl.2 appears to be proximal to phospholipase C activation as direct G protein activation by A1F4- triggers PI hydrolysis and bypassing PI hydrolysis using phorbol diester stimulation of protein kinase C restores the inhibitable phenotype and the ability to upregulate c-fos. Even more interesting, sIg-linked Ca2+ responses by VS2.12-cl.2 are equivalent to these observed in the wildtype WEHI-231. These latter results suggest that contrary to current thought, sIg-generated signals may not be coupled to Ca2+ fluxes entirely via inositol phospholipid hydrolysis. Thus, VS2.12-cl.2 is a new and powerful tool with which to analyze signaling through sIg at the molecular level.     

A role for lipid rafts in B cell antigen receptor signaling and antigen targeting

The B cell antigen receptor (BCR) serves both to initiate signal transduction cascades and to target antigen for processing and presentation by MHC class II molecules. How these two BCR functions are coordinated is not known. Recently, sphingolipid- and cholesterol-rich plasma membrane lipid microdomains, termed lipid rafts, have been identified and proposed to function as platforms for both receptor signaling and membrane trafficking. Here we show that upon cross-linking, the BCR rapidly translocates into ganglioside G(M1)-enriched lipid rafts that contain the Src family kinase Lyn and exclude the phosphatase CD45R. Both Igalpha and Lyn in the lipid rafts become phosphorylated, and subsequently the BCR and a portion of G(M1) are targeted to the class II peptide loading compartment. Entry into lipid rafts, however, is not sufficient for targeting to the antigen processing compartments, as a mutant surface Ig containing a deletion of the cytoplasmic domain is constitutively present in rafts but when cross-linked does not internalize to the antigen processing compartment. Taken together, these results provide evidence for a role for lipid rafts in the initial steps of BCR signaling and antigen targeting.     

Functional characterization of a signal transducing motif present in the T cell antigen receptor zeta chain

A conserved sequence motif has been identified in a number of signaling subunits associated with hematopoietic cell antigen receptors. Here, we characterize signaling by a 17 amino acid motif that is triplicated in the T cell antigen receptor zeta chain. Analysis of zeta truncations and constructs containing the isolated motif demonstrates that this motif is sufficient for the induction of both proximal and distal events associated with T cell activation. Stimulation of truncations that contain either one, two, or three copies of the motif results in induction of an identical pattern of tyrosine phosphoproteins. Moreover, triplication of the NH2-terminal zeta motif results in enhanced signaling, suggesting a redundant role in signal amplification for the three motifs in zeta. Finally, we demonstrate the association of a recently identified protein tyrosine kinase ZAP-70 with this motif, and provide evidence for its involvement in zeta function.     

Signaling capacity of the T cell antigen receptor is negatively regulated by the PTP1C tyrosine phosphatase

The association of PTP1C deficiency with the multiplicity of lymphoid cell abnormalities manifested by motheaten (me) and viable motheaten (me(v)) mice suggests a pivotal role for this tyrosine phosphatase in the regulation of lymphocyte differentiation and function. To delineate the relevance of PTP1C to T cell physiology, we have examined me and me(v) T cells with regards to their capacity to transduce activating signals through the T cell antigen receptor (TCR). Although thymocyte maturation appeared normal in the mutant mice, both thymocytes and peripheral T cells from these animals exhibited proliferative response to TCR stimulation that were markedly increased relative to those elicited in normal cells. Compared to normal thymocytes, PTP1C- deficient thymocytes also showed increased constitutive tyrosine phosphorylation of the TCR complex and enhanced and prolonged TCR- induced tyrosine phosphorylation of the TCR-zeta and CD3-epsilon, as well as a number of cytosolic proteins, most notably a 38-kD phosphoprotein found to associate with the Grb2 adaptor SH2 domain in activated thymocytes. These latter phosphoproteins also associated with the Vav guanine nucleotide exchange factor upon TCR ligation, and were dephosphorylated by recombinant PTP1C in vitro. In conjunction with the finding of PTP1C-TCR association in unstimulated normal thymocytes, these results reveal the capacity of PTP1C to interact with and likely dephosphorylate resting and activated TCR complex components, as well as more distal signaling effectors that are normally recruited to the Vav and Grb2 SH2 domains after TCR stimulation. These data therefore strongly implicate PTP1C in the downregulation of TCR signaling capacity and, taken together with the aberrant prolongation of TCR- induced, mitogen-associated kinase (MAPK) activation observed in PTP1C- deficient thymocytes, these findings suggest that the inhibitory influence of PTP1C on TCR signal relay is realized through its effects on both the TCR complex and downstream signaling elements that couple the activated antigen receptor to the Ras/MAPK response pathway.     

Schisandrin B suppresses glioma cell metastasis mediated by inhibition of mTOR/MMP-9 signal pathway

BackgroundMalignant glioma is the aggressive tumor in the brain and is characterized by high morbidity, high mortality. The main purpose of the present study was to investigate the therapeutic effects of Schisandrin B on glioma cells and preliminary explore the possible mechanism underlying anti-metastasis of Schisandrin B.MethodsTwo glioma cell lines, U251 and U87, were used in present study. The ability of metastasis of glioma cells was evaluated using transwell migration assay and invasion assay. Expression of Akt, mTOR, MMP-2 and MMP-9 was determined using Western blotting. Antagonist or agonist was used to activated or inactivated signal molecules.ResultsSchisandrin B suppressed cell migration and invasion in manner of dose dependent as well as inhibited expression of p-Akt, p-mTOR and MMP-9. Activation of PI3K by 740Y-P treatment leaded to upregulation of p-Akt, mTOR and MMP-9; inactivation of mTOR by Rapamycin treatment inhibited expression MMP-9 while activation of mTOR by l-Leucine treatment enhanced MMP-9 expression in Schisandrin B incubated cells. Anti-migration and invasion action of Schisandrin B was also reversed by mTOR activation.ConclusionOur findings demonstrate that Schisandrin B can suppress migration and invasion of glioma cell via PI3K/Akt-mTOR-MMP-9 signaling pathway.     

Sialic acid binding domains of CD22 are required for negative regulation of B cell receptor signaling

CD22, a negative regulator of B cell antigen receptor signaling, binds glycoconjugates terminating in alpha2, 6 sialic acid. The physiological ligand(s) for CD22 remain unknown. We asked whether the sialic acid binding domains are necessary for CD22 to function as a negative regulator. We generated two mutants that lack sialic acid binding activity and expressed them in a novel CD22(-/-) murine B cell line. Anti-IgM activated B cells expressing either CD22 mutant had greater Ca(2+) responses than cells expressing wild-type CD22. Each variant also had reduced CD22 tyrosine phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1 association. These data suggest that the alpha2, 6 sialic acid ligand binding activity of CD22 is critical for its negative regulatory functions.     

Coupling between B cell receptor and phospholipase C-gamma2 is essential for mature B cell development

Two signaling pathways known to be essential for progression from immature to mature B cells are BAFF receptor (BAFF-R) and the B cell receptor (BCR). Here, we first show that phospholipase C (PLC)-gamma2 is required for a BAFF-R-mediated survival signal. Then, we have examined the question of whether the reduced number of mature B cells in PLC-gamma2-/- mice is caused by a defect in either BCR or BAFF-R signaling. We find that a PLC-gamma2 SH2 mutant, which inhibits coupling between BCR and PLC-gamma2, fails to restore B cell maturation, despite supporting BAFF-dependent survival. Therefore, our data suggest that the BAFF-R-mediated survival signal, provided by PLC-gamma2, is not sufficient to promote B cell maturation, and that, in addition, activation of PLC-gamma2 by BCR is required for B cell development.     

The B cell antigen receptor controls integrin activity through Btk and PLCgamma2

Integrin-mediated adhesion and B cell antigen receptor (BCR) signaling play a critical role in B cell development and function, including antigen-specific B cell differentiation. Here we show that the BCR controls integrin alpha4beta1 (VLA-4)-mediated adhesion of B cells to vascular cell adhesion molecule-1 and fibronectin. Molecular dissection of the underlying signaling mechanism by a combined biochemical, pharmacological, and genetic approach demonstrates that this BCR-controlled integrin-mediated adhesion requires the (consecutive) activation of Lyn, Syk, phosphatidylinositol 3-kinase, Bruton's tyrosine kinase (Btk), phospholipase C (PLC)gamma2, IP3R-mediated Ca2+ release, and PKC. In contrast, activation of mitogen-activated protein kinase kinase (MEK) or extracellular signal-regulated kinase (ERK) is not required, and simultaneous activation of MEK, ERK, and PKB is not sufficient either. Furthermore, Btk is also involved in the control of integrin-mediated adhesion of preB cells. The control of integrin alpha4beta1-mediated B cell adhesion by the BCR involves cytoskeletal reorganization and integrin clustering. These results reveal a novel function for the BCR and Btk, i.e., regulation of integrin alpha4beta1 activity, thereby providing new insights into the control of B cell development and differentiation, as well as into the pathogenesis of the immunodeficiency disease X-linked agammaglobulineamia (XLA).     

T cell accumulation in B cell follicles is regulated by dendritic cells and is independent of B cell activation

We investigated the mechanism of CD4 T cell accumulation in B cell follicles after immunization. Follicular T cell numbers were correlated with the number of B cells, indicating B cell control of the niche that T cells occupy. Despite this, we found no role for B cells in the follicular migration of T cells. Instead, T cells are induced to migrate into B cell follicles entirely as a result of interaction with dendritic cells (DCs). Migration relies on CD40-dependent maturation of DCs, as it did not occur in CD40-deficient mice but was reconstituted with CD40(+) DCs. Restoration was not achieved by the activation of DCs with bacterial activators (e.g., lipopolysaccharide, CpG), but was by the injection of OX40L-huIgG1 fusion protein. Crucially, the up-regulation of OX40L (on antigen-presenting cells) and CXCR-5 (on T cells) are CD40-dependent events and we show that T cells do not migrate to follicles in immunized OX40-deficient mice.