Beth A. Graf

Cutting Edge: CD28-Mediated Transcriptional and Posttranscriptional Regulation of IL-2 Expression Are Controlled through Different Signaling Pathways

Despite the clear functional importance of CD28 costimulation, the signaling pathways transduced through CD28 have remained controversial. PI3K was identified early as a candidate for CD28 signaling, but conflicting data during the past decade has left the role of PI3K unresolved. In this report, we have resolved this controversy. We show that mutation of the PI3K interaction site in the cytosolic tail of CD28 site disrupts the ability of CD28 to recruit protein kinase C-θ to the central supramolecular activation cluster (c-SMAC) region of the immunological synapse, promote NF-κB nuclear translocation, and enhance IL-2 gene transcription. In contrast, mutation of the PI3K interaction site had no effect on the ability of CD28 to enhance IL-2 mRNA stability. These results suggest that two distinct pathways mediate CD28-induced up-regulation of IL-2 expression, a PI3K-dependent pathway that may function through the immunological synapse to enhance IL-2 transcription and a PI3K-independent pathway that induces IL-2 mRNA stability.

LFA-1-Mediated T Cell Costimulation through Increased Localization of TCR/Class II Complexes to the Central Supramolecular Activation Cluster and Exclusion of CD45 from the Immunological Synapse

T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.

Prostaglandin E2 and cAMP promote B lymphocyte class switching to IgG1

Prostaglandins of the E series (PGE) have traditionally been considered as suppressive for immune responses; however, recent data suggest that PGE channels the immune response towards a T helper 2 type response and production of selected immunoglobulin isotypes. Herein, we present data showing that PGE(2) and other agents that induce intracellular rises in cAMP significantly increased B lymphocyte IgG1 production (up to sevenfold). PGE(2) acted on small resting B cells and on uncommitted B cells expressing high levels of surface IgM to increase the number of cells secreting IgG1. PGE(2) even increased IgG1 synthesis by purified B cells in the absence of exogenous IL-4. Finally, PGE(2) synergized with IL-4 to induce germline gamma1 transcripts through the switch region. This transcription is required for isotype switching. These data support the hypothesis that PGE(2) acts on uncommitted resting B cells at the level of germline gamma1 transcription to promote class switching to IgG1. PGE(2) is an important regulator of the immune response, shifting the balance towards a T helper type 2 response, directing selection of the isotypes produced, and promoting memory cell formation.

Biphenotypic B / macrophage cells express COX‐1 and up‐regulate COX‐2 expression and prostaglandin E2 production in response to pro‐inflammatory signals

B / macrophage cells are biphenotypic leukocytes of unknown function that simultaneously express B lymphocyte (IgM, IgD, B220, CD5) and macrophage (phagocytosis, F4 / 80, Mac‐1) characteristics. B / macrophage cells can be generated from purified mouse B lymphocytes incubated in fibroblast‐conditioned medium. A potential role for B / macrophage cells in inflammation was shown by their ability to express prostaglandin H synthase‐1 (COX‐1) and prostaglandin H synthase‐2 (COX‐2) and by their production of prostaglandin (PG) E2. COX‐1 and COX‐2 mRNA expression is not observed in the precursor B lymphocytes and is not known to be a property of B lineage cells. In contrast, COX‐2 and the prostanoids PGE2, PGF2α and PGD2 are highly inducible in B / macrophage cells upon stimulation with lipopolysaccharide, CD40 ligand, or via engagement of surface IgM, supporting a role for these cells in inflammation. PGD2 and its metabolites are of interest because they activate the nuclear receptor PPARγ that regulates lipid metabolism. The B / macrophage represents the first instance of a normal B‐lineage cell capable of expressing COX‐2. Importantly, B / macrophage cells were identified in vivo, providing evidence that they may play a significant role in immune responses. Since PGE2 blunts IL‐12 production, its synthesis by B / macrophage cells may shift the balance of an immune response towards Th2 and humoral immunity.

Signals and Sequences That Control CD28 Localization to the Central Region of the Immunological Synapse

During T cell interaction with APC, CD28 is recruited to the central region (cSMAC) of the immunological synapse. CD28-mediated signaling through PI3K results in the recruitment of protein kinase C-θ (PKCθ) to the cSMAC, activation of NF-κB, and up-regulation of IL-2 transcription. However, the mechanism that mediates CD28 localization to the cSMAC and the functional consequences of CD28 localization to the cSMAC are not understood. In this report, we show that CD28 recruitment and persistence at the immunological synapse requires TCR signals and CD80 engagement. Addition of mAb to either MHC class II or CD80 results in the rapid displacement of CD28 from the immunological synapse. Ligand binding is not sufficient for CD28 localization to the immunological synapse, as truncation of the cytosolic tail of CD28 disrupts synapse localization without effecting the ability of CD28 to bind CD80. Furthermore, a single point mutation in the CD28 cytosolic tail (tyrosine 188) interferes with the ability of CD28 to preferentially accumulate at the cSMAC. PKCθ distribution at the immunological synapse mirrors the distribution of tyrosine 188-mutated CD28, indicating that CD28 drives the localization of PKCθ even when CD28 is not localized to the cSMAC. Mutation of tyrosine 188 also results in diminished activation of NF-κB, suggesting that CD28-mediated localization of PKCθ to the cSMAC is important for efficient signal transduction. These data reinforce the importance of the interplay of signals between TCR and CD28 and suggest that CD28 signaling through PCKθ may be mediated through localization to the cSMAC region of the immunological synapse.

Prostaglandin E2 as a modulator of lymphocyte mediated inflammatory and humoral responses.

Prostaglandins are a family of structurally related small lipid molecules that can regulate cellular growth, differentiation, and homeostasis. Prostaglandins are primarily derived from arachidonic acid, which is released from the membrane by phospholipases in response to a variety of extrinsic stimuli. Constitutive and inducible forms of cyclooxygenase in conceit with isomerses convert arachidonic acid into different prostaglandins including prostaglandin E2 (PGE2). PGE2 was previously known as an immunosuppressive prostaglandin as it inhibits T cell production of IL-2 and B lymphocyte production of IgM. Recently a new concept emerged that PGE2 is a critical modulator of B and T cell function and is not necessarily immunosuppressive. The focus of this paper is to present new developments that indicate PGE2 functions by promoting antibody driven responses at the expense of inflammatory responses.

Two pathways of costimulation through CD28.

CD28 is recognized as the primary costimulatory molecule involved in the activation of naïve T cells. However, the biochemical signaling pathways that are activated by CD28 and how these pathways are integrated with TCR signaling are still not understood. We have recently shown that there are at least two independent activation pathways induced by CD28 costimulation. One is integrated with TCR signaling in the context of the immunological synapse and is mediated through transcriptional enhancement and the second is mediated through the induction of mRNA stability. Here, we review the immunological consequences and biochemical mechanisms associated with CD28 costimulation and discuss the major questions that need to be resolved to understand the molecular mechanisms that transduce CD28 costimulation.

Proinflammatory Signals Upregulate COX-2 and Increase PGE2 Production in Biphenotypic B/Macrophage Cells

B-lymphocytes and macrophages are considered to be derived from separate lineages and to have specialized functions. However, it has been reported that certain malignant CD5+ B-lymphocytes are capable of differentiating to macrophage-like cells that express characteristics of both cell types.1 Differentiation to such biphenotypic cells has been termed lineage infidelity and has been found to be associated with a variety of diseases such as AIDS, Hodgkin’s lymphoma, and Sjögren’s syndrome.2 While all previous reports of cells exhibiting lineage infidelity had been confined to malignancies and other disease states, our lab has isolated a biphenotypic cell from normal mouse B-lymphocytes.3,4 These cells, called B/macrophage cells, are biphenotypic leukocytes generated from purified mouse B-lymphocytes incubated in mouse splenic fibroblast conditioned medium. B/Macrophage cells simultaneously express CD5+ B-lymphocyte (IgM, IgD, B220, CD5) and macrophage (phagocytosis, F4/80, Mac-1) characteristics. The function of these cells is unknown. The potential role of B/macrophage cells in inflammation and immune responses was explored by evaluating the expression of prostaglandin H synthase-1 (COX-1) and prostaglandin H synthase-2 (COX-2) and the production of PGE2. PGE2 is an important mediator in inflammatory responses. Synthesis of PGE2 is catalyzed by the cyclooxygenases, of which there are two isoforms, COX-1 and COX-2.5 COX-1 is constitutively expressed and is considered to be a housekeeping enzyme important for physiologic regulation. COX-2, on the other hand, is an inducible enzyme that can be upregulated after stimulation with certain cytokines, hormones, and lipid mediators. COX-1 and COX-2 expression are not detected in the starting population of B-lymphocytes. However, COX-1 and COX-2 mRNA are detectable after differentiation to the biphenotypic B/macrophage cells (FIG. 1). To confirm functional cyclooxygenase activity, PGE2 production was evaluated by immunoassay. B/Macrophage cells stimulated with LPS, anti-IgM, anti-IgM plus CD40L, and IFN-γ plus CD40L showed a significant increase in PGE2 production over the untreated sample (TABLE 1). To determine whether the PGE2 produced by these cells was synthesized mainly by COX-2, stimulated cultures were treated with