Cutting edge: CD40 engagement eliminates the need for Bruton's tyrosine kinase in B cell receptor signaling for NF-kappa B

The Tec kinase Bruton's tyrosine kinase (Btk) represents a key intermediary for B cell receptor (BCR) signaling. Btk mutation produces B cell deficiency in mice with X-linked immunodeficiency (xid), and surface Ig-mediated responses of mature B cells are seriously deranged. The central role that Btk plays in directing downstream events produced by BCR engagement is demonstrated by the complete failure of NF-kappa B induction and cellular proliferation following anti-Ig treatment of B cells obtained from xid mice. In this study, we report that the block in BCR signaling produced by Btk mutation is reversed by CD40 engagement. Prior treatment with CD40 ligand normalized subsequent responses of xid B cells to BCR cross-linking, so that typical outcomes of BCR signaling such as NF-kappa B activation and cell cycle progression occurred in a Btk-independent fashion. These results demonstrate that a specific genetic lesion interrupting BCR-mediated intracellular signaling is circumvented through stimulation of CD40.     

The adaptor protein BLNK is required for b cell antigen receptor-induced activation of nuclear factor-kappa B and cell cycle entry and survival of B lymphocytes

B lymphocytes lacking the adaptor protein B cell linker (BLNK) do not proliferate in response to B cell antigen receptor (BCR) engagement. We demonstrate here that BCR-activated BLNK(-)/- B cells fail to enter the cell cycle, and this is due to their inability to induce the expression of the cell cycle regulatory proteins such as cyclin D2 and cyclin-dependent kinase 4. BCR-stimulated BLNK(-)/- B cells also do not up-regulate the cell survival protein Bcl-x(L), which may be necessary for the cells to complete the cell cycle. In addition, BLNK(-)/- B cells exhibit a high rate of spontaneous apoptosis in culture. Examination of the various BCR-activated signaling pathways in mouse BLNK(-)/- B cells reveals the intact activation of Akt and mitogen-activated protein kinases but the impaired activation of nuclear factor (NF)-kappaB that is known to regulate genes involved in cell proliferation and survival. The inability to activate NF-kappaB in BCR-stimulated BLNK(-)/- B cells is due to a failure to induce the degradation of the inhibitory kappaB protein. In all these aspects, BLNK(-)/- B cells resemble xid B cells that have a mutation in Bruton's tyrosine kinase (Btk). Recently, phospholipase C (PLC)-gamma2 has also been demonstrated to be essential for NF-kappaB activation. Since BLNK has been shown separately to interact with both Btk and PLC-gamma2, our finding of normal Btk but impaired PLC-gamma2 activation in BCR-stimulated BLNK(-)/- B cells strongly suggests that BLNK orchestrates the formation of a Btk-PLC-gamma2 signaling axis that regulates NF-kappaB activation. Taken together, the NF-kappaB activation defect may be sufficient to explain the similar defects in BCR-induced B cell proliferation and T cell-independent immune responses in BLNK(-)/-, Btk(-)/-, and PLC-gamma2(-)/- mice.     

IL-7-regulated homeostatic maintenance of recent thymic emigrants in association with caspase-mediated cell proliferation and apoptotic cell death

Homeostasis of T cells is essential to the maintenance of the T cell pool and TCR diversity. In this study, mechanisms involved in the regulation of cytokine-mediated expansion of naive T cells in the absence of Ag, in particular the role of caspase activation and susceptibility to apoptosis of recent thymic emigrants (RTEs), were examined. Low level caspase-8 and caspase-3 activation was detected in proliferating IL-7-treated cells in the absence of cell death during the first days of culture. Caspase inhibitors suppressed IL-7-induced expansion of RTEs. Low level expression of CD95 and blocking Ab experiments indicated that this early caspase activation was CD95 independent. However, CD95 levels subsequently became dramatically up-regulated on proliferating naive T cells, and these cells became susceptible to CD95 ligation, resulting in high level caspase activation and apoptotic cell death. These results show a dual role for caspases in proliferation and in CD95-induced cell death during Ag-independent expansion of RTEs. This method of cell death in IL-7-expanded RTEs is a previously unrecognized mechanism for the homeostatic control of expanded T cells.     

Il-12 enhances CD8 T cell homeostatic expansion

The size of the T lymphocyte pool is maintained by regulation of T cell production, proliferation, and survival. Under the pressure of a T lymphopenic environment, mature naive T cells begin to proliferate in the absence of Ag, a process called homeostatic expansion. Homeostatic expansion involves TCR recognition of self peptide/MHC ligands, but less is known about the soluble factors that regulate this process. Here we show that IL-12 dramatically enhanced the homeostatic proliferation of CD8 T cells. In contrast, IL-2 had no beneficial effect on homeostatic expansion and, in fact, inhibited T cell expansion induced by IL-12. Using gene-targeted mice, we showed that IL-12 acted directly on the T cells to enhance homeostatic expansion, but that IL-12 cannot override the requirement for TCR interaction with self peptide/MHC ligands in homeostatic expansion. These data indicate that inflammatory cytokines may modulate T cell homeostasis after lymphopenia and have implications for regulation of the T cell repertoire and autoimmunity.     

Bruton's tyrosine kinase mediates NF-kappa B activation and B cell survival by B cell-activating factor receptor of the TNF-R family

Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-kappaB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IkappaB kinase gamma-containing complexes and defective IkappaBalpha degradation. In addition, Btk-deficient B cells produce reduced levels of NF-kappaB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-kappaB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-kappaB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-kappaB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-kappaB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.     

Cutting edge: naive T cells masquerading as memory cells

This study shows that naive CD8 T cells can acquire characteristics of memory T cells in the absence of stimulation with specific Ag simply by the process of homeostatic proliferation under lymphopenic conditions. This Ag-independent T cell differentiation pathway did not result in up-regulation of early activation markers (CD69, CD25, CD71), but expression of several memory markers (CD44, CD122, Ly6C) increased progressively with successive divisions. These markers were then stably expressed, and these cells also became more responsive functionally to specific Ag. Thus, all "memory" phenotype T cells in an individual may not be true Ag-experienced cells and may include naive cells masquerading as memory cells. These findings are specially relevant in cases of disease or treatment-induced lymphopenia such as in HIV-infected individuals or transplant recipients. In addition, this study may have implications for autoimmunity because homeostatic proliferation of naive T cells requires interaction with self peptide plus MHC molecules.     

Crystal structure of Bruton's tyrosine kinase domain suggests a novel pathway for activation and provides insights into the molecular basis of X-linked agammaglobulinemia

Bruton's tyrosine kinase is intimately involved in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B lineage lymphoid cells. Mutations in the human btk gene are the cause of X-linked agammaglobulinemia, a male immune deficiency disorder characterized by a lack of mature, immunoglobulin-producing B lymphocytes. We have determined the x-ray crystal structure of the Bruton's tyrosine kinase kinase domain in its unphosphorylated state to a 2.1 A resolution. A comparison with the structures of other tyrosine kinases and a possible mechanism of activation unique to Bruton's tyrosine kinase are provided.     

Unique signaling properties of B cell antigen receptor in mature and immature B cells: implications for tolerance and activation

Immature B cells display increased sensitivity to tolerance induction compared with their mature counterparts. The molecular mechanisms underlying these differences are poorly defined. In this study, we demonstrate unique maturation stage-dependent differences in B cell Ag receptor (BCR) signaling, including BCR-mediated calcium mobilization responses. Immature B cells display greater increases in intracellular calcium concentrations following Ag stimulation. This has consequences for the induction of biologically relevant responses: immature B cells require lower Ag concentrations for activation than mature B cells, as measured by induction of receptor editing and CD86 expression, respectively. BCR-induced tyrosine phosphorylation of CD79a, Lyn, B cell linker protein, and phospholipase Cgamma2 is enhanced in immature B cells and they exhibit greater capacitative calcium entry in response to Ag. Moreover, B cell linker protein, Bruton's tyrosine kinase, and phospholipase Cgamma2, which are crucial for the induction of calcium mobilization responses, are present at approximately 3-fold higher levels in immature B cells, potentially contributing to increased mobilization of calcium. Consistent with this possibility, we found that the previously reported lack of inositol-1,4,5-triphosphate production in immature B cells may be explained by enhanced inositol-1,4,5-triphosphate breakdown. These data demonstrate that multiple mechanisms guarantee increased Ag-induced mobilization of calcium in immature B cells and presumably ensure elimination of autoreactive B cells from the repertoire.     

A role for TCR affinity in regulating naive T cell homeostasis

Homeostatic signals that control the overall size and composition of the naive T cell pool have recently been identified to arise from contact with self-MHC/peptide ligands and a cytokine, IL-7. IL-7 presumably serves as a survival factor to keep a finite number of naive cells alive by preventing the onset of apoptosis, but how TCR signaling from contact with self-MHC/peptide ligands regulates homeostasis is unknown. To address this issue, murine polyclonal and TCR-transgenic CD8+ cells expressing TCR with different affinities for self-MHC/peptide ligands, as depicted by the CD5 expression level, were analyzed for their ability to respond to and compete for homeostatic factors under normal and lymphopenic conditions. The results suggest that the strength of the TCR affinity determines the relative "fitness" of naive T cells to compete for factors that support cell survival and homeostatic proliferation.     

Naive human T cells are activated and proliferate in response to the heme oxygenase-1 inhibitor tin mesoporphyrin

Heme oxygenase-1 (HO-1) and its catabolic by-products have potent anti-inflammatory activity in many models of disease. It is not known, however, if HO-1 also plays a role in the homeostatic control of T cell activation and proliferation. We demonstrate here that the HO-1 inhibitor tin mesoporphyrin (SnMP) induces activation, proliferation, and maturation of naive CD4(+) and CD8(+) T cells via interactions with CD14(+) monocytes in vitro. This response is dependent upon interactions of T cells with MHC class I and II on the surface of CD14(+) monocytes. Furthermore, CD4(+)CD25(+)FoxP3(+) regulatory T cells were able to suppress this proliferation, even though their suppressive activity was itself impaired by SnMP. Given the magnitude of the Ag-independent T cell response induced by SnMP, we speculate that HO-1 plays an important role in dampening nonspecific T cell activation. Based on these findings, we propose a potential role for HO-1 in the control of naive T cell homeostatic proliferation.     

Requirement for B cells for activation of contrasuppressor T cells by type III pneumococcal polysaccharide

Type III pneumococcal polysaccharide (S3) is unable to activate S3-specific contrasuppressor T cells (Tcs) in mice depleted of B cells by chronic anti-IgM treatment or in immune defective xid mice that lack the B cell subset required for anti-S3 antibody responses. The inability of S3 to activate Tcs in xid mice was shown to be due to a requirement of B cells for Tcs activation rather than to an absence of Tcs in xid mice. The B cells from normal mice that are required for Tcs activation apparently function to present the S3 Ag to Tcs. S3 physically coupled to spleen cells (S3-SC) prepared from normal BCF1 SC could activate Tcs in both xid and BCF1 mice whereas S3-SC prepared from xid SC or B cell-depleted BCF1 SC could not activate Tcs in either strain. B cell APC function was abrogated by 3000 R irradiation and by treatment of the B cells with either chloroquine or paraformaldehyde. Interestingly, B cells from mice previously immunized with S3 were unable to function in Tcs activation; preimmunization of B cell donors with an irrelevant Ag or with a T-dependent form of S3 had no effect on their ability to function as APC. These latter observations are discussed in terms of the in vivo persistence of polysaccharide Ag and their ability to induce B cell tolerance under the experimental conditions used for these experiments. The results of this study provide evidence that B cells play an important and apparently obligatory role in the activation of Tcs by S3; B cells apparently function to present Ag to Tcs, resulting in the activation of this regulatory T cell subset.     

PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization

Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates the threshold for immunoreceptor signaling and thereby modulates lymphocyte activation.     

Synergistic B cell activation by CD40 and the B cell antigen receptor: role of B lymphocyte antigen receptor-mediated kinase activation and tumor necrosis factor receptor-associated factor regulation

Optimal activation of B-lymphocytes depends both upon expression of various cell surface receptors and adequate integration of signaling pathways. This requires signals generated upon recognition of antigen by the B lymphocyte antigen receptor (BCR) as well as additional signals provided by cognate interaction with T helper cells, including the CD40-CD154 interaction. Engagement of both the BCR and CD40 results in synergistic activation of B cells. Previous studies identified tumor necrosis factor receptor-associated factor (TRAF)-2 and TRAF3 in the CD40-signaling pathway together with BCR-activated protein kinase D (PKD) as important cooperative factors in this synergy. To better understand the role of these factors in bridging the BCR and CD40 signaling pathways, BCR signal regulation of TRAF function was examined. Results show that phosphorylation of TRAF2 is increased upon BCR but not CD40 engagement and that of the potentially phosphorylated residues of TRAF2, tyrosine 484 is crucial for BCR-CD40 synergy. Additionally, wild type or constitutively active Bruton's tyrosine kinase (Btk) enhanced, whereas the xid mutant form of Btk prevented, BCR-CD40 synergy. These effects were dependent upon TRAF2 and PKD activity. These findings suggest a model in which Btk contributes to the enhancement of the CD40 response by TRAF2 in a PKD-dependent manner.     

A closer look at homeostatic proliferation of CD4+ T cells: costimulatory requirements and role in memory formation

Ag-specific proliferation of CD4+ T cells is regulated, in part, by costimulatory signals through CD28. The proliferative response during primary activation is an important determinant of the ability of the T cell to respond to Ag re-encounter. Proliferation of mature CD4+ T cells during lymphopenia (homeostatic proliferation) requires interaction with endogenous peptide MHC. However, the role of costimulation during homeostatic proliferation is unclear, as is the ability of homeostatic proliferation to regulate secondary T cell responses. Using a TCR transgenic system and serial adoptive transfers we find that homeostatic proliferation of CD4+ T cells occurs for at least 5 wk after adoptive transfer into recombination-activating gene (RAG)-/- recipients. Two discrete populations of proliferating T cells can be resolved, one that is highly proliferative and dependent on CD28 signaling, and the other that contains cells undergoing low levels of CD28-independent proliferation. Importantly, naive CD4+ T cells that have undergone homeostatic proliferation acquire both phenotypic and functional characteristics of true memory cells. These studies indicate that functional memory T cells can be generated by encounters with endogenous Ags only. This mechanism of T cell regeneration is possibly active during lymphopenia due to viral infections, such as HIV, transplantation, or cancer therapy, and may explain selected autoimmune diseases.