Memory B cells are generated during an individual's first encounter with a foreign antigen and respond to re-encounter with the same antigen through cell surface immunoglobulin G (IgG) B cell receptors (BCRs) resulting in rapid, high-titered IgG antibody responses. Despite a central role for IgG BCRs in B cell memory, our understanding of the molecular mechanism by which IgG BCRs enhance antibody responses is incomplete. Here, we showed that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ-binding motif, associated with synapse-associated protein 97 (SAP97), a member of the PDZ domain–containing, membrane-associated guanylate-kinase family of scaffolding molecules that play key roles in controlling receptor density and signal strength at neuronal synapses. We showed that SAP97 accumulated and bound to IgG BCRs in the immune synapses that formed in response to engagement of the B cell with antigen. Knocking down SAP97 in IgG-expressing B cells or mutating the putative PDZ-binding motif in the tail impaired immune synapse formation, the initiation of IgG BCR signaling, and downstream activation of p38 mitogen-activated protein kinase. Thus, heightened B cell memory responses are encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immune synapse.

Functionally diverse CD8+ T cells develop in response to antigenic stimulation with differing capacities to couple TCR engagement to downstream signals and functions. However, mechanisms of diversifying TCR signaling are largely uncharacterized. Here we identified two alternative splice variants of scaffold protein Dlg1, Dlg1AB and Dlg1B, that diversify signaling to regulate p38 –dependent and –independent effector functions in CD8+ T cells. Dlg1AB, but not Dlg1B associated with Lck, coupling TCR stimulation to p38 activation and proinflammatory cytokine production. Conversely, both Dlg1AB and Dlg1B mediated p38-independent degranulation. Degranulation depended on a Dlg1 fragment containing an intact Dlg1SH3-domain and required the SH3-ligand WASp. Further, Dlg1 controlled WASp activation by promoting TCR-triggered conformational opening of WASp. Collectively, our data support a model where Dlg1 regulates p38-dependent proinflammatory cytokine production and p38-independent cytotoxic granule release through the utilization of alternative splice variants, providing a mechanism whereby TCR engagement couples downstream signals to unique effector functions in CD8+ T cells.

CD8+ T cells respond to TCR stimulation by producing proinflammatory cytokines, and destroying infected or malignant cells through the production and release of cytotoxic granules. Scaffold protein Discs large homolog 1 (Dlg1) specifies TCR-dependent functions by channeling proximal signals toward the activation of p38-dependent proinflammatory cytokine gene expression and/or p38-independent cytotoxic granule release. Two Dlg1 variants are expressed in CD8+ T cells via alternative splicing, Dlg1AB and Dlg1B, which have differing abilities coordinate TCR-dependent functions. Although both variants facilitate p38-independent cytotoxicity, only Dlg1AB coordinates p38-dependent proinflammatory cytokine expression. In this study, we identify TCR-induced Dlg1 tyrosine phosphorylation as a key regulatory step required for Dlg1AB-mediated p38-dependent functions, including proinflammatory cytokine expression. We find that Dlg1AB but not Dlg1B is tyrosine phosphorylated by proximal tyrosine kinase Lck in response to TCR stimulation. Furthermore, we identify Dlg1 tyrosine 222 (Y222) as a major site of Dlg1 phosphorylation required for TCR-triggered p38 activation and NFAT-dependent expression of proinflammatory cytokines, but not for p38-independent cytotoxicity. Taken together, our data support a model where TCR-induced phosphorylation of Dlg1 Y222 is a key point of control that endows Dlg1AB with the ability to coordinate p38 activation and proinflammatory cytokine production. We propose blocking Dlg1AB phosphorylation as a novel therapeutic target to specifically block proinflammatory cytokine production but not cytotoxicity.

The recognition of antigen by the TCR triggers the polarized recruitment of receptors, signal transducers and cytoskeletal components to the T-cell:APC interface, providing a molecular platform for selectively instructing T effector transcription and polarized secretion. How TCR signals are specified to regulate distinct downstream signaling and functional outputs remains poorly understood. We report that T cells utilize distinct protein variants of Dlgh1, a scaffold that regulates synaptic polarity and signaling, to independently regulate pathways controlling TCR-induced transcriptional activation and secretion of effector molecules. T cells express two Dlgh1 protein variants, Dlgh1 i1A-i1B and Dlgh1 i1B, which differ by inclusion of the alternatively spliced i1A-domain, hypothesized to control binding of the TCR proximal kinase, Lck. Over-expression of Dlgh1 i1A-i1B in T cells selectively enhanced TCR-induced TNFα and NFATc1, but not IκBα or IL-2 mRNA expression, while over-expression of Dlgh1 i1B did not. In contrast, knockdown of the Dlgh1 i1B variant in CD8+ T cells attenuated TCR-induced granzyme B secretion, but did not alter TCR induced granzyme B mRNA upregulation. Knockdown of Dlgh1 i1A-i1B reduced TCR-induced TNFα and NFATc1 mRNA upregulation, but not granzyme B secretion. Therefore, alternative splicing specializes Dlgh1 variants to orchestrate TCR signal specificity, selectively coupling TCR engagement to transcriptional activation or secretion of effectors.

In cytotoxic T lymphocytes (CTLs), engagement of the T cell receptor (TCR) triggers signaling pathways that culminate in cytokine production and cytotoxic killing. TCR signal strength is linked to CTL function with low signal triggering cytotoxicity while high signal leads to both cytotoxicity and cytokine production. Scaffold protein Dlg1 (Discs Large Homolog 1) is a key point of control downstream of the TCR that coordinates both cytokine production and cytotoxic functional output. However, the molecular mechanism by which Dlg1 regulates cytokine production and cytotoxicity in response to TCR signal strength remains elusive. We report that Dlgh1 is tyrosine phosphorylated in response to TCR stimulation, and that phosphorylation is a key step in Dlg1-mediated activation of the alternative p38 pathway and downstream cytokine gene expression. We demonstrate that Dlg1 phosphorylation is mediated by Dlg1-associated Lck, not Zap70; and that Dlg1 Tyr222 is a major site of TCR-induced phosphorylation. Importantly, T cells expressing Dlg1 with a tyrosine to phenylalanine mutation (Y222F) show a defect in TCR-induced activation of the alternative p38 pathway, activation of NFAT and transcriptional upregulation of TNFα and IFNγ genes, but not the transcriptional upregulation of the granzyme B gene or CTL degranulation. These data position Dlg1 phosphorylation as a molecular switch allowing selective activation of p38-dependent events in response to the strength of the TCR signal.

CD8+ cytotoxic T lymphocytes (CTLs) are a critical component of the adaptive immune system because they mediate contact-dependent cytolysis and produce proinflammatory cytokines. Although both of these CTL activities require T cell receptor (TCR) activation these functions are not always coordinately invoked, suggesting that signaling pathways downstream of the TCR can be selectively activated to guide specific functional outcomes. Here we investigated the role of Discs large homolog 1 (DLG1) alternative splice variants, DLG1 i1A-i1B and DLG1 i1B, in coupling proximal TCR signals to select downstream pathways to specify cytotoxicity and proinflammatory cytokine transcription in CD8+ T cells. We found that DLG1 i1A-i1B, but not DLG1 i1B selectively directs TCR-induced p38/NFAT-dependent proinflammatory cytokine production by binding Lck, promoting alternative p38 activation and downstream NFAT mediated upregultion of IFNγ and TNFα gene expression. Conversely, DLG1 i1A-i1B mediated alternative p38 activation had no effect on actin polymerization, granzyme B gene expression, granzyme B release, or degranulation. Rather, either DLG1 variant expressed in CD8+ T cells promoted antigen-induced degranulation, suggesting that both DLG1 variants have p38-independent activity guiding granule-dependent cytotoxicity. Thus CD8+ T cells contain two distinct DLG1 scaffolds that act as molecular conduits for proximal TCR signals to allow differential CTL functional responses.

Stimulation through the T cell receptor (TCR) leads to rapid activation of proximal tyrosine kinases, Lck and ZAP70, which couple to distinct downstream signaling cascades and functional outcomes. We have shown that MAGUK family scaffold protein Discs Large Homolog 1 (Dlgh1) juxtaposes activated Lck and ZAP70 with p38 MAP kinase to selectively activate the alternative p38 pathway and downstream NFAT; but not NFκB. Lck, ZAP70 and p38 are known to complex with Dlgh1 in response TCR engagement. However, Lck and ZAP70 binding sites within Dlgh1 and the mechanism by which Dlgh1 positions and activates Lck and ZAP70 allowing p38 activation remain incompletely understood. Knowing YxxL/I is a preferred motif for Lck tyrosine phosphorylation and that SH2 domains of ZAP70 and Lck prefer to bind Lck-phosphorylated substrates; we identified Dlgh1 Y222 as a potential target of phosphorylation and/or binding. We subsequently assessed the potential role of Dlgh1 Y222 phosphorylation in coordinating the alternative p38 pathway. Preliminary studies using rLck, rZAP70 and GST-Dlgh1 Y222F fusion proteins demonstrate that Y222 is a primary site of Lck phosphorylation required for optimal alternative p38 activation. Experiments are underway to assess the role of Dlgh1 Y222 in Lck and ZAP70 binding and alternative p38 activation in primary T cells. These experiments will elucidate molecular mechanisms behind Dlgh1 coordination of the alternative p38 pathway and TCR signal specificity.