Monomer hapten and hapten-specific IgG inhibit mast cell activation evoked by multivalent hapten with different mechanisms.

Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co-ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2-containing inositol 5'-phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP-alanine) to multivalent antigen (TNP-OVA)-activated rat basophilic leukemia cells and mouse bone marrow-derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP-OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca2+ concentrations ([Ca2+ ]i ), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten-specific IgG monoclonal antibody (anti-TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co-ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca2+ ]i , weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.

The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor.

Early events of B cell activation after B cell receptor (BCR) triggering have been well characterized. However, little is known about the steady state of the BCR on the cell surface. Here, we simultaneously visualize single BCR particles and components of the membrane skeleton. We show that an ezrin- and actin-defined network influenced steady-state BCR diffusion by creating boundaries that restrict BCR diffusion. We identified the intracellular domain of Igbeta as important in mediating this restriction in diffusion. Importantly, alteration of this network was sufficient to induce robust intracellular signaling and concomitant increase in BCR mobility. Moreover, by using B cells deficient in key signaling molecules, we show that this signaling was most probably initiated by the BCR. Thus, our results suggest the membrane skeleton plays a crucial function in controlling BCR dynamics and thereby signaling, in a way that could be important for understanding tonic signaling necessary for B cell development and survival.

Immobile BCRs: the safety on the signal trigger.

B cell receptor (BCR)-mediated tonic signals are important for B cell survival and development. In this issue of Immunity, Treanor et al. (2010) show that triggering of downstream responses is kept in check with BCR immobilization by actin.

Differentiation between control subjects and patients with chronic spontaneous urticaria based on the ability of anti-IgE autoantibodies (AAbs) to induce FcεRI crosslinking, as compared to anti-FcεRIα AAbs.

BACKGROUND: The reported prevalences of IgG autoantibodies (AAbs) to FcεRIα and IgE in sera from patients with chronic spontaneous urticaria (CSU) have varied, and these AAbs are also often observed in healthy control subjects. Regarding the histamine release activity of purified IgG from patients with CSU, the number of examined patients has been small. Thus, we sought to determine the prevalence and FcεRI crosslinking ability of these AAbs in a large number of patients with CSU and non-atopic control (NC) subjects. METHODS: We compared the concentrations of anti-IgE and anti-FcεRIα AAbs and the abilities of these AAbs to cause FcεRI aggregation in patients with CSU (n = 134) and NC subjects (n = 55) using ELISA and an in vitro elicitation test, respectively. RESULTS: The concentration of anti-IgE AAbs was significantly different between the NC subjects and the CSU patients (P < 0.0001, cutoff value: 0.558 µg/mL), whereas the concentration of anti-FcεRIα AAbs was not. A significant difference in the duration of illness was noted between patients with lower and those with higher concentrations of anti-IgE AAbs relative to the cutoff value. The abilities of anti-IgE AAbs, but not anti-FcεRIα AAbs, to induce FcεRI crosslinking were significantly higher in CSU patients than in NC subjects (P = 0.0106). CONCLUSIONS: In the Japanese population of CSU patients studied, the ability of the anti-IgE AAbs to induce FcεRI crosslinking differed significantly between NC subjects and CSU patients, suggesting the involvement of anti-IgE AAbs in the pathogenesis of CSU in the Japanese population.

IL-15 dependent induction of IL-18 secretion as a feedback mechanism controlling human MAIT-cell effector functions.

Mucosal-associated invariant T (MAIT) cells are characterized by an invariant TCRVα7.2 chain recognizing microbial vitamin B metabolites presented by the MHC-Ib molecule MR1. They are mainly detectable in the CD8(+) and CD8(-) CD4(-) "double negative" T-cell compartments of mammals and exhibit both Th1- and Th17-associated features. As MAIT cells show a tissue-homing phenotype and operate at mucosal surfaces with myriads of pathogenic encounters, we wondered how IL-15, a multifaceted cytokine being part of the intestinal mucosal barrier, impacts on their functions. We demonstrate that in the absence of TCR cross-linking, human MAIT cells secrete IFN-γ, increase perforin expression and switch on granzyme B production in response to IL-15. As this mechanism was dependent on the presence of CD14(+) cells and sensitive to IL-18 blockade, we identified IL-15 induced IL-18 production by monocytes as an inflammatory, STAT5-dependent feedback mechanism predominantly activating the MAIT-cell population. IL-15 equally affects TCR-mediated MAIT-cell functions since it dramatically amplifies bacteria-induced IFN-γ secretion, granzyme production, and cytolytic activity at early time points, an effect being most pronounced under suboptimal TCR stimulation conditions. Our data reveal a new quality of IL-15 as player in an inflammatory cytokine network impacting on multiple MAIT-cell functions.

B-cell inhibition by cross-linking CD79b is superior to B-cell depletion with anti-CD20 antibodies in treating murine collagen-induced arthritis.

Depletion of B cells with the anti-CD20 antibody rituximab is an established therapy for rheumatoid arthritis. However, rituximab has only moderate efficacy, most likely due to insufficient depletion of B cells in lymphoid organs and expansion of pathogenic B cells. We found that an antibody against mouse CD79b profoundly blocks B-cell proliferation induced via the B-cell receptor, CD40, CD180, and chondroitin sulfate, but not via TLR4 or TLR9. Treatment with anti-CD79b also induces death in resting and activated B cells. B-cell inhibition is mediated by cross-linkage of CD79b, but independent of Fc-receptor engagement. In the model of collagen-induced arthritis, an antibody against mouse CD20 depletes B cells very efficiently but fails to suppress the humoral immune response against collagen and the development of arthritis. In contrast, the antibody against CD79b, and a deglycosylated variant of this antibody, almost completely inhibits the increase in anti-collagen antibodies and the development of arthritis. In mice with established arthritis only the fully glycosylated antibody against CD79b is effective. Our data show that targeting B cells via CD79b is much more effective than B-cell depletion with anti-CD20 antibodies for therapy of arthritis. These findings may have important implications for treatment of B-cell-mediated autoimmune diseases.

T-cell immunoglobulin and ITIM domain (TIGIT) receptor/poliovirus receptor (PVR) ligand engagement suppresses interferon-γ production of natural killer cells via ß-arrestin 2-mediated negative signaling.

Natural killer (NK) cell activation is well orchestrated by a wide array of NK cell receptor repertoire. T-cell immunoglobulin and ITIM domain (TIGIT) receptor was recently defined as an inhibitory receptor that is expressed on NK cells and T cells. TIGIT receptor/poliovirus receptor (PVR) ligand engagement signaling inhibits cytotoxicity mediated by NK and CD8(+) T cells. However, it is unclear how TIGIT/PVR signaling regulates cytokine secretion in NK cells. Here we show that TIGIT/PVR engagement suppresses interferon-γ (IFN-γ) production of NK cells. TIGIT transgenic NK cells generate less IFN-γ undergoing TIGIT/PVR ligation. Moreover, TIGIT knock-out NK cells produce much more IFN-γ. TIGIT/PVR ligation signaling mediates suppression of IFN-γ production via the NF-κB pathway. We identified a novel adaptor ß-arrestin 2 that associates with phosphorylated TIGIT for further recruitment of SHIP1 (SH2-containing inositol phosphatase 1) through the ITT-like motif. Importantly, SHIP1, but not other phosphatases, impairs the TNF receptor-associated factor 6 (TRAF6) autoubiquitination to abolish NF-κB activation, leading to suppression of IFN-γ production in NK cells.

NKG2D ligation relieves 2B4-mediated NK-cell self-tolerance in mice.

Along with MHC class I (MHCI), 2B4 provides nonredundant NK-cell inhibition in mice. The immunoregulatory role of 2B4 has been increasingly appreciated in models of tumor and viral infection, however, the interactions among 2B4, MHCI, and other activating NK-cell receptors remain uncertain. Here, we dissect the influence of two distinct inhibitory pathways in modulating NK-cell-mediated control of tumors expressing strong activating ligands, including RAE-1γ. In vitro cytotoxicity and in vivo peritoneal clearance assays using MHCI(+) CD48(+) (RMA-neo), MHCI(+) CD48(+) RAE-1γ (RMA-RAE-1γ), MHCI(-) CD48(+) (RMA-S-neo), and MHCI(-) CD48(+) RAE-1γ (RMA-S-RAE-1γ) tumor lines demonstrated that NKG2D activation supersedes the inhibitory effect of both 2B4- and MHCI-mediated immune-tolerance systems. Furthermore, 2B4KO mice subcutaneously challenged with RMA-neo and RMA-S-neo exhibited reduced tumor growth and significantly prolonged survival compared with WT mice, implying that 2B4 is constitutively engaged in the NK-cell tolerance mechanism in vivo. Nevertheless, the inhibitory effect of 2B4 is significantly attenuated when NK cells encountered highly stressed tumor cells expressing RAE-1γ, resulting in an immune response shift toward NK-cell activation and tumor regression. Therefore, our data highlight the importance of the 2B4-mediated inhibitory system as an alternate self-tolerance mechanism, whose role can be modulated by the strength of activating receptor signaling within the tumor microenvironment.

CR1-mediated ATP release by human red blood cells promotes CR1 clustering and modulates the immune transfer process.

Humans and other higher primates are unique among mammals in using complement receptor 1 (CR1, CD35) on red blood cells (RBC) to ligate complement-tagged inflammatory particles (immune complexes, apoptotic/necrotic debris, and microbes) in the circulation for quiet transport to the sinusoids of spleen and liver where resident macrophages remove the particles, but allow the RBC to return unharmed to the circulation. This process is called immune-adherence clearance. In this study we found using luminometric- and fluorescence-based methods that ligation of CR1 on human RBC promotes ATP release. Our data show that CR1-mediated ATP release does not depend on Ca(2+) or enzymes previously shown to mediate an increase in membrane deformability promoted by CR1 ligation. Furthermore, ATP release following CR1 ligation increases the mobility of the lipid fraction of RBC membranes, which in turn facilitates CR1 clustering, and thereby enhances the binding avidity of complement-opsonized particles to the RBC CR1. Finally, we have found that RBC-derived ATP has a stimulatory effect on phagocytosis of immune-adherent immune complexes.

Influence of PD-L1 cross-linking on cell death in PD-L1-expressing cell lines and bovine lymphocytes.

Programmed death-ligand 1 (PD-L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death-1 (PD-1). However, the mechanism of PD-L1-mediated inhibitory signalling after PD-L1 cross-linking by anti-PD-L1 monoclonal antibody (mAb) or PD-1-immunogloblin fusion protein (PD-1-Ig) is still unknown, although it may induce cell death of PD-L1(+) cells required for regular immune reactions. In this study, PD-1-Ig or anti-PD-L1 mAb treatment was tested in cell lines that expressed PD-L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD-L1-mediated cell death. PD-L1 cross-linking by PD-1-Ig or anti-PD-L1 mAb primarily increased the number of dead cells in PD-L1(high) cells, but not in PD-L1(low) cells; these cells were prepared from Cos-7 cells in which bovine PD-L1 expression was induced by transfection. The PD-L1-mediated cell death also occurred in Cos-7 and HeLa cells transfected with vectors only encoding the extracellular region of PD-L1. In bovine lymphocytes, the anti-PD-L1 mAb treatment up-regulated interferon-γ (IFN-γ) production, whereas PD-1-Ig treatment decreased this cytokine production and cell proliferation. The IFN-γ production in B-cell-depleted peripheral blood mononuclear cells was not reduced by PD-1-Ig treatment and the percentages of dead cells in PD-L1(+) B cells were increased by PD-1-Ig treatment, indicating that PD-1-Ig-induced immunosuppression in bovine lymphocytes could be caused by PD-L1-mediated B-cell death. This study provides novel information for the understanding of signalling through PD-L1.

NKG2D regulates production of soluble TRAIL by ex vivo expanded human γδ T cells.

Soluble TRAIL (sTRAIL) can be produced by myeloid-derived cells to kill cancer cells. Whether this mechanism is used by T cells, and if so, how sTRAIL production is regulated, remains unclear. Our previous studies showed that ex vivo expanded human γδ T cells express TRAIL and NK receptor group 2 (R2), member D (NKG2D), and possess potent anticancer activities both in vitro and in vivo. Here, we investigated in greater detail the mechanisms by which γδ T cells utilize TRAIL and NKG2D to kill lung cancer cells. We demonstrate that human lung cancer cells express TRAIL R2 and NKG2D ligands. Blocking TRAIL or NKG2D during γδ T-cell-lung cancer cell co-cultures significantly reduced γδ T-cell-mediated cytotoxicity. Cross-linking NKG2D with anti-NKG2D antibody to mimic ligand binding promoted γδ T cells to produce sTRAIL, which induced apoptosis in lung cancer cells through TRAIL R2. Either neutralizing sTRAIL or blocking lung cancer cell TRAIL R2 significantly reduced γδ T-cell-mediated cytotoxicity to lung cancer cells. This study demonstrates that γδ T cells can mediate anticancer immunity via NKG2D-regulated production of sTRAIL.

The Mertk receptor tyrosine kinase promotes T-B interaction stimulated by IgD B-cell receptor cross-linking.

The Mertk receptor tyrosine kinase facilitates macrophage and DC apoptotic-cell clearance and regulates immune tolerance. Mertk may also contribute to B-cell activation, because Mertk-KO mice fail to develop autoantibodies when allo-activated by T cells. We investigated this possibility with a well-characterized model in which injection of mice with goat anti-IgD antibody causes membrane IgD cross-linking that induces T-independent B cell activation and antigen presentation to T cells. Goat anti-mouse IgD antibody-injected C57BL/6 Mertk-KO mice had normal initial B cell activation and proliferation, but significantly lower T cell activation and proliferation, as well as lower IgE and IgG anti-goat IgG responses, as compared to C57BL/6 WT controls. B cell antigen processing, analyzed by evaluating B cell fluorescence following injection of monoclonal anti-IgD antibody labeled with biotin or FITC, was comparable between Mertk-KO mice and WT mice. IgD Ab primed B cells from Mertk-KO mice exhibited significantly lower ability in activating memory T cells isolated from WT mice injected with the same antigen 10 days before. These observations suggest that Mertk expression is required for optimal B-cell antigen presentation, which is, in turn, required in this model for optimal T cell activation and subsequent T cell-dependent B cell differentiation.

Soluble CD23 controls IgE synthesis and homeostasis in human B cells.

CD23, the low-affinity receptor for IgE, exists in membrane and soluble forms. Soluble CD23 (sCD23) fragments are released from membrane (m)CD23 by the endogenous metalloprotease a disintegrin and metalloprotease 10. When purified tonsil B cells are incubated with IL-4 and anti-CD40 to induce class switching to IgE in vitro, mCD23 is upregulated, and sCD23 accumulates in the medium prior to IgE synthesis. We have uncoupled the effects of mCD23 cleavage and accumulation of sCD23 on IgE synthesis in this system. We show that small interfering RNA inhibition of CD23 synthesis or inhibition of mCD23 cleavage by an a disintegrin and metalloprotease 10 inhibitor, GI254023X, suppresses IL-4 and anti-CD40-stimulated IgE synthesis. Addition of a recombinant trimeric sCD23 enhances IgE synthesis in this system. This occurs even when endogenous mCD23 is protected from cleavage by GI254023X, indicating that IgE synthesis is positively controlled by sCD23. We show that recombinant trimeric sCD23 binds to cells coexpressing mIgE and mCD21 and caps these proteins on the B cell membrane. Upregulation of IgE by sCD23 occurs after class-switch recombination, and its effects are isotype-specific. These results suggest that mIgE and mCD21 cooperate in the sCD23-mediated positive regulation of IgE synthesis on cells committed to IgE synthesis. Feedback regulation may occur when the concentration of secreted IgE becomes great enough to allow binding to mCD23, thus preventing further release of sCD23. We interpret these results with the aid of a model for the upregulation of IgE by sCD23.

Insights into the regulatory mechanism controlling the inhibition of vaccine-induced seroconversion by maternal antibodies.

The inhibition of vaccination by maternal antibodies is a widely observed phenomenon in human and veterinary medicine. Maternal antibodies are known to suppress the B-cell response. This is similar to antibody feedback mechanism studies where passively transferred antibody inhibits the B-cell response against particulate antigens because of epitope masking. In the absence of experimental data addressing the mechanism underlying inhibition by maternal antibodies, it has been suggested that epitope masking explains the inhibition by maternal antibodies, too. Here we report that in the cotton rat model of measles virus (MV) vaccination passively transferred MV-specific immunoglobulin G inhibit B-cell responses through cross-linking of the B-cell receptor with FcγRIIB. The extent of inhibition increases with the number of antibodies engaging FcγRIIB and depends on the Fc region of antibody and its isotype. This inhibition can be partially overcome by injection of MV-specific monoclonal IgM antibody. IgM stimulates the B-cell directly through cross-linking the B-cell receptor via complement protein 3d and antigen to the complement receptor 2 signaling complex. These data demonstrate that maternal antibodies inhibit B-cell responses by interaction with the inhibitory/regulatory FcγRIIB receptor and not through epitope masking.

Interaction with FcγRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody.

A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10-100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.

Polyubiquitination of insulin-like growth factor I receptor (IGF-IR) activation loop promotes antibody-induced receptor internalization and down-regulation.

Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer.

CCAAT/enhancer-binding protein alpha (C/EBPalpha) is critical for interleukin-4 expression in response to FcepsilonRI receptor cross-linking.

Basophils mediate many of their biological functions by producing IL-4. However, it is unknown how the Il4 gene is regulated in basophils. Here, we report that CCAAT/enhancer-binding protein α (C/EBPα), a major myeloid transcription factor, was highly expressed in basophils. We show that C/EBPα selectively activated Il4 promoter-luciferase reporter gene transcription in response to IgE cross-linking, but C/EBPα did not activate other known Th2 or mast cell enhancers. We found that the PI3K pathway and calcineurin were essential in C/EBPα-driven Il4 promoter-luciferase gene transcription. Our mutation analyses revealed that C/EBPα drove Il4 promoter-luciferase activity depending on its DNA binding domain. Mutation of the C/EBPα-binding site in the Il4 promoter region abolished C/EBPα-driven Il4 promoter-luciferase activity. Our results further showed that a mutation in nuclear factor of activated T cells (NFAT)-binding sites in the Il4 promoter also negated C/EBPα-driven Il4 promoter-luciferase activity. Our study demonstrates that C/EBPα, in cooperation with NFAT, directly regulates Il4 gene transcription.

A conserved enhancer element differentially regulates developmental expression of CD5 in B and T cells.

We previously identified an enhancer element upstream of the mouse cd5 gene that was required in reporter assays for the induction of cd5 promoter activity by BCR cross-linking. This element is highly conserved in placental mammals. To determine its physiological role, we have now generated mice with a targeted deletion of the enhancer. The result is the loss of CD5 expression in peritoneal and splenic B-1a cells of adult mice and an inability to induce CD5 by cross-linking of the BCR on splenic B-2 cells. Surprisingly, CD5 expression on B-1a cells of neonatal mice was only minimally compromised. Cd5 enhancer deletion also had only a modest effect on CD5 expression in the T lineage. Thus, this enhancer provides age- and tissue-specific regulation of CD5 expression and is an example of the utilization of different modes of regulation of expression in T and B cells.

T-cell activation through immunological synapses and kinapses.

T-cell activation requires 'contact' with antigen-presenting cells (APCs) to bring the T-cell receptor (TCR) and antigenic major histocompatibility complex (MHC)-peptide complex together. Contact is defined by the size of the TCR and MHC-peptide complex, which at approximately 13 nm requires extensive interdigitation of the glycocalyx of the T cell and APC. T cells may be activated through formation of a stable T cell-APC junction, referred to as an immunological synapse. It has also been shown in vitro that T cells can integrate signals from APCs without a stable interaction. In vivo imaging studies supported the importance of both motile and stable T cell-APC interactions in T-cell priming. We have found that stability depends not upon turning off motile machinery but by symmetrization of force-generating structures to balance forces and hold the cell in place. Motility is induced by breaking this symmetry, which may be necessary to maintain the differentiation potential of the T cell. Recently, we also discovered a mode of T-cell signaling leading to tolerance in vivo based purely on motile interactions. Because this entire process takes place in a state of continuous T-cell kinesis, I propose the term 'kinapse' for motile T cell-APC contacts leading to signaling. Synapses and kinapses are inter-convertible by symmetrization/symmetry breaking processes, and both modes appear to be involved in normal T-cell priming. Imbalance of synapse/kinapse states may lead to immunopathology.

An essential role for SKAP-55 in LFA-1 clustering on T cells that cannot be substituted by SKAP-55R.

Lymphocyte function-associated antigen (LFA)-1 clustering, which is needed for high avidity binding to intercellular adhesion molecule (ICAM)-1 and -2, regulates T cell motility and T cell-antigen-presenting cell (APC) conjugation. In this study, down-regulation of SKAP-55 by small interfering RNAs (siRNAs) identified an essential role for this adaptor molecule in the T cell receptor (TCR)-mediated "inside-out signaling" that is needed for LFA-1 clustering and T cell-APC conjugation. In contrast, down-regulation of SKAP-55 had no effect on TCR-CD3 clustering. Furthermore, the expression of the related protein SKAP-55R failed to compensate for the loss of SKAP-55 in LFA-1 clustering, indicating that SKAP-55 has a unique function that cannot be replaced by this closely related protein. Our findings therefore indicate that SKAP-55, unlike SKAP-55R, is specifically tailored as an essential component of the inside-out signaling events that couple the TCR to LFA-1 clustering and T cell-APC conjugation.