Prolonged costimulation is required for naive T cell activation

Costimulation by members of the B7 family of molecules is critical for the activation of naive CD4+ T cells. While prolonged TCR signaling is necessary for T cell activation, the duration of costimulatory signals required has not been established. In this study, murine bone marrow-derived dendritic cells (DC) and na?ve CD4+ T cells were used to determine the temporal costimulatory requirements for naive T cell activation. By blocking CD80/CD86 costimulation at various time points during DC-T cell interaction and using the CFSE technique to assess the dynamics of T cell proliferation, we found that prolonged costimulation was required for naive T cells to enter and progress through the cell cycle over a wide range of peptide concentrations. Prolonged costimulation was also important for IL-2 production and CD25/CD69 expression by naive T cells. Video microscopy demonstrated that DC and naive T cells formed stable conjugates that persisted for more than 6 h. Thus, persistent CD80/CD86 signaling during prolonged interactions with DC allows naive T cells to enter the cell cycle and programs the daughter cells to undergo subsequent divisions.     

T cell receptor α-chain tail is required for protein kinase C-mediated down-regulation,but not for signaling

Antigen stimulation through the T cell receptor (TCR) induces phosphorylation of the associated CD3 γδσ- and ζ-chain cytoplasmic tails. These events lead to the induction of the intracellular signaling pathways with concomitant receptor down-regulation. The TCR is down-regulated from the cell surface by the activation of protein kinase C (PKC) and subsequent serine phosphorylation of the CD3 γ-chain. We report here that the TCR α-chain cytoplasmic tail is also necessary for PKC-mediated internalization of the TCR complex. The requirement for the TCR α-chain cytoplasmic tail is specific for internalization of the TCR complex, since down-regulation of CD4 is still intact in hybridoma cells expressing a tailless TCR α-chain. The absence of TCR internalization directly correlates with defective PKC-mediated phosphorylation of the CD3 γ-chain. Despite deficient PKC-mediated TCR down-regulation, the tailless αβ TCR still transduces antigenic signals resulting in the production of interleukin-2. Although the TCR tails are not obviously required for signal transduction, the TCR α-tail may serve as a targeting domain for PKC-mediated down-regulation of the TCR complex.     

IL-1 alpha, but not IL-1 beta, is required for contact-allergen-specific T cell activation during the sensitization phase in contact hypersensitivity.

Contact hypersensitivity (CHS) is a T cell-mediated cellular immune response caused by epicutaneous exposure to contact allergens. In this reaction, after the first epicutaneous allergen sensitization, Langerhans cells (LC) catch allergens and migrate from the skin to draining lymph nodes (LN) and activate naive T cells. Although IL-1 is suggested to be involved in these processes, the mechanisms have not been elucidated completely. In this report, to elucidate roles of IL-1alpha and IL-1beta in CHS, we analyzed ear swelling in 2,4,6-trinitrochlorobenzene (TNCB)-induced CHS using gene-targeted mice. We found that ear swelling was suppressed in IL-1alpha-deficient (IL-1alpha(-/-)) mice but not in IL-1beta(-/-) mice. LC migration from the skin into LN was delayed in both IL-1alpha(-/-) and IL-1beta(-/-) mice, suggesting that this defect was not the direct cause for the reduced CHS in these mice. However, we found that the proliferative response of trinitrophenyl (TNP)-specific T cells after sensitization with TNCB was specifically reduced in IL-1alpha(-/-) mice. Furthermore, adoptive transfer of TNP-conjugated IL-1-deficient epidermal cells (EC) into wild-type mice indicated that only IL-1alpha, but not IL-1beta, produced by antigen-presenting cells in EC could prime allergen-specific T cells. These observations indicate that IL-1alpha, but not IL-1beta, plays a crucial role in TNCB-induced CHS by sensitizing TNP-specific T cells.     

Multidrug-resistance-associated protein 1 (Mrp1) is probably not required for murine Th cell activation

Previously, we have demonstrated that multidrug-resistance-associated protein 1 (Mrp1) represents an activation marker for murine Th1 cells and is constitutively expressed by Th2 cells. Using the inhibitor MK571, we and others also suggested that Mrp1 is necessary for Th cell activation. However, herein, we show that Mrp1-deficient Th cells can be differentiated to a similar extent to Th1 and Th2 cells in vitro and, upon re-stimulation, produce comparable amounts of IL-2, IFNgamma and IL-4. Mrp1-deficient mice are equally susceptible than wild-type mice to infection with the protozoan parasite Leishmania major, a well-respected model for in vivo Th1 and Th2 cell differentiation. Intriguingly, MK571 is able to completely block activation of Mrp1-deficient Th cells. Most likely, therefore, the molecule relevant for Th cell activation which is blocked by MK571 is different from Mrp1. While these results are compatible with our previously reported data on Mrp1 expression, they contradict our previous conclusions about Mrp1 function in murine Th1 cells as well as those published in a very recent report in this journal on human Th cells.     

Notch 1 signaling regulates peripheral T cell activation

Notch signaling has been identified as an important regulator of leukocyte differentiation and thymic maturation. Less is known about the role of Notch signaling in regulating mature T cells. We examined the role of Notch 1 in regulating peripheral T cell activity in vitro and in vivo. Coligation of Notch 1 together with TCR and CD28 resulted in a dramatic inhibition of T cell activation, proliferation, and cytokine production. This effect was dependent on presenilin activity and induced the expression of HES-1, suggestive of Notch 1 signaling. Biochemical analysis demonstrated an inhibition of AKT and GSK3beta phosphorylation following Notch 1 engagement while other biochemical signals such as TCR and ERK phosphorylation remained intact. Similar effects were observed in vivo in an adoptive transfer model. Therefore, Notch 1 signaling may play an important role in regulating naive T cell activation and homeostasis.     

A novel functional interaction between Vav and PKCtheta is required for TCR-induced T cell activation

Vav and PKCtheta play an early and important role in the TCR/CD28-induced stimulation of MAP kinases and activation of the IL-2 gene. Vav is also essential for actin cytoskeleton reorganization and TCR capping. Here, we report that PKCtheta function was selectively required in a Vav signaling pathway that mediates the TCR/CD28-induced activation of JNK and the IL-2 gene and the upregulation of CD69 expression. Vav also promoted PKCtheta translocation from the cytosol to the membrane and cytoskeleton and induced its enzymatic activation in a CD3/CD28-initiated pathway that was dependent on Rac and on actin cytoskeleton reorganization. These findings reveal that the Vav/Rac pathway promotes the recruitment of PKCtheta to the T cell synapse and its activation, essential processes for T cell activation and IL-2 production.     

Accessory cell-derived signals required for T cell activation

Various populations of accessory cells differ in their abilities to function as effective antigen-presenting cells (APC) and stimulate CD4+ T cells to produce interleukin-2. Three important factors directly related to APC potency are the expression of class II major histocompatibility complex molecules and the ability to present peptide antigens to the T cell antigen receptor, the expression of costimulatory ligands which deliver important activation signals independent of T cell receptor occupancy and the expression of adhesion molecules which promote conjugate formation so that these activation signals can be effectively delivered to the T cells. The relative importance of these accessory cell functions in T cell activation will be discussed, with an emphasis on costimulation and the CD28/B7 receptor/ligand pair. The consequence of inadequate costimulation by an otherwise effective APC in inducting T cell anergy will also be discussed. *** DIRECT SUPPORT *** A02GS021 00006     

MHC-restricted T cell receptor signaling is required for alpha beta TCR replacement of the pre T cell receptor

A developmental block is imposed on CD25(+)CD44(-) thymocytes at the beta-selection checkpoint in the absence of the pre T cell receptor (preTCR) alpha-chain, pTalpha. Early surface expression of a transgenic alphabeta TCR has been shown to partially circumvent this block, such that thymocytes progress to the CD4(+)CD8(+) double-positive stage. We wanted to analyze whether a restricting MHC element is required for alphabeta TCR-expressing double-negative (DN) thymocytes to overcome the developmental block in pTalpha-deficient animals. We used the HY-I knock-in model that endows thymocytes with alphabeta TCR expression in the DN compartment but has the advantage of physiological expression levels, in contrast to conventional TCR transgenes. On a pTalpha-deficient background, this HY-I TCR transgene 'rescued' CD25(+)CD44(-) thymocytes from apoptosis and enabled progression to later differentiation stages. On a non-selecting MHC background, however, pTalpha-deficient HY-I mice presented a pronounced reduction in numbers of splenocytes and thymocytes when compared to animals of selecting MHC genotype, showing that MHC restriction is necessary to drive HY-TCR-mediated rescue of pTalpha-deficient thymocytes.     

Extracellular but not intracellular calcium mobilization is required for Epstein-Barr virus-containing supernatant-induced B cell activation

Changes in intracytosolic free calcium concentration ([Ca2+]i) are though to be an important trigger for the initiation of the cellular events culminating in B cell activation. After exposure of human B lymphocytes loaded with the fluorescent indicator quin-2 to the Epstein-Barr virus (EBV)-containing supernatant of B95-8 cell line, a rise in [Ca2+]i is observed. To determine the respective contribution of the intra- and extracellular Ca2+ pools in EBV-induced B cell activation, the pharmacologic modulation of these processes was investigated using an intracellular calcium chelator, 1,2-bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic (BAPTA) or the calcium channel blocking drugs. When the extracellular Ca2+ contribution was minimized in the presence of calcium channel-blocking drugs or incubation of the cells in Ca2+-free medium, the EBV-induced human B cell activation was fully inhibited. When the calcium channel-blocking drugs, either verapamil or diltiazem, were withdrawn or when exogenous Ca2+ was added to the Ca2+-free medium, EBV-induced B cell activation was noted, demonstrating the reversibility of the inhibition. On the contrary, when the intracellular Ca2+ contribution was reduced after loading the cell with BAPTA, no alteration of the EBV-induced B cell activation was observed. Thus, the EBV-induced rise of [Ca2+]i required in the activation of human B cells appears to be essentially related to the entry of extracellular Ca2+ and not to the release of Ca2+ from internal stores.     

Regulation of immune cell signaling by SHIP1: A phosphatase,scaffold protein,and potential therapeutic target

The phosphoinositide phosphatase SHIP is a critical regulator of immune cell activation. Despite considerable study, the mechanisms controlling SHIP activity to ensure balanced cell activation remain incompletely understood. SHIP dampens BCR signaling in part through its association with the inhibitory coreceptor Fc gamma receptor IIB, and serves as an effector for other inhibitory receptors in various immune cell types. The established paradigm emphasizes SHIP's inhibitory receptor‐dependent function in regulating phosphoinositide 3‐kinase signaling by dephosphorylating the phosphoinositide PI(3,4,5)P₃; however, substantial evidence indicates that SHIP can be activated independently of inhibitory receptors and can function as an intrinsic brake on activation signaling. Here, we integrate historical and recent reports addressing the regulation and function of SHIP in immune cells, which together indicate that SHIP acts as a multifunctional protein controlled by multiple regulatory inputs, and influences downstream signaling via both phosphatase‐dependent and ‐independent means. We further summarize accumulated evidence regarding the functions of SHIP in B cells, T cells, NK cells, dendritic cells, mast cells, and macrophages, and data suggesting defective expression or activity of SHIP in autoimmune and malignant disorders. Lastly, we discuss the biological activities, therapeutic promise, and limitations of small molecule modulators of SHIP enzymatic activity.     

KSR is a scaffold required for activation of the ERK/MAPK module

Mechanisms that regulate signal propagation through the ERK/MAPK pathway are still poorly understood. Several proteins are suspected to play critical roles in this process. One of these is Kinase Suppressor of Ras (KSR), a component previously identified in RAS-dependent genetic screens in Drosophila and Caenorhabditis elegans. Here, we show that KSR functions upstream of MEK within the ERK/MAPK module. In agreement with this, we found that KSR facilitates the phosphorylation of MEK by RAF. We further show that KSR associates independently with RAF and MEK, and that these interactions lead to the formation of a RAF/MEK complex, thereby positioning RAF in close proximity to its substrate MEK. These findings suggest that KSR functions as a scaffold that assembles the RAF/MEK functional pair.     

Co-stimulation by anti-immunoglobulin is required for B cell activation by CD40Llow T cells

During cognate B : T interactions, B cells encounter antigen (Ag) through surface immuno-globulin (sIg) and present antigenic peptides to T helper (Th) cells. However, most in vitro systems used to study contact events involved in the delivery of T help for B cells circumvent the requirement for T cell Ag specificity by using anti-CD3/T cell receptor (TcR) monoclonal antibodies (mAb) to activate T cells. To study the role of sIg engagement in the responsiveness of B cells to T help, we pre-treated small resting B cells with soluble anti-χ mAb prior to contact with an activated Th1 clone. By reducing the concentration of anti-TcR mAb we obtained low levels of CD40 ligand (CD40L^low) on Th cells, comparable to those expressed by lymph node T cells activated in vitro (ex vivo T cells). In contrast to untreated B cells, which did not respond to CD40L^low Th, anti-Ig-treated B cells responded strongly. Low buoyant density B cells also responded to CD40L^low Th cells. There was no B cell response to resting Th cells. mAb against CD54/intercellular adhesion molecule-1 or major histocompatibility complex (MHC) class II completely inhibited B cell responses to CD40L^low Th1 cells, equivalent to the effects of blocking CD40 interactions. This contrasts with mAb blocking responses to CD40L^highTh, where CD40 effects predominate. Our data show that sIg engagement is necessary for the induction of B cell response to CD40L^low Th cells. Anti-CD3-activated ex vivo T cells that were also CD40L^low did not provide help to small resting B cells, but did induce responses from sIg-stimulated B cells. Thus, our data support a requirement for sIg signaling in physiological B cell activation, and further confirm previous work showing CD40 ligation to be necessary but not sufficient for delivery of T help to B cells.     

Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1

Leukocyte functional antigen 1 (LFA-1), with intercellular adhesion molecule ligands, mediates T cell adhesion, but the signaling pathways and functional effects imparted by LFA-1 are unclear. Here, intracellular phosphoprotein staining with 13-dimensional flow cytometry showed that LFA-1 activation induced phosphorylation of the beta(2) integrin chain and release of Jun-activating binding protein 1 (JAB-1), and mediated signaling of kinase Erk1/2 through cytohesin-1. Dominant negatives of both JAB-1 and cytohesin-1 inhibited interleukin 2 production and impaired T helper type 1 differentiation. LFA-1 stimulation lowered the threshold of T cell activation. Thus, LFA-1 signaling contributes to T cell activation and effects T cell differentiation.     

ZAP-70 is required for calcium mobilization but is dispensable for mitogen-activated protein kinase (MAPK) superfamily activation induced via CD2 in human T cells

Stimulation with specific pairs of anti-CD2 antibodies can induce T cell activation and proliferation. In this study, we investigate the significance of ZAP-70 in CD2 signaling using ZAP-70-deficient T cells derived from a CD8-deficient patient and show that ZAP-70 is necessary for cellular proliferation and cytokine production in T cells stimulated via CD2. Biochemical analyses show that CD2 stimulation induces activation of mitogen-activated protein kinase (MAPK) superfamily in ZAP-70-deficient T cells, indicating that a ZAP-70-independent pathway(s) exists for MAPK superfamily activation via CD2. In contrast, intracellular Ca(2+) mobilization and activation of nuclear factor of activated T cells (NFAT) upon CD2 triggering were impaired in T cells lacking ZAP-70. Furthermore, we found that pharmacological Ca(2+) elevation combined with CD2 stimulation restored NFAT activation and subsequent cytokine production in ZAP-70-deficient T cells. These results indicate that in CD2 signaling, ZAP-70 plays an essential role in Ca(2+) mobilization and NFAT activation.     

Coronin-1 expression in T lymphocytes: insights into protein function during T cell development and activation

Coronin has been described as an actin-binding protein of Dictyostelium discoideum, and it has been demonstrated to play a role in cell migration, cytokinesis and phagocytosis. Coronin-related proteins are found in many eukaryotic species, including Coronin-1 in mammals whose expression is enriched in the hematopoietic tissues. Here, we characterize Coronin-1 gene and protein expression in mouse embryonic and adult T lymphocytes. Coronin-1 is expressed throughout T cell ontogeny and in peripheral alphabeta T cells. Expression varies along thymic cell development, with maximum levels observed in embryonic early thymocytes and, in the adults, the selected TCRalphabeta(+) single-positive thymocytes. Subcellular localization analysis indicates that Coronin-1 is in equilibrium between the cytosol and the cell cortex, where it accumulates in F-actin-rich membrane protrusions induced by polarized activation of TCR-CD3-stimulated T cells. These data are consistent with a role of Coronin-1 in T cell differentiation/activation events involving membrane dynamisms and the cortical actin cytoskeleton.     

The novel SAM domain protein Aveugle is required for Raf activation in the Drosophila EGF receptor signaling pathway

Activation of the Raf kinase by GTP-bound Ras is a poorly understood step in receptor tyrosine kinase signaling pathways. One such pathway, the epidermal growth factor receptor (EGFR) pathway, is critical for cell differentiation, survival, and cell cycle regulation in many systems, including the Drosophila eye. We have identified a mutation in a novel gene, aveugle, based on its requirement for normal photoreceptor differentiation. The phenotypes of aveugle mutant cells in the eye and wing imaginal discs resemble those caused by reduction of EGFR pathway function. We show that aveugle is required between ras and raf for EGFR signaling in the eye and for mitogen-activated protein kinase phosphorylation in cell culture. aveugle encodes a small protein with a sterile alpha motif (SAM) domain that can physically interact with the scaffold protein connector enhancer of Ksr (Cnk). We propose that Aveugle acts together with Cnk to promote Raf activation, perhaps by recruiting an activating kinase.     

Suppressor of cytokine signaling 1 is required for the differentiation of CD4+ T cells

Suppressor of cytokine signaling 1 (Socs1) is critical for the regulation of interferon-gamma responses and T cell homeostasis. Although the presentation of the inflammatory disease of Socs1-deficient mice is complex, we have tested here the hypothesis that it originates from inappropriate T cell development and the appearance of autoreactive T cells. Socs1-deficient T cell receptor-transgenic mice showed severely impaired positive selection and a substantial alteration in CD4-CD8 T cell fate specification. These defects were dependent on interferon-gamma. Moreover, negative selection was also impaired, suggesting that autoimmunity contributes to the disease observed in Socs1(-/-) mice. We conclude that the constitutive expression of Socs1 in the thymus protects the process of thymic development and selection from the effects of systemic inflammation.     

Requirement of the JIP1 scaffold protein for stress-induced JNK activation

The c-Jun N-terminal kinase (JNK) signal transduction pathway is activated in response to the exposure of cells to environmental stress. Components of the JNK signaling pathway interact with the JIP1 scaffold protein. JIP1 is located in the neurites of primary hippocampal neurons. However, in response to stress, JIP1 accumulates in the soma together with activated JNK and phosphorylated c-Jun. Disruption of the Jip1 gene in mice by homologous recombination prevented JNK activation caused by exposure to excitotoxic stress and anoxic stress in vivo and in vitro. These data show that the JIP1 scaffold protein is a critical component of a MAP-kinase signal transduction pathway.