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Background Proteasomes play a central role in the major histocompatibility class I (MHCI) antigen processing pathway. They conduct the proteolytic degradation of proteins in the cytosol, generating the C-terminus of CD8 T cell epitopes and MHCI-peptide ligands (P1 residue of cleavage site). There are two types of proteasomes, the constitutive form, expressed in most cell types, and the immunoproteasome, which is constitutively expressed in mature dendritic cells. Protective CD8 T cell epitopes are likely generated by the immunoproteasome and the constitutive proteasome, and here we have modeled and analyzed the cleavage by these two proteases. Results We have modeled the immunoproteasome and proteasome cleavage sites upon two non-overlapping sets of peptides consisting of 553 CD8 T cell epitopes, naturally processed and restricted by human MHCI molecules, and 382 peptides eluted from human MHCI molecules, respectively, using N-grams. Cleavage models were generated considering different epitope and MHCI-eluted fragment lengths and the same number of C-terminal flanking residues. Models were evaluated in 5-fold cross-validation. Judging by the Mathew's Correlation Coefficient (MCC), optimal cleavage models for the proteasome (MCC = 0.43 ± 0.07) and the immunoproteasome (MCC = 0.36 ± 0.06) were obtained from 12-residue peptide fragments. Using an independent dataset consisting of 137 HIV1-specific CD8 T cell epitopes, the immunoproteasome and proteasome cleavage models achieved MCC values of 0.30 and 0.18, respectively, comparatively better than those achieved by related methods. Using ROC analyses, we have also shown that, combined with MHCI-peptide binding predictions, cleavage predictions by the immunoproteasome and proteasome models significantly increase the discovery rate of CD8 T cell epitopes restricted by different MHCI molecules, including A*0201, A*0301, A*2402, B*0702, B*2705. Conclusions We have developed models that are specific to predict cleavage by the proteasome and the immunoproteasome. These models ought to be instrumental to identify protective CD8 T cell epitopes and are readily available for free public use at http://imed.med.ucm.es/Tools/PCPS/.

Rap1-GTP-interacting adaptor molecule (RIAM), an adaptor molecule of the Mig-10/RIAM/Lamellipodin (MRL) family, plays a critical role in actin reorganization and inside-out activation of integrins in lymphocytes and platelets. We investigated the role of RIAM in T cell receptor (TCR)-mediated signaling. Elimination of endogenous RIAM by short hairpin RNA (shRNA) resulted in impaired generation of inositol 1,4,5-trisphosphate (IP3) and mobilization of intracellular Ca2+, whereas phosphorylation of ζ chain–associated protein kinase of 70 kD (ZAP-70) and formation of the linker of activated T cells (LAT) signalosome were unaffected. Knockdown of RIAM also resulted in defective nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT) and activation of Ras guanine nucleotide-releasing protein 1 (RasGRP)1, which led to the diminished transcription of Il2. These events were associated with the impaired translocation of phosphorylated phospholipase C γ1 (PLC-γ1) to the actin cytoskeleton, which was required for the recruitment of PLC-γ1 to the immediate proximity of its substrate phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5)P2], and were reversed by reconstitution of cells with RIAM. Thus, by regulating the activation of PLC-γ1, RIAM has a central role in the activation of T cells and the transcription of target genes.

T cell immune responses are driven by the recognition of peptide antigens (T cell epitopes) that are bound to major histocompatibility complex (MHC) molecules. T cell epitope immunogenicity is thus contingent on several events, including appropriate and effective processing of the peptide from its protein source, stable peptide binding to the MHC molecule, and recognition of the MHC-bound peptide by the T cell receptor. Of these three hallmarks, MHC-peptide binding is the most selective event that determines T cell epitopes. Therefore, prediction of MHC-peptide binding constitutes the principal basis for anticipating potential T cell epitopes. The tremendous relevance of epitope identification in vaccine design and in the monitoring of T cell responses has spurred the development of many computational methods for predicting MHC-peptide binding that improve the efficiency and economics of T cell epitope identification. In this report, we will systematically examine the available methods for predicting MHC-peptide binding and discuss their most relevant advantages and drawbacks.

The small GTPase Rap1 has been involved in different cellular processes. Rap1 is known to increase cell adhesion by means of integrin activation, to induce cell spreading, and to regulate adherent junctions at cell–cell contacts. How Rap1 mediates these cell responses is poorly known, but currently developing evidence points to the involvement of different effector pathways. Recently, we described RIAM, a Rap1 interacting adaptor protein that regulates integrin activation and hence cell adhesion. RIAM is required for Rap1‐induced adhesion and seems to control Rap1 localization at the plasma membrane, where Rap1 regulates integrin activation. In this chapter, we focus in the role of RIAM in regulating Rap1‐mediated cell adhesion. We describe the method for studying the Rap1–RIAM interaction using in vitro and in vivo approaches such as yeast two hybrids, pull‐down assays. and coimmunoprecipitation. The role of Rap1 and RIAM in integrin‐mediated adhesion is studied by cell adhesion assays to immobilized integrin substrates and by changes in integrin activation as determined by activation epitope exposure. Finally, we describe an approach to determine the role of RIAM in regulating intracellular localization of active Rap1.

The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.

Efficient internal initiation of translation from the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires sequences of domain II, but the precise role of these sequences is still unknown. In this study, the formation of RNA–RNA complexes in the HCV IRES was evaluated. Using transcripts that contain the sequences of the structural HCV IRES domains II, IIIabcd, IIIabc, IV and IIIef-IV, specific long-range interactions between domains II and IV, as well as domains II and IIIabcd, have been found. These interactions were readily detected in a gel mobility-shift assay and required the presence of magnesium ions. A high concentration of nonspecific competitors, an 80 nt fragment of 18S rRNA or poly(I:C), did not interfere with the formation of RNA complexes. Interestingly, an RNA oligonucleotide bearing the sequence of stem–loop IIId interacted with domain II but not with domain IV or IIIef-IV, strongly suggesting that the interaction between domains II and IIIabcd was mediated by the IIId hairpin. Interaction between domains IIIabcd and IV was barely detected, consistent with the result that the apical part of domain III folds independently of the rest of the IRES. Moreover, the addition of stem–loop IIIef sequences to domain IV significantly reduced its ability to interact, which is in agreement with the formation of a compact RNA structure of domain IV with IIIef. The interactions observed in the absence of proteins between domains II and IV as well as stem–loop IIId and domain II may be transient, having a regulatory role in the translation efficiency of the HCV IRES.

Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.

Abstract 1118 Rap1, a small GTPase of the Ras superfamily, is a regulator of cytoskeletal reorganization, adherens junctions positioning and cell adhesion. The best-studied function of Rap1 is inside-out activation of integrins and cell adhesion. Two newly identified Rap1 effectors, RapL and RIAM, have been implicated in Rap1-mediated inside-out activation of integrins and cell adhesion. However, significant differences in the structure of these molecules indicate that they may regulate distinct signaling events. RIAM has a N-terminal coiled-coil region, central RA and pleckstrin homology (PH) domains, and proline-rich N-terminal and C-terminal regions, with multiple FPPPP motifs capable of interacting with the EVH1 domains of the actin regulatory proteins Ena/VASP and multiple XPPPP motifs interacting with Profilin. RIAM interacts with PLC-γ1 and regulates PLC-γ1 spatiotemporal distribution and activation during TCR-mediated stimulation. RapL has an RBD (Ras binding domain-structurally similar to RA domain) and a C-terminal coiled-coil region and interacts with Rap1 via its RBD domain and its N-terminal region and with the aL subunit of LFA-1 via its C-terminal domain. In the present study we investigated the role of RIAM and RapL in regulating TCR-mediated signaling and IL-2 production. We generated RIAM-knockdown (KD) and RapL-KD Jurkat T cells in which endogenous RIAM and RapL, respectively, were depleted by shRNA. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 was impaired by depletion of RIAM, activation of these kinases was unaffected by depletion of RapL. In contrast, activation of p38 and JNK was unaltered in RIAM-KD cells but was abrogated in RapL-KD cells. RIAM knockdown resulted in impaired activation of PLC-γ1, leading to impaired calcium release and activation of calcium and diacylglycerol-dependent GEFs, RasGRP1 and CalDAG-GEFI, thereby inhibiting activation of Ras and Rap1 and abrogating IL-2 production. In contrast, RapL knockdown did not affect PLC-γ1 activation but resulted in dramatic increase in IL-2 production. Because activation of p38 and JNK has been associated with suppression of IL-2, we examined whether inhibition of these pathways in RapL-KD cells might have a causative role in the increased IL-2 production. Forced expression of enzymatically active p38, JNK, or their combination suppressed IL-2 transcription and protein production to the levels of control T cells. To examine whether these two distinct Rap1 effectors display signal hierarchy or independently mediate their opposing effects on MAPKs and IL-2 transcription, we examined whether elimination of RapL in RIAM-KD T cells might restore their ability to produce IL-2. Elimination of RapL did not restore the ability of RIAM-KD cells to produce IL-2. Taken together our results indicate that although RIAM and RapL can mediate LFA-1 activation downstream of Rap1, these Rap1 effectors regulate distinct signaling events and have opposing roles on IL-2 production upon TCR triggering. Moreover, RIAM-mediated signaling is a pre-requisite for RapL to exert its inhibitory effect on IL-2 production. These results indicate that the selective and temporally regulated interaction of Rap1 with these two distinct effectors might have significant implications on the outcome of TCR triggering by inducing initial activation and subsequent suppression of IL-2 production. Disclosures: No relevant conflicts of interest to declare.

Abstract 3683 Poster Board III-619 Rap1, a small GTPase of the Ras superfamily, originally identified by its ability to reverse Ras-mediated transformation, is now known to regulate cytoskeletal reorganization, cell morphology, adherens junction positioning and adhesion. The best-studied function of Rap1 is inside-out activation of integrins and cell adhesion. Two newly identified Rap1 effectors, RapL and RIAM, have been implicated in Rap1 mediated in inside-out activation of integrins and cell adhesion. However, significant differences in the structure and interactions of these molecules indicate that they may mediate distinct signaling events. RIAM has a N-terminal coiled-coil region, central RA and pleckstrin homology (PH) domains, and proline-rich N-terminal and C-terminal regions, with multiple FPPPP motifs capable of interacting with the EVH1 domains of the actin regulatory proteins Ena/VASP, multiple XPPPP motifs interacting with Profilin and multiple PXXP motifs capable of interacting with SH3 domain containing proteins. Because of these properties RIAM is a regulator of actin polymerization but also interacts with components of the T cell signaling machinery. In contrast, RapL has an RBD (Ras binding domain-structurally similar to RA domain) and a C-terminal coiled-coil region and interacts with Rap1 via its RBD domain and its N-terminal region, and with the aL subunit of LFA-1 via its C-terminal domain. In the present study we investigated the role of RIAM and RapL in regulating signaling and functional events activated via the TCR. For this purpose we used RIAM-knockdown (KD) and RapL-KD Jurkat T cells in which endogenous RIAM and RapL, respectively, had been depleted by siRNA. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 was impaired by depletion of RIAM, activation of these kinases was unaffected by depletion of RapL. In contrast, activation of p38 was unaltered in RIAM-KD cells but was abrogated in RapL-KD cells. Moreover, RIAM knockdown resulted in impaired activation of Ras and Rap1 due to defective activation of the calcium and diacylglycerole-dependent GEFs, RasGRP1 and CalDAG-GEFI. In contrast, RapL knockdown had no effect on these events compared to control Jurkat T cells. Strikingly, RIAM-KD cells displayed impaired IL-2 production in response to stimulation with SEE-loaded APC or to TCR/CD3-plus-CD28 crosslinking, whereas RapL-KD cells displayed a dramatic increase in IL-2 production upon stimulation under the same conditions. These results indicate that although both RIAM and RapL regulate Rap1-dependent LFA-1 activation, these molecules have distinct roles in regulating signaling and functional outcomes of T cell responses after T cell receptor triggering. Disclosures: No relevant conflicts of interest to declare.

T cell receptor (TCR) ligation induces rapid polarization of the actin cytoskeleton resulting in the formation of the immunological synapse (IS), recruitment of signaling molecules, and initiation of signaling cascades leading to T cell activation. Specific recruitment, redistribution and organization of signaling molecules in the IS is facilitated by lipid raft microdomains, which provide a scaffold for focal protein assembly. Fyn and ZAP-70 are the most proximal TCR signaling molecules that localize in the IS and are redistributed in the lipid rafts during T cell activation. Currently, it is poorly understood how signals originating from the TCR are linked to specific mechanisms that regulate T cell activation. We have identified RIAM, an adaptor molecule that contains a RA (Ras Association) domain, a PH (Plekstrin Homology) domain and proline-rich motifs. RIAM interacts with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. RIAM also interacts with ADAP/SKAP-55 and, thereby, is recruited to the plasma membrane during T cell activation. We have previously determined that, during TCR ligation by antigen, RIAM localizes at the IS and the lipid rafts and serves as a substrate for Fyn and ZAP-70. Because of these properties, we examined whether RIAM might be involved in regulating the molecular and functional outcome of T cell activation. Using RIAM-knock down (KD) T cells in which endogenous RIAM was depleted by siRNA, we determined that RIAM was necessary for IL-2 transcription and RIAM-KD cells had impaired capacity for IL-2 production in response to stimulation with SEE-loaded APC or to TCR/CD3-plus-CD28 crosslinking. However, despite the impaired IL-2 production, analysis of TCR-proximal signaling events did not show impairment of ZAP-70 phosphorylation or formation of the LAT signalosome comprised of phosphorylated PLC-γ1, SLP-76 and Vav1. TCR triggering of both control and RIAM-KD cells also resulted in similar phosphorylation of PLC-γ1. Activation of PLC-γ1 leads to the generation of InsP3 and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PtdIns (4,5)P2). InsP3 binds to InsP3 receptors and triggers Ca2+ release from intracellular stores. Strikingly, TCR triggering of RIAM-KD cells resulted in markedly reduced upregulation of InsP3 compared to that in control T cells. Consistent with the defective upregulation of InsP3, calcium flux of RIAM-KD cells was dramatically impaired. This event was due to the impaired InsP3-mediated calcium release from the endoplasmic reticulum and not due to impaired store content or impaired calcium release-activated calcium (CRAC) channel entry as determined by using the Ca2+ ATPase blocker thapsigargin, which resulted in abundant calcium release in RIAM-KD cells. To analyze the consequences of deregulated InsP3 production and to investigate whether RIAM is specifically involved in PLC-γ1-mediated processes we evaluated activation of several signaling events on which PLC-γ1 activation has distinct effects. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 that are PLC-γ1 and Ca2+-dependent, was impaired in the absence of RIAM, activation of p38 and IKK was unaltered compared to control T cells. These results are consistent with a specific role of RIAM in InsP3-mediated Ca2+ release and indicate that deletion of RIAM does not result in a generalized defect in TCR-mediated signaling. Activation of PLC-γ1 at the cell membrane for proper generation of InsP3 requires appropriate docking and positioning of PLC-γ1. For this reason, we examined whether RIAM interacted with PLC-γ1 and regulated its subcellular localization after T cell activation. Detailed analysis by in vivo co-precipitation experiments in cell lysates and by in vitro association assays of purified proteins revealed a direct RIAM-PLC-γ1 interaction that was mediated via the SH3 domain of PLC-γ1. Furthermore, subcellular fractionation into cytoplasmic and cytoskeletal fractions revealed that PLC-γ1 translocated to the cytoskeleton upon T cell activation and this event was abrogated in RIAM-KD cells. These results indicate a novel and unexpected role of RIAM in T cell responses that involves regulation of spatio-temporal distribution and activation of PLC-γ1, leading to generation of InsP3 and Ca2+ mobilization after T cell receptor triggering.

Engagement of immune recognition subunits on lymphocytes results in cytoskeletal reorganization, polarization of polymerized actin, conjugate formation between T cells and APC, stabilization of the immunological synapse (IS), and initiation of signaling cascades leading to T cell activation. Actin remodeling is essential for these events and is mandatory for T cell activation. We have identified RIAM, a novel adaptor molecule that interacts specifically with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. Profilin associates with G-actin and promotes actin polymerization by adding actin monomers to the barbed ends of F-actin. Ena/VASP family proteins are cytoskeletal proteins and regulate actin dynamics. Via these interactions, RIAM functions as a regulator of the actin cytoskeleton. RIAM also has a mandatory role in regulating activation of integrins downstream of Rap1. The ability of a T cell to recognize and conjugate with an APC is dependent on cytoskeletal dynamics and “inside out” signaling leading to integrin-mediated adhesion. Activation of the b2 integrin LFA-1 leads to high avidity binding to ICAM proteins, which is crucial for stable T cell-APC conjugation. Because RIAM is involved both in cytoskeletal dynamics and integrin activation, we examined whether RIAM had a role in this process. Stable GFP-transfected Jurkat T cells were incubated with Raji B cells as APC, in the presence or absence of SEE. T cell:APC conjugates and formation of IS were examined by confocal microscopy. Synapse formation between GFP-Jurkat cells and Raji B cells was detected only in the presence of SEE. Under these conditions, polymerized (F) actin was highly recruited at the IS, as determined by phalloidin staining. Staining with RIAM-specific antibody indicated that in the absence of antigen, RIAM was diffusely expressed in the cytoplasm and at the plasma membrane. Impressively, upon incubation with SEE loaded Raji cells, RIAM was redistributed at the IS where it co-localized with polymerized (F) actin and ZAP-70. Knockdown of RIAM in Jurkat T cells resulted in significant defects in conjugate formation with SEE loaded B cells. Thus RIAM is involved in actin reorganization in the IS and in the formation of T cell:APC conjugates. Because appropriate actin reorganization is required for T cell activation we next explored whether RIAM knockdown leads to defects in TCR-mediated signaling events. RIAM-knockdown Jurkat T cells displayed significantly impaired IL-2 production in response to SEE loaded Raji and in decreased IL-2 promoter driven transcriptional activity. Surprisingly, despite the significant decrease in IL-2 transcription, RIAM-knockdown Jurkat cells did not show altered TCR-induced phosphorylation of PLCg1, or activation of MAP kinases including MEK, Erk1/2 and JNK, compared to control cells. Moreover, AP-1 and NF-kB-mediated gene transcription induced by CD3-plus-CD28 was unaffected. In contrast, NFAT-driven transcription was significantly impaired. Assessment of intracellular calcium by flow cytometry indicated that RIAM-knockdown cells had significantly reduced TCR-mediated intracellular calcium signal. These results indicate that RIAM is an important component of a signaling pathway that regulates calcium mobilization and NFAT mediated gene transcription in T lymphocytes.

In Oz, the yellow brick road led to dreams come true. In platelets, the road from agonist receptors to αIIbβ3 leads to thrombus formation, a response necessary for hemostasis and thrombosis. Upon platelet stimulation, agonist receptor pathways converge on the cytoplasmic and transmembrane domains of αIIbβ3, causing conformational changes that are propagated to the extracellular domain to effect high affinity ligand binding (activation). Numerous intracellular proteins have been implicated inαIIbβ3 activation, including protein kinase C (PKC), the Rap1 GTPase and talin. Indeed, talin recruitment to the β3 cytoplasmic domain is a final common step in αIIbβ3 activation. However, the relationships of these signaling proteins to each other, and the mechanism(s) of talin recruitment to β3 have not been established. Here we used forward, reverse, and synthetic genetics to engineer and order an αIIbβ3 activation pathway. In a CHO cell model system, increased expression of PKCαand talin to levels approximating those in platelets reconstituted αIIbβ3 activation in response to phorbol myristate acetate (PMA). This response was detectable by monitoring fibrinogen binding or the binding of a monovalent, ligand-mimetic antibody Fab fragment (PAC-1 Fab) that is specific for high-affinity αIIbβ3. PMA-induced activation of αIIbβ3 could be blocked by over-expression of Rap1GAP, which inhibits Rap1 GTPase activity. In contrast, over-expression of constitutively-active Rap1A(G12V) caused αIIbβ3 activation that bypassed the requirement for PKCα, indicating that Rap1 is downstream of PKCαin this integrin activation pathway. αIIbβ3 activation by Rap1 was promoted by a specific Rap1 effector, RIAM, an adapter molecule with a PH domain and proline-rich and EVH-1 binding regions. For example, over-expression of RIAM, but not another Rap1 effector (RapL), caused activation of αIIbβ3 and bypassed the requirement for PKCαand Rap1. Furthermore, siRNA-mediated knockdown of RIAM blocked αIIbβ3 activation by PMA or Rap1A(G12V), and this effect could be rescued by an siRNA-resistant form of RIAM. The integrin activating effects of PKCα, Rap1A(G12V) and RIAM were not observed if an integrin binding-defective talin mutant (W359A) was expressed instead of talin. In both CHO cells and platelets, activated Rap1 formed an integrin-associated complex with talin and RIAM, as assessed by deconvolution microscopy and co-immunoprecipitation. In addition, when RIAM was knocked down by siRNA in ES cell-derived murine megakaryocytes, αIIbβ3 activation in response to ADP or thrombin receptor agonists was impaired. These results identify and order a signaling pathway from agonist stimulation to αIIbβ3 activation. At the end of this road, Rap1 organizes an integrin activating complex composed of RIAM and talin. The ability to synthetically reconstruct an αIIbβ3 activation pathway and validate it in platelets and megakaryocytes now provides molecular targets to facilitate characterization of clinical platelet activation defects and development of better anti-platelet drugs. We are not in Kansas anymore.

T cell receptor (TCR) ligation induces rapid polarization of the actin cytoskeleton resulting in the formation and stabilization of the immunological synapse (IS), recruitment of signaling molecules, and initiation of signaling cascades of T cell activation. Actin remodeling is essential for these events and is mandatory for T cell activation. Specific recruitment, redistribution and organization of signaling molecules in the IS is facilitated by lipid raft microdomains, which provide a scafold for focal protein assembly. Fyn and ZAP-70 are the most proximal TCR signaling molecules that localized in the IS and are redistributed in the lipid rafts during T cell activation. Currently, it is poorly understood how signals originating from the activated TCR are linked to the cytoskeletal rearrangement and the focal redistribution of signaling proteins. Recently, we identified RIAM, a novel adaptor molecule that contains a RA (Ras Association) domain, a PH (Plekstrin Homology) domain and proline-rich motifs. RIAM interacts specifically with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. Profilin associates with G-actin and promotes actin polymerization by adding actin monomers to the barbed ends of F-actin. Ena/VASP family proteins are cytoskeletal proteins and regulate actin dynamics. Via these interactions, RIAM functions as a regulator of the actin cytoskeleton. To examine whether RIAM plays a role in the local reorganization of the actin cytoskeleton during IS formation, stable GFP-transfected Jurkat T cells were incubated with Raji B cells as APC, in the presence or absence of SEE. T cell:APC conjugates and formation of IS were examined by confocal microscopy. Synapse formation between GFP-Jurkat cells and Raji B cells was detected only in the presence of SEE. Under these conditions, polymerized (F) actin was highly recruited at the IS, as determined by phalloidin staining. Staining with RIAM-specific antibody indicated that in the absence of antigen, RIAM was diffusely expressed in the cytoplasm and at the plasma membrane. Impressively, upon incubation with SEE loaded Raji cells, RIAM was redistributed at the IS where it co-localized with polymerized (F) actin. Staining with ZAP-70 specific antibody revealed that ZAP-70 was also recruited at the IS upon activation and co-localized with RIAM and polymerized (F) actin. Sucrose gradient centrifugation indicated that RIAM translocated to the lipid rafts and co-localized in the same raft fraction with fyn and ZAP-70. RIAM was also a substrate of fyn and ZAP-70, which induced its tyrosine phosphorylation. Co-expression studies, by transfection of RIAM with fyn or ZAP-70 cDNA in COS cells, indicated that RIAM was a specific substrate of fyn and ZAP-70 but not lck. In vivo association of endogenous RIAM with fyn and ZAP-70 was also observed in primary T cells and in RIAM-transfected Jurkat cells and this interaction was highly upregulated upon activation. In the same complex, RIAM associated with grb2, crkL and the cytoskeletal proteins VASP and Profilin, indicating that RIAM functions as a link between the actin cytoskeleton and TCR signaling. Taken together these observations indicate a unique role of RIAM as an adaptor protein integrating TCR-mediated events that result in focal redistribution of polymerized actin, recruitment of signaling molecules at the IS and initiation of downstream signaling pathways.

We have previously determined that quiescent T cells express Tob, a member of the novel APRO gene family, which functions to repress T cell activation. Tob is as a transcriptional regulator and may regulate mRNA transcription. To identify genes that are regulated by Tob, Jurkat T cells were transfected with Tob cDNA or empty vector. Differential gene expression was determined by suppression subtractive hybridization. Twisted Gastrulation (Tsg) was one of the genes suppressed by Tob. Tsg is an evolutionarily conserved secreted protein that interacts with Drosophila Decapentaplegic (Dpp) and its vertebrate orthologs BMP2/4, and also the extracellular Dpp/BMP inhibitors short gastrulation (sog) and chordin. As a result, Tsg affects the binding of Dpp/BMP2/4 to their receptors and subsequent downstream signaling. BMPs belong to a family of secreted signaling molecules the founder of which, TGF-b, is essential for immune homeostasis and maintenance of T cell quiescence. In the thymus, Tsg functions as a regulator of thymocyte development by inhibiting BMP2/4 signaling which arrests thymocyte differentiation. Here, we examined Tsg expression and function in mature CD4+ human T cells. Tsg mRNA was expressed at very low levels in unstimulated T cells and was highly upregulated after activation by TCR/CD3 and either CD28, IL-2 or PMA. To determine the function of Tsg, we generated recombinant human Tsg tagged with V5 epitope. Based on its effects on thymocytes, we hypothesized that Tsg may regulate BMP2/4-mediated effects on mature T cells. Culture of CD4+ T cells with various concentrations of BMP2 or BMP4 in the presence or absence of anti-CD3 mAb did not affect T cell proliferation or cytokine production. Addition of Tsg to cultures containing BMP2/4 did not alter T cell responses. Because our results showed that Tsg mRNA was induced after activation, we tested pre-activated, polyclonal CD4+ alloreactive T cell lines. Neither BMP2 nor BMP4 had any effect on proliferation of alloreactive T cell lines in the presence or absence of anti-CD3 mAb. Strikingly, Tsg induced a significant inhibitory effect on CD3-mediated proliferation and cytokine production, including IL-2, IL-4, IFN-g and IL-10. This effect was not altered by the presence of BMP2 or BMP4. In contrast, Tsg enhanced the inhibitory effect of TGF-b1 suggesting that Tsg regulates TGF-b and not BMP downstream signaling in pre-activated, mature CD4+ T cells. Consistent with this hypothesis, Tsg did not affect phosphorylation of the BMP-specific Smad1, but induced phosphorylation of the TGF-b-specific Smad2 and mediated DNA binding on Smad3/4 consensus sites. In vitro association assays using recombinant Tsg-V5 and TGF-b revealed a direct interaction of these proteins as determined by co-immunoprecipitation followed by western blot with V5-specific and TGF-b-specific antibodies. Moreover, soluble anti-TGF-b receptor reversed the inhibitory effect of Tsg on pre-activated T cells either in the presence or in the absence of TGF-b, providing functional evidence for the biological significance of the Tsg/TGF-b interaction. Our results show that Tsg is a potent agonist of TGF-b downstream signaling in human CD4+ T cells and suggest that enhancement of TGF-b mediating signaling by Tsg may represent a novel target for molecular intervention for induction of transplantation tolerance.

The small GTPase Rap1, originally identified by its ability to reverse Ras-mediated transformation, regulates cytoskeleton organization, adherens junction positioning and cell adhesion. In T cells, Rap1 is activated upon TCR ligation and recruited to the plasma membrane where it regulates activation of β1 (VLA4, VLA5) and β2 (LFA-1) integrins. However, it is poorly understood how active Rap1 is localized at the plasma membrane and how it mediates integrin activation. Recently, we identified RIAM, a novel adaptor molecule that interacts specifically with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. Because Rap1-GTP induces integrin mediated adhesion, we examined whether RIAM might have a similar effect. Stable Jurkat T cell lines transfected with either RIAM, the GTP-bound mutant Rap1E63 or empty vector, were added in plates coated with fibronectin or ICAM-1. Cells transfected with RIAM had increased adhesion, which was comparable to that induced by Rap1E63. RIAM- and Rap1E63-induced adhesion on fibronectin was blocked by an antibody against the β1 chain of VLA-4, whereas adhesion on ICAM-1 was blocked by a blocking LFA-1 mAb. To determine whether RIAM induced active conformational changes in beta1 integrins, we analyzed activation epitope exposure by incubation with HUTS4 antibody that specifically binds to β1 integrins in their active conformation. RIAM transfected cells showed increased binding to HUTS4. RIAM also induced active conformation of LFA-1 as determined by using KIM127 antibody that specifically binds to β2 integrins in their active conformation. The fact that RIAM interacts selectively with Rap1-GTP and induces integrin activation suggests that RIAM functions as a Rap1-downstream effector. To test this hypothesis we generated stable Jurkat T cell lines expressing Rap1E63, and either specific RIAM shRNAs to knockdown expression of endogenous RIAM (Rap1E63-RIAM-KD) or shRNA control (Rap1E63-control-KD). Compared to Rap1E63, Rap1E63-RIAM-KD cells lost their enhanced adhesion properties and displayed reduced adhesion to fibronectin and ICAM-1 to levels similar to parental control Jurkat cells. In contrast, Rap1E63-control-KD retained increased adhesion to fibronectin and ICAM-1, to levels similar to Rap1E63 cells. Rap1 is recruited to the plasma membrane upon activation and this localization is required for Rap1-GTP to mediate cell adhesion. Therefore, we examined whether, similarly to adhesion, distribution of Rap1-GTP was affected by depletion of RIAM. Rap1E63-RIAM-KD and Rap1E63-control-KD Jurkat cells were seeded on anti-CD3 coated slides, fixed, probed with GST-RalGDS-RBD that interacts specifically with Rap1-GTP, and analyzed by confocal microscopy. In Rap1E63-control-KD cells Rap1-GTP was detected at the perinuclear region and at the plasma membrane, where it co-localized with polymerized (F)-actin. Strikingly, in Rap1E63-RIAM-KD cells Rap1-GTP was displaced from the plasma membrane and was detected only in the perinuclear region. In these cells no co-localization of Rap1-GTP with F-actin was detected. Thus, RIAM is a critical downstream effector of Rap1, which regulates Rap1-GTP localization at the plasma membrane and mediates active conformation of integrins and Rap1-induced adhesion.

Elucidating the mechanisms that regulate T cell activation and tolerance in vivo will provide insights into the maintenance of physiologic homeostasis and will facilitate development novel strategies for induction of transplantation tolerance. Transient activation of the small GTPase Rap1 is one of the physiologic consequences of TCR ligation and is mandatory for β1 and β2 integrin-mediated adhesion. In contrast, sustained increase of active Rap1 inhibits T cell activation and IL-2 transcription in vitro. In order to understand the role of Rap1 in the immune responses of the intact host we generated transgenic (Tg) mice, which express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg mice had no defects in thymocyte development or maturation. Rap1E63-Tg thymocytes were capable of activating Ras and Erk1/2 and, compared to wild type (WT) thymocytes, displayed enhanced LFA-1:ICAM-1-mediated adhesion and increased proliferation in response to anti-CD3. Surprisingly, although lymph node and splenic CD4+ cells from the Rap1E63-Tg mice also displayed increased LFA-1:ICAM-1-mediated adhesion, they had significantly impaired activation of Erk1/2 and dramatically reduced proliferation and IL-2 production in response to anti-CD3 and WT antigen presenting cells (APC). The defective responses of CD4+ T cells suggest that Rap1E63-Tg mice may have impaired helper function in vivo. To address this issue we immunized Rap1E63-Tg and WT mice with TNP-OVA, a T-cell dependent antigen. Total IgG, IgG1 and IgG2a were dramatically reduced, indicating that Rap1E63-Tg mice had a defect in immunoglobulin class switching, consistent with defective helper T cell-dependent B cell activation. Because these results suggest that Rap1E63-Tg CD4+ cells may have an anergic phenotype, we tested rechallenge responses. We immunized Rap1E63-Tg and WT mice with TNP-OVA in vivo and subsequently we rechallenged T cells in vitro with WT APC pulsed with OVA. Compared with WT, Rap1E63-Tg T cells had dramatically reduced proliferation, IFN- γ and IL-2 production on rechallenge, findings consistent with T cell anergy. Using suppression subtraction hybridization we determined that Rap1E63 induced mRNA expression of CD103, a marker that defines a potent subset of regulatory T cells (Treg). Strikingly, Rap1E63-Tg mice had a 5-fold increase of CD103+CD25+CD4+ Treg compared to WT mice. Rap1E63-Tg CD103+CD25+CD4+ Treg expressed the highest level of Foxp3 among all T cell subsets and had the most potent inhibitory effect on proliferation and IL-2 production when added into cultures of WT CD4+CD25− cells. Importantly, removal of the CD103+ cells significantly restored Erk1/2 activation, proliferation and IL-2 production of Rap1E63-Tg CD4+ T cells. Generation of CD103+ Treg occurs after thymic development and requires encounter of peripheral autoantigen. Consistent with this, differences in CD103+ Treg were detected only between lymph node and splenic cells and not between thymocytes from Rap1E63-Tg and WT mice. Since generation of CD103+ Treg depends on the strength of TCR signal, these results suggest that by enhancing adhesion, active Rap1 regulates the generation of Treg. Moreover, these results provide evidence that active Rap1 is a potent negative regulator of immune responses in vivo and have significant implications for the development of immune-based therapies geared towards tolerance induction.

Ligation of the T cell receptor (TCR) and costimulatory receptors leads to cytokine secretion and clonal expansion, whereas ligation of TCR alone leads to anergy. We have previously determined that anergic cells express Tob, a member of the novel APRO gene family, which inhibits T cell activation. The precise molecular mechanisms via which Tob mediates its effects in T cells are not fully understood. Tob functions as transcriptional coactivator and enhances DNA binding of Smads. Therefore, Tob may regulate de novo mRNA synthesis or gene transcription. To identify genes that are induced by Tob, Jurkat T cells that lack endogenous Tob, were transfected with Tob cDNA or empty vector and differential gene expression was determined by suppression subtractive hybridization. TRIM36 was one of the genes induced by Tob. TRIM36 is a RING finger E3 ubiquitin ligase. It belongs to a recently identified tripartite motif (TRIM) gene family which also includes Pyrin/Marenosrtin, MID1, MUL, PML, RFP and TIF1, proteins implicated in familial human diseases and cancer. E3 proteins confer substrate specificity to the ubiquitin system. Previous studies have shown that the trancriptional profile of anergic cells includes the E3 ubiquitin ligases Cbl-b, GRAIL and Itch. Therefore, the finding that Tob, a transcriptional regulator expressed in anergic cells, induces expression of TRIM36 E3 ubiquitin ligase is very intriguing. Northern blot analysis confirmed that TRIM36 mRNA was selectively upregulated in anergic T cells. To determine the role of TRIM36 on IL-2 gene transcription, Jurkat T cells were transfected with full-length TRIM36 cDNA along with the IL-2 promoter/enhancer cDNA (2kb) linked to the luciferase gene. TRIM36 inhibited CD3+CD28-mediated IL-2 transcription by 90%. Interestingly, when cells were stimulated with PMA+Ionomycin, which bypass the TCR proximal signals, IL-2 transcription was almost unaffected. These results prompted us to search for candidate ubiquitination substrates among signaling molecules that have a critical role on TCR-mediated T cell activation and IL-2 transcription. Previous studies have shown that among T cell signaling molecules, TCRζ, ZAP70, PLC-γ1 and PKC-𝛉 undergo ubiquitin-targeted degradation. For this reason, we investigated whether any of these proteins might be substrates for TRIM36-mediated ubiquitination. V5-tagged TRIM36 or empty vector was expressed in Jurkat T cells followed by stimulation with anti-CD3+anti-CD28 mAbs in the presence of ubiquitin aldehyde that prevents substrate deubiquitination. Immunoblot with antibodies specific for TCR ζ, ZAP70, PLC-γ1 and PKC-𝛉 showed that expression of PLC-γ1 and PKC-𝛉 was selectively reduced in the presence of TRIM36. Immunoprecipitation with V5 mAb followed by immunoblot with substrate-specific antibodies revealed that PLC- γ1 and PKC-𝛉 coprecipitated with TRIM36. Immunoblot with ubiquitin-specific antibody revealed that PLC-γ1 and PKC- 𝛉 were substrates for ubiquitination by TRIM36. Our results show that at least one molecular mechanism via which Tob mediates its inhibitory effect on T cell activation involves the induction of TRIM36 ubiquitin ligase, which mediates degradation of two key signaling proteins, PLC- γ1 and PKC-𝛉. Moreover, these results suggest that TRIM36 may represent a novel target of molecular intervention for induction of transplantation tolerance.

Lamellipodial protrusion is regulated by Ena/VASP proteins. We identified Lamellipodin (Lpd) as an Ena/VASP binding protein. Both proteins colocalize at the tips of lamellipodia and filopodia. Lpd is recruited to EPEC and Vaccinia, pathogens that exploit the actin cytoskeleton for their own motility. Lpd contains a PH domain that binds specifically to PI(3,4)P2, an asymmetrically localized signal in chemotactic cells. Lpd's PH domain can localize to ruffles in PDGF-treated fibroblasts. Lpd overexpression increases lamellipodial protrusion velocity, an effect observed when Ena/VASP proteins are overexpressed or artificially targeted to the plasma membrane. Conversely, knockdown of Lpd expression impairs lamellipodia formation, reduces velocity of residual lamellipodial protrusion, and decreases F-actin content. These phenotypes are more severe than loss of Ena/VASP, suggesting that Lpd regulates other effectors of the actin cytoskeleton in addition to Ena/VASP.