A requirement for IL-2/IL-2 receptor signaling in intrathymic negative selection

The nature of the signals that influence thymocyte selection and determine the fate of CD4(+)8(+) (double positive) thymocytes remains unclear. Cytokines produced locally in the thymus may modulate signals delivered by TCR-MHC/peptide interactions and thereby influence the fate of double-positive thymocytes. Because the IL-2/IL-2R signaling pathway has been implicated in thymocyte and peripheral T cell survival, we investigated the possibility that IL-2/IL-2R interactions contribute to the deletion of self-reactive, Ag-specific thymocytes. By using nontransgenic and transgenic IL-2-sufficient and -deficient animal model systems, we have shown that during TCR-mediated thymocyte apoptosis, IL-2 protein is expressed in situ in the thymus, and apoptotic thymocytes up-regulate expression of IL-2RS: IL-2R(+) double-positive and CD4 single-positive thymocytes undergoing activation-induced cell death bind and internalize IL-2. IL-2-deficient thymocytes are resistant to TCR/CD3-mediated apoptotic death, which is overcome by providing exogenous IL-2 to IL-2(-/-) mice. Furthermore, disruption or blockade of IL-2/IL-2R interactions in vivo during Ag-mediated selection rescues some MHC class II-restricted thymocytes from apoptosis. Collectively, these findings provide evidence for the direct involvement of the IL-2/IL-2R signaling pathway in the deletion of Ag-specific thymocyte populations and suggest that CD4 T cell hyperplasia and autoimmunity in IL-2(-/-) mice is a consequence of ineffective deletion of self-reactive T cells.     

Selective regulation of TCR signaling pathways by the CD45 protein tyrosine phosphatase during thymocyte development

In CD45-deficient animals, there is a severe defect in thymocyte-positive selection, resulting in an absence of mature T cells and the accumulation of thymocytes at the DP stage of development. However, the signaling defect(s) responsible for the block in development of mature single-positive T cells is not well characterized. Previous studies have found that early signal transduction events in CD45-deficient cell lines and thymocytes are markedly diminished following stimulation with anti-CD3. Nevertheless, there are also situations in which T cell activation and TCR signaling events can be induced in the absence of CD45. For example, CD45-independent TCR signaling can be recovered upon simultaneous Ab cross-linking of CD3 and CD4 compared with cross-linking of CD3 alone. These data suggest that CD45 may differentially regulate TCR signaling events depending on the nature of the signal and/or on the differentiation state of the cell. In the current study, we have assessed the role of CD45 in regulating primary thymocyte activation following physiologic stimulation with peptide. Unlike CD3-mediated stimulation, peptide stimulation of CD45-deficient thymocytes induces diminished, but readily detectable TCR-mediated signaling events, such as phosphorylation of TCR-associated zeta, ZAP70, linker for activation of T cells, and Akt, and increased intracellular calcium concentration. In contrast, phosphorylation of ERK, which is essential for positive selection, is more severely affected in the absence of CD45. These data suggest that CD45 has a selective role in regulating different aspects of T cell activation.     

Cell type-specific regulation of ITAM-mediated NF-kappaB activation by the adaptors, CARMA1 and CARD9

Activating NK cell receptors transduce signals through ITAM-containing adaptors, including FcRgamma and DAP12. Although the caspase recruitment domain (CARD)9-Bcl10 complex is essential for FcRgamma/DAP12-mediated NF-kappaB activation in myeloid cells, its involvement in NK cell receptor signaling is unknown. Herein we show that the deficiency of CARMA1 or Bcl10, but not CARD9, resulted in severe impairment of cytokine/chemokine production mediated by activating NK cell receptors due to a selective defect in NF-kappaB activation, whereas cytotoxicity mediated by the same receptors did not require CARMA1-Bcl10-mediated signaling. IkappaB kinase (IKK) activation by direct protein kinase C (PKC) stimulation with PMA plus ionomycin (P/I) was abrogated in CARMA1-deficient NK cells, similar to T and B lymphocytes, whereas CARD9-deficient dendritic cells (DCs) exhibited normal P/I-induced IKK activation. Surprisingly, CARMA1 deficiency also abrogated P/I-induced IKK activation in DCs, indicating that CARMA1 is essential for PKC-mediated NF-kappaB activation in all cell types, although the PKC-CARMA1 axis is not used downstream of myeloid ITAM receptors. Consistently, PKC inhibition abrogated ITAM receptor-mediated activation only in NK cells but not in DCs, suggesting PKC-CARMA1-independent, CARD9-dependent ITAM receptor signaling in myeloid cells. Conversely, the overexpression of CARD9 in CARMA1-deficient cells failed to restore the PKC-mediated NF-kappaB activation. Thus, NF-kappaB activation signaling through ITAM receptors is regulated by a cell type-specific mechanism depending on the usage of adaptors CARMA1 and CARD9, which determines the PKC dependence of the signaling.     

IL-1 receptor-associated kinase 4 is essential for IL-18-mediated NK and Th1 cell responses

IL-18 is an important cytokine for both innate and adaptive immunity. NK T cells and Th1 cells depend on IL-18 for their divergent functions. The IL-18R, IL-1R, and mammalian Toll-like receptors (TLRs) share homologous intracellular domains known as the TLR/IL-1R/plant R domain. Previously, we reported that IL-1R-associated kinase (IRAK)-4 plays a critical role in IL-1R and TLR signaling cascades and is essential for the innate immune response. Because TLR/IL-1R/plant R-containing receptors mediate signal transduction in a similar fashion, we investigated the role of IRAK-4 in IL-18R signaling. In this study, we show that IL-18-induced responses such as NK cell activity, Th1 IFN-gamma production, and Th1 cell proliferation are severely impaired in IRAK-4-deficient mice. IRAK-4(-/-) Th1 cells also do not exhibit NF-kappaB activation or IkappaB degradation in response to IL-18. Moreover, AP-1 activation which is triggered by c-Jun N-terminal kinase activation is also completely inhibited in IRAK-4(-/-) Th1 cells. These results suggest that IRAK-4 is an essential component of the IL-18 signaling cascade.     

The EphB6 receptor inhibits JNK activation in T lymphocytes and modulates T cell receptor-mediated responses

EphB6 is the most recently identified member of the Eph receptor tyrosine kinase family. EphB6 is primarily expressed in thymocytes and a subpopulation of T cells, suggesting that it may be involved in regulation of T lymphocyte differentiation and functions. We show here that overexpression of EphB6 in Jurkat T cells and stimulation with the EphB6 ligand, ephrin-B1, results in the selective inhibition of TCR-mediated activation of JNK but not the MAPK pathway. EphB6 appears to suppress the JNK pathway by preventing T cell receptor (TCR)-induced activation of the small GTPase Rac1, a critical event in initiating the JNK cascade. Furthermore, EphB6 blocked anti-CD3-induced secretion of IL-2 and CD25 expression in a ligand-dependent manner. Dominant negative EphB6 suppressed the inhibitory activity of the endogenous receptor and enhanced anti-CD3-induced JNK activation, CD25 expression, and IL-2 secretion, confirming the requirement for EphB6-specific signaling. Activation of the JNK pathway and the establishment of an IL-2/IL-2R autocrine loop have been shown to play a role in the negative selection of CD4(+)CD8(+) self-reacting thymocytes. In agreement, stimulation of murine thymocytes with ephrin-B1 not only blocked anti-CD3-induced CD25 up-regulation and IL-2 production, but also inhibited TCR-mediated apoptosis. Thus, EphB6 may play an important role in regulating thymocyte differentiation and modulating responses of mature T cells.     

Intercellular adhesion molecule 1 is critical for activation of CD28-deficient T cells

Presentation of Ag to T lymphocytes in the absence of the requisite costimulatory signals leads to an Ag-specific unresponsiveness termed anergy, whereas Ag presentation in conjunction with costimulation leads to clonal expansion. B7/CD28 signaling has been shown to provide this critical costimulatory signal and blockade of this pathway may inhibit in vitro and in vivo immune responses. Although T cells from CD28-deficient mice are lacking in a variety of responses, they nonetheless are capable of various primary and secondary responses without the induction of anergy expected in the absence of costimulation. This suggests that there may be alternative costimulatory pathways that can replace CD28 signaling under certain circumstances. In this paper, we show that ICAM-1becomes a dominant costimulatory molecule for CD28-deficient T cells. ICAM-1 costimulates anti-CD3-mediated T cell proliferation and IL-2 secretion in CD28-deficient murine T cells. Furthermore, splenocytes from ICAM-1-deficient mice could not activate CD28-deficient T cells and splenocytes lacking both ICAM and CD28 fail to proliferate in response to anti-CD3-induced T cell signals. This confirms that not only can ICAM-1 act as a CD28-independent costimulator, but it is the dominant, requisite costimulatory molecule for the activation of T cells in the absence of B7/CD28 costimulation.     

Defective thymic T cell activation by concanavalin A and anti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis.

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.     

Suppression of T cell signaling by polyunsaturated fatty acids: selectivity in inhibition of mitogen-activated protein kinase and nuclear factor activation

Polyunsaturated fatty acids (PUFAs) are known to suppress inflammatory and autoimmune responses and, therefore, clinical applications of PUFAs as immunomodulatory substances are extensively studied. PUFAs are known to inhibit T cell responses, but with respect to TCR/CD3-mediated signal transduction only a block in CD3-induced phospholipase Cgamma1/calcium signaling has been shown so far. In this study, we investigated PUFA-mediated changes in downstream T cell signal transduction. We show that among the mitogen-activated protein kinase families activation of c-Jun NH(2)-terminal kinase, but not phosphorylation of extracellular signal-regulated kinase-1/-2 or p38 is inhibited. CD3/CD28-induced activity of NF-AT was markedly reduced by PUFA treatment, while activation of other nuclear receptors (AP-1 and NF-kappaB) remained unaltered. Furthermore, IL-2 promoter activity, IL-2 and IL-13 mRNA levels, IL-2 secretion, and IL-2R alpha-chain expression were significantly diminished by PUFA treatment, whereas the expression of IFN-gamma, IL-4, IL-10, and CD69 remained essentially unaffected by PUFAs. In conclusion, PUFA treatment of T cells inhibits selectively c-Jun NH(2)-terminal kinase and NF-AT activation, resulting in diminished production of IL-2 and IL-13.     

Disruption of Mekk2 in mice reveals an unexpected role for MEKK2 in modulating T-cell receptor signal transduction

MEKK2 is a member of the mitogen-activated protein kinase (MAPK) kinase kinase gene family involved in regulating multiple MAPK signaling pathways. To elucidate the in vivo function of MEKK2, we generated mice carrying a targeted mutation in the Mekk2 locus. Mekk2(-/-) mice are viable and fertile. Major subsets of thymic and spleen T cells in Mekk2-deficient mice were indistinguishable from those in wild-type mice. B-cell development appeared to proceed similarly in the bone marrow of Mekk2-deficient and wild-type mice. However, Mekk2(-/-) T-cell proliferation was augmented in response to anti-CD3 monoclonal antibody (MAb) stimulation, and these T cells produced more interleukin 2 and gamma interferon than did the wild-type T cells, suggesting that MEKK2 may be involved in controlling the strength of T-cell receptor (TCR) signaling. Consistently, Mekk2(-/-) thymocytes were more susceptible than wild-type thymocytes to anti-CD3 MAb-induced cell death. Furthermore, TCR-mediated c-Jun N-terminal kinase activation was not blocked but moderately enhanced in Mekk2(-/-) T cells. Neither extracellular signal-regulated kinase nor p38 MAPK activation was affected in Mekk2(-/-) T cells. In conclusion, we found that MEKK2 may be required for controlling the strength of TCR/CD3 signaling.     

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.     

Responsiveness of fetal and adult CD4-, CD8- thymocytes to T cell activation

Day-14 fetal CD4-, CD8- thymocytes showed a greater proliferative response to PMA + IL-4 than did adult double-negative thymocytes. In contrast, adult double-negative thymocytes were more responsive to PMA + IL-1 + IL-2 or to IL-1 + IL-2 alone. The adult double-negative thymocytes showed significantly greater proliferation than fetal thymocytes after stimulation via anti-CD3 or anti-Thy-1 in the presence or absence of interleukins (IL-1 + IL-2 or IL-4). Adult CD4-, CD8- thymocytes also exhibited greater calcium mobilization following anti-CD3 stimulation IL-2-dependent activation with anti-Thy-1 or IL-1 + IL-2 in the absence of PMA resulted in marked expansion of CD 3+, F23.1+, CD4-, CD8- thymocytes, a population absent in fetal thymocytes but constituting 4% of pre-cultured CD4-, CD8- adult thymocytes. IL-4 + PMA failed to expand this CD 3+ population. It is hypothesized that before expression of functional TCR, T cell development may be more dependent on activation pathways not using IL-2; after TCR expression, IL-2-dependent pathways, including Thy-1-mediated stimulation, become functional.     

Regulation of mature T lymphocyte proliferation and differentiation by Par-4

The genetic inactivation of the atypical protein kinase C (aPKC) inhibitor, Par-4, gives rise to increased NF-kappaB activation and decreased stimulation of JNK in embryo fibroblasts. Here we have characterized the immunological phenotype of the Par-4(-/-) mice and found that the loss of this gene leads to an increased proliferative response of peripheral T cells when challenged through the TCR. This is accompanied by a higher increase in cell cycle entry and inhibition of apoptosis, with enhanced IL-2 secretion but normal CD25 synthesis. Interestingly, the TCR-triggered activation of NF-kappaB was augmented and that of JNK was severely abrogated. Consistent with previous data from knock outs of different JNKs, NFATc1 activation and IL-4 secretion were augmented in the Par-4-deficient CD4+ T cells, suggesting that the loss of Par-4 drives T-cell differentiation towards a Th2 response. This is compelling evidence that Par-4 is a novel modulator of the immune response through its ability to impact aPKC activity, which translates into lower JNK signaling.     

E2 Polyubiquitin-conjugating Enzyme Ubc13 in Keratinocytes Is Essential for Epidermal Integrity

The E2 polyubiquitin-conjugating enzyme Ubc13 is a mediator of innate immune reactions. Ubc13 mediates the conjugation of keratin (K)63-linked polyubiquitin chains onto TNF receptor-associated factor 6 and IKKγ during NF-κB activation. In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains function in nonproteasomal biological processes. Although Ubc13 has been shown to be critical for Toll-like receptor (TLR) and IL-1 receptor signaling, the function of Ubc13 in the epidermis has not been studied. We generated keratinocyte-specific Ubc13-deficient mice (Ubc13^flox/floxK5-Cre). At birth, the skin of the Ubc13^flox/floxK5-Cre mice was abnormally shiny and smooth; in addition, the mice did not grow and died by postnatal day 2. Histological analysis showed atrophy of the epidermis with keratinocyte apoptosis. Immunohistochemical analyses revealed reduced proliferation, abnormal differentiation, and apoptosis of keratinocytes in the Ubc13^flox/floxK5-Cre mouse epidermis. In culture, Ubc13^flox/floxK5-Cre keratinocyte growth was impaired, and spontaneous cell death occurred. Moreover, the deletion of Ubc13 from cultured Ubc13^flox/flox keratinocytes by means of an adenoviral vector carrying Cre recombinase also resulted in spontaneous cell death. Therefore, Ubc13 is essential for keratinocyte growth, differentiation, and survival. Analyses of intracellular signaling revealed that the IL-1 and TNF-induced activation of JNK, p38, and NF-κB pathways was impaired in Ubc13^flox/floxK5-Cre keratinocytes. In conclusion, Ubc13 appears to be essential for epidermal integrity in mice.     

Modulation of CREB and NF-kappaB signal transduction by cannabinol in activated thymocytes

Cannabinoid compounds inhibit the cAMP signalling cascade in leukocytes. One of these compounds, cannabinol (CBN) has been shown to inhibit interleukin-2 (IL-2) expression and the activation of cAMP response element binding protein (CREB) and nuclear factor for immunoglobulin kappa chain in B cells (NF-kappaB) following phorbol-12-myristate-13 acetate (PMA) plus ionomycin (Io) treatment of thymocytes. Therefore, the objective of the present studies was to determine the role of cAMP and protein kinase A (PKA) in the CBN-mediated inhibition of IL-2, CREB, and NF-kappaB in PMA/Io-activated thymocytes. The inhibition of CREB/ATF-1 phosphorylation, or cAMP response element (CRE) or kappaB DNA binding activity produced by CBN in PMA/Io-activated thymocytes, could not be reversed by DBcAMP costimulation. Furthermore, DBcAMP failed to reverse the concentration-dependent inhibition of IL-2 protein secretion by CBN. Pretreatment of thymocytes with H89 produced a modest inhibition of PMA/Io-induced CREB/ATF-1 phosphorylation and CRE DNA binding activity but H89 had no effect on protein binding to a kappaB motif. Additionally, H89 modestly inhibited PMA/Io-induced IL-2 secretion. In light of the modest involvement of the cAMP pathway in CBN-mediated inhibition of CREB and IL-2 in PMA/Io-activated thymocytes, PD098059 (PD), the MEK inhibitor, was utilized to determine the role of ERK MAP kinases in thymocytes. ERKs play a critical role in IL-2 production but not for CREB phsophorylation. Collectively, these findings suggest that CBN may modulate several signalling pathways in activated T cells.     

TCR-independent CD30 signaling selectively induces IL-13 production via a TNF receptor-associated factor/p38 mitogen-activated protein kinase-dependent mechanism

Initiation of T lymphocyte responses to most Ags requires concurrent stimulation through the TCR and costimulatory receptors such as CD28. Following initial activation, secondary receptors are up-regulated that can costimulate T cells in concert with TCR engagement. One such receptor is the TNFR family member CD30. In this study, we report that unlike CD28, ligation of CD30 on normal effector T cells induces IL-13 production in the absence of concurrent TCR engagement. TCR-independent CD30-mediated IL-13 release correlated with activation of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase (MAPK), and NF-kappaB, and was completely inhibited by the expression of a TNFR-associated factor 2 (TRAF2) dominant-negative transgene (TRAF2.DN-Tg), but not by that of an I-kappaBalpha dominant-negative transgene. In parallel, expression of the TRAF2.DN-Tg selectively prevented the induction of c-Jun N-terminal kinase and p38 MAPK, but not that of NF-kappaB. Furthermore, IL-13 production was reduced in a dose-dependent manner by the p38 MAPK inhibitor SB203580. Together, these results suggest that TCR-independent CD30-mediated production of IL-13 is triggered by association of CD30 with TRAF family members and subsequent activation of p38 MAPK. Inasmuch as IL-13 can promote airway inflammation and cancer progression, production of IL-13 in a TCR-independent manner has important pathological implications in vivo.     

Inhibition of NF-kappaB activation reduces the tissue effects of transgenic IL-13

IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4Ralpha/IL-13Ralpha1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-kappaB. As a result, we hypothesized that NF-kappaB activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-kappaB activity. Three pharmacologic approaches were used to inhibit NF-kappaB including 1) PS1145, a small molecule inhibitor of IkappaBalpha kinase (IKK2), 2) antennapedia-linked NF-kappaB essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-kappaB component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-kappaB is an attractive target for immunotherapy of IL-13-mediated diseases.     

CD3/CD28 costimulation-induced NF-kappaB activation is mediated by recruitment of protein kinase C-theta, Bcl10, and IkappaB kinase beta to the immunological synapse through CARMA1

CARMA1 (also known as CARD11) is a scaffold molecule and contains a caspase-recruitment domain (CARD) and a membrane-associated guanylate kinase-like (MAGUK) domain. It plays an essential role in mediating CD3/CD28 costimulation-induced NF-kappaB activation. However, the molecular mechanism by which CARMA1 mediates costimulatory signals remains to be determined. Here, we show that CARMA1 is constitutively associated with the cytoplasmic membrane. This membrane association is essential for the function of CARMA1, since a mutant of CARMA1, CARMA1(L808P), that is defective in the membrane association cannot rescue CD3/CD28 costimulation-induced NF-kappaB activation in JPM50.6 CARMA1-deficient T cells. Although CD3/CD28 costimulation effectively induces the formation of the immunological synapse in CARMA1-deficient T cells, the recruitment of protein kinase C-theta (PKC-theta), Bcl10, and IkappaB kinase beta (IKKbeta) into lipid rafts of the immunological synapse is defective. Moreover, expression of wild-type CARMA1, but not CARMA1(L808P), restores the recruitment of PKC-theta, Bcl10, and IKKbeta into lipid rafts in CARMA1-deficient T cells. Consistently, expression of a mutant CARMA1, CARMA1(DeltaCD), that cannot associate with Bcl10 failed to restore CD3/CD28 costimulation-induced NF-kappaB activation in JPM50.6 cells, whereas expression of Bcl10-CARMA(DeltaCD) fusion protein effectively restored this NF-kappaB activation. Together, these results indicate that CARMA1 mediates CD3/CD28 costimulation-induced NF-kappaB activation by recruiting downstream signaling components into the immunological synapse.