Tespa1 is involved in late thymocyte development through the regulation of TCR-mediated signaling.

Signaling via the T cell antigen receptor (TCR) during the CD4(+)CD8(+) double-positive developmental stage determines thymocyte selection and lineage commitment. Here we describe a previously uncharacterized T cell-expressed protein, Tespa1, with critical functions during the positive selection of thymocytes. Tespa1(-/-) mice had fewer mature thymic CD4(+) and CD8(+) T cells, which reflected impaired thymocyte development. Tespa1 associated with the TCR signaling components PLC-γ1 and Grb2, and Tespa1 deficiency resulted in attenuated TCR signaling, as reflected by defective activation of the Erk-AP-1 and Ca(2+)-NFAT pathways. Our findings demonstrate that Tespa1 is a component of the TCR signalosome and is essential for T cell selection and maturation through the regulation of TCR signaling during T cell development.

Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation.

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

The role of PLCγ2 in immunological disorders, cancer, and neurodegeneration.

Phosphatidylinositol-specific phospholipase Cγ2 (PLCγ2) is a critical signaling molecule activated downstream from a variety of cell surface receptors that contain an intracellular immunoreceptor tyrosine-based activation motif. These receptors recruit kinases such as Syk, BTK, and BLNK to phosphorylate and activate PLCγ2, which then generates 1D-myo-inositol 1,4,5-trisphosphate and diacylglycerol. These well-known second messengers are required for diverse membrane functionality including cellular proliferation, endocytosis, and calcium flux. As a result, PLCγ2 dysfunction is associated with a variety of diseases including cancer, neurodegeneration, and immune disorders. The diverse pathologies associated with PLCγ2 are exemplified by distinct genetic variants. Inherited mutations at this locus cause PLCγ2-associated antibody deficiency and immune dysregulation, in some cases with autoinflammation. Acquired mutations at this locus, which often arise as a result of BTK inhibition to treat chronic lymphocytic leukemia, result in constitutive downstream signaling and lymphocyte proliferation. Finally, a third group of PLCγ2 variants actually has a protective effect in a variety of neurodegenerative disorders, presumably by increased uptake and degradation of deleterious neurological aggregates. Therefore, manipulating PLCγ2 activity either up or down could have therapeutic benefit; however, we require a better understanding of the signaling pathways propagated by these variants before such clinical utility can be realized. Here, we review the signaling roles of PLCγ2 in hematopoietic cells to help understand the effect of mutations driving immune disorders and cancer and extrapolate from this to roles which may relate to protection against neurodegeneration.

Vitamin D controls T cell antigen receptor signaling and activation of human T cells.

Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation.

OSBP-Related Protein 5L Maintains Intracellular IP3/Ca2+ Signaling and Proliferation in T Cells by Facilitating PIP2 Hydrolysis.

Phospholipase C (PLC) isoforms play central roles in signaling cascades by cleaving PIP2 into the second messengers IP3 and DAG. In this study, to our knowledge, we uncover that ORP5L interacts physically with PLCγ1 in T cells, extracts PIP2 from the plasma membrane via its ORD domain (OSBP-related domain), presents it to PLCγ1 (enabling IP3 generation), and eventually maintains intracellular Ca2+ homeostasis. Through this mechanism, ORP5L promotes T cell proliferation in a Ca2+-activated NFAT2-dependent manner. To our knowledge, our study uncovers a new key function of ORP5L as a critical cofactor for PLCγ1 catalysis and its crucial role in human T cell proliferation.

BCALM (AC099524.1) Is a Human B Lymphocyte-Specific Long Noncoding RNA That Modulates B Cell Receptor-Mediated Calcium Signaling.

Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell-specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell-specific phospholipase C γ 2 (PLCG2), a B cell-specific enzyme that stimulates intracellular Ca2+ signaling in response to BCR activation. AC099524.1 (B cell-associated lncRNA modulator of BCR-mediated Ca+ signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca+ flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that downmodulates BCR-mediated Ca+ signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca+ signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR-dependent cancers.

Type of PaperY192 within the SH2 Domain of Lck Regulates TCR Signaling Downstream of PLC-γ1 and Thymic Selection.

Signaling via the TCR, which is initiated by the Src-family tyrosine kinase Lck, is crucial for the determination of cell fates in the thymus. Because of its pivotal role, ablation of Lck results in a profound block of T-cell development. Here, we show that, in addition to its well-known function in the initiation of TCR signaling, Lck also acts at a more downstream level. This novel function of Lck is determined by the tyrosine residue (Y192) located in its SH2 domain. Thymocytes from knock-in mice expressing a phosphomimetic Y192E mutant of Lck initiate TCR signaling upon CD3 cross-linking up to the level of PLC-γ1 phosphorylation. However, the activation of downstream pathways including Ca2+ influx and phosphorylation of Erk1/2 are impaired. Accordingly, positive and negative selections are blocked in LckY192E knock-in mice. Collectively, our data indicate that Lck has a novel function downstream of PLCγ-1 in the regulation of thymocyte differentiation and selection.

PGE2 deficiency predisposes to anaphylaxis by causing mast cell hyperresponsiveness.

BACKGROUND: Reduced levels of prostaglandin E2 (PGE2) contribute to aspirin-induced hypersensitivity. COX inhibitors are also frequent cofactors in anaphylaxis. Whether alterations in the PGE2 system contribute to anaphylaxis independently of COX inhibitor intake is unclear. OBJECTIVE: Our aim was to test the hypothesis that relative PGE2 deficiency predisposes to anaphylaxis. METHODS: Sera from 48 patients with anaphylaxis and 27 healthy subjects were analyzed for PGE2 levels and correlated against severity; 9α,11ß-PGF2 and PGI2 metabolites were measured for control purposes. PGE2 stabilization by 15-hydroxyprostaglandin dehydrogenase inhibitor or EP2 or EP4 receptor agonists were used in a murine model of passive systemic anaphylaxis. FcεRI-triggered mediator release was determined in bone marrow-derived cultured mast cells (MCs) and human skin-derived MCs. Signaling was studied by Western blot analysis. RESULTS: Patients with anaphylaxis were characterized by markedly reduced PGE2 levels vis-à-vis healthy subjects, whereas prostacyclin metabolite levels were diminished only weakly, and 9α,11ß-PGF2 levels conversely increased. PGE2 was negatively correlated with severity. Lower PGE2 levels and higher susceptibility to anaphylaxis were also found in C57BL/6 mice vis-à-vis in Balb/c mice. Stabilization of PGE2 level by 15-hydroxyprostaglandin dehydrogenase inhibitor protected mice against anaphylaxis. Exogenous PGE2 attenuated bone marrow-derived cultured MC activation through EP2 and EP4 receptors. EP2 and EP4 agonism also curbed FcεRI-mediated degranulation of human MCs. Mechanistically, PGE2 interfered with the phosphorylation of phospholipase C gamma-1 and extracellular signal-regulated kinase. CONCLUSIONS: Homeostatic levels of PGE2 attenuate MC activation via EP2/EP4 and protect against anaphylaxis. Relative deficiency of PGE2 predisposes to anaphylaxis in humans and mice, whereas PGE2 stabilization protects against anaphylactic reactions.

AIMP1 regulates TCR signaling and induces differentiation of regulatory T cells by interfering with lipid raft association.

In addition to a role in translation, AIMP1 is secreted to affect various immune cells, such as macrophages, dendritic cells, B cells, and natural killer cells. However, the direct effects of AIMP1 on T cells have not yet been reported. In this study, we investigated whether AIMP1 could modulate T cell responses directly. Results revealed that AIMP1 significantly inhibited T cell receptor (TCR)-dependent activation and proliferation of CD4 T cells, as well as decreased TCR stimuli-induced Ca2+ influx in CD4 T cells. In addition, microscopic analysis revealed that lipid raft association in response to TCR engagement was significantly reduced in the presence of AIMP1, and the phosphorylation of PLCγ and PI3K was also down-regulated in CD4 T cells by AIMP1. Furthermore, AIMP1 specifically enhanced the differentiation of regulatory T (Treg) cells, while it had no effect on T helper type 1 (Th1), type 2 (Th2), and type 17 (Th17) cell differentiation. Collectively, these results indicate that AIMP1 affects T cells directly by down-regulating TCR signaling complex formation and inducing Treg cell differentiation in CD4 T cells.

Plcγ2/Tmem178 dependent pathway in myeloid cells modulates the pathogenesis of cytokine storm syndrome.

Cytokine storm syndrome (CSS) is a life-threatening condition characterized by excessive activation of T cells and uncontrolled inflammation, mostly described in patients with familial hemophagocytic lymphohistiocytosis and certain systemic auto-inflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA). Defects in T cell cytotoxicity as a mechanism for uncontrolled inflammation following viral infections fail to represent the whole spectrum of CSS. Evidence implicates dysregulated innate immune responses, especially activation of monocytes and macrophages, in patients with CSS. However, the direct contribution of monocytes/macrophages to CSS development and the signaling pathways involved in their activation have not been formally investigated. We find that depletion of monocytes/macrophages during early stages of CSS development, by clodronate-liposomes or neutralizing anti-CSF1 antibody, reduces mortality and inflammatory cytokine levels in two CSS mouse models, one dependent on T cells and the second induced by repeated TLR9 stimulation. We further demonstrate that activation of Plcγ2 in myeloid cells controls CSS development by driving macrophage pro-inflammatory responses. Intriguingly, the Plcγ2 downstream effector Tmem178, a negative modulator of calcium levels, acts in a negative feedback loop to restrain inflammatory cytokine production. Genetic deletion of Tmem178 leads to pro-inflammatory macrophage polarization in vitro and more severe CSS in vivo. Importantly, Tmem178 levels are reduced in macrophages from mice with CSS and after exposure to plasma from sJIA patients with active disease. Our data identify a novel Plcγ2/Tmem178 axis as a modulator of inflammatory cytokine production by monocytes/macrophages. We also find that loss of Tmem178 accentuates the pro-inflammatory responses in CSS.

Expanding the clinical features of autoinflammation and phospholipase Cγ2-associated antibody deficiency and immune dysregulation by description of a novel patient.

BACKGROUND: Autoinflammation and phospholipase Cγ2-associated antibody deficiency and immune dysregulation (APLAID) is an exceedingly rare monogenic autoinflammatory disease. To date, only five cases have been reported with four distinct pathogenic mutations. OBJECTIVES: We present a novel case of APLAID, corroborated by molecular analysis, with newly described clinical findings including central nervous system vasculitis (CNSV); and distinctive histopathological characteristics that may expand our knowledge of this rare disease's phenotype. METHODS: This is a case report presentation of a 3-year-old boy, seen at a reference paediatric hospital in Mexico. His parents authorized the use of his clinical information and photographs. RESULTS: A 3-day-old boy presented to the emergency department with a vesiculo-pustular rash that resolved within 1 week. Two months later, he developed widespread papules and pseudovesicles that evolved into infiltrated plaques. He also had periodical flares of conjunctivitis, diarrhoea and erythematous blistering acral plaques triggered by upper respiratory infections. By the age of 10 months, he experienced seizures and CNSV. Laboratory work-up showed mild neutropenia, decreased serum levels of immunoglobulins and B-cell lymphopenia. A skin biopsy revealed a dense, perivascular and interstitial histiocytic and granulomatous infiltrate, with palisading granulomas, and leucocytoclastic vasculitis with karyorrhexis. APLAID syndrome was confirmed by Sanger sequencing of PLCG2 gene [heterozygous genotype LRG_376t1:c.2543T>C or p.(Leu848Pro)]. CONCLUSIONS: Presence of CNSV has not been previously described in APLAID, however as the number of reported patients with APLAID is very small, it is possible that the overall spectrum of clinical manifestations has not been completely elucidated. The herein identified p.(Leu848Pro) variant was also documented in a Portuguese patient, suggesting that it could be a PLCG2 gene 'hot-spot'.

NF-κB inducing kinase (NIK) is an essential post-transcriptional regulator of T-cell activation affecting F-actin dynamics and TCR signaling.

NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCγ upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.

Differential expression of interleukin-17A and -17F is coupled to T cell receptor signaling via inducible T cell kinase.

T helper 17 (Th17) cells play major roles in autoimmunity and bacterial infections, yet how T cell receptor (TCR) signaling affects Th17 cell differentiation is relatively unknown. We demonstrate that CD4(+) T cells lacking Itk, a tyrosine kinase required for full TCR-induced phospholipase C-gamma (PLC-gamma1) activation, exhibit decreased interleukin-17A (IL-17A) expression in vitro and in vivo, despite relatively normal expression of retinoic acid receptor-related orphan receptor-gammaT (ROR-gammaT) and IL-17F. IL-17A expression was rescued by pharmacologically induced Ca(2+) influx or constitutively activated nuclear factor of activated T cells (NFAT). Conversely, decreased TCR stimulation or calcineurin inhibition preferentially reduced IL-17A expression. We further found that the promoter of Il17a but not Il17f has a conserved NFAT binding site that bound NFATc1 in wild-type but not Itk-deficient cells, even though both exhibited open chromatin conformations. Finally, Itk(-/-) mice also showed differential regulation of IL-17A and IL-17F in vivo. Our results suggest that Itk specifically couples TCR signaling to Il17a expression and the differential regulation of Th17 cell cytokines through NFATc1.

Lectin binding to surface Ig variable regions provides a universal persistent activating signal for follicular lymphoma cells.

The vast majority of cases of follicular lymphoma (FL), but not normal B cells, acquire N-glycosylation sites in the immunoglobulin variable regions during somatic hypermutation. Glycans added to sites are unusual in terminating at high mannoses. We showed previously that the C-type lectins, dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose receptor, bound to FL surface immunoglobulin (sIg), generating an intracellular Ca(2+) flux. We have now mapped further intracellular pathways activated by DC-SIGN in a range of primary FL cells with detection of phosphorylated ERK1/2, AKT, and PLCγ2. The SYK inhibitor (tamatinib) or the BTK inhibitor (ibrutinib) each blocked phosphorylation. Activation by DC-SIGN occurred in both IgM(+) and IgG(+) cases and led to upregulation of MYC expression, with detection in vivo observed in lymph nodes. Unlike cells of chronic lymphocytic leukemia, FL cells expressed relatively high levels of sIg, unchanged by long-term incubation in vitro, indicating no antigen-mediated downregulation in vivo. In contrast, expression of CXCR4 increased in vitro. Engagement of sIg in FL cells or normal B cells by anti-Ig led to endocytosis in vitro as expected, but DC-SIGN, even when cross-linked, did not lead to significant endocytosis of sIg. These findings indicate that lectin binding generates signals via sIg but does not mediate endocytosis, potentially maintaining a supportive antigen-independent signal in vivo. Location of DC-SIGN in FL tissue revealed high levels in sinusoidlike structures and in some colocalized mononuclear cells, suggesting a role for lectin-expressing cells at this site.

The Importance of IL-6 in the Development of LAT-Mediated Autoimmunity.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is highly tyrosine phosphorylated upon engagement of the TCR. Phosphorylated LAT binds Grb2, Gads, and phospholipase C (PLC)γ1 to mediate T cell activation, proliferation, and cytokine production. T cells from mice harboring a mutation at the PLCγ1 binding site of LAT (Y136F) have impaired calcium flux and Erk activation. Interestingly, these T cells are highly activated, resulting in the development of a lymphoproliferative syndrome in these mice. CD4(+) T cells in LATY136F mice are Th2 skewed, producing large amounts of IL-4. In this study, we showed that the LATY136F T cells could also overproduce IL-6 due to activated NF-κB, AKT, and p38 pathways. By crossing LATY136F mice with IL-6-deficient mice, we demonstrated that IL-6 is required for uncontrolled T cell expansion during the early stage of disease development. Reduced CD4(+) T cell expansion was not due to a further block in thymocyte development or an increase in the number of regulatory T cells, but was caused by reduction in cell survival. In aged IL-6(-/-) LATY136F mice, CD4(+) T cells began to hyperproliferate and induced splenomegaly; however, isotype switching and autoantibody production were diminished. Our data indicated that the LAT-PLCγ1 interaction is important for controlling IL-6 production by T cells and demonstrated a critical role of IL-6 in the development of this lymphoproliferative syndrome.

The role of LAT-PLCγ1 interaction in γδ T cell development and homeostasis.

LAT is a transmembrane adaptor protein that is vital for integrating TCR-mediated signals to modulate T cell development, activation, and proliferation. Upon T cell activation, LAT is phosphorylated and associates with Grb2, Gads, and PLCγ1 through its four distal tyrosine residues. Mutation of one of these tyrosines, Y136, abolishes LAT binding to PLCγ1. This results in impaired TCR-mediated calcium mobilization and Erk activation. CD4 αß T cells in LATY136F knock-in mice undergo uncontrolled expansion, resulting in a severe autoimmune syndrome. In this study, we investigated the importance of the LAT-PLCγ1 interaction in γδ T cells by crossing LATY136F mice with TCRß(-/-) mice. Our data showed that the LATY136F mutation had no major effect on homeostasis of epithelial γδ T cells, which could be found in the skin and small intestine. Interestingly, a population of CD4(+) γδ T cells in the spleen and lymph nodes underwent continuous expansion and produced elevated amounts of IL-4, resulting in an autoimmune syndrome similar to that caused by αß T cells in LATY136F mice. Development of these hyperproliferative γδ T cells was not dependent on MHC class II expression or CD4, and their proliferation could be suppressed, in part, by regulatory T cells. Our data indicated that a unique subset of CD4 γδ T cells can hyperproliferate in LATY136F mice and suggested that LAT-PLCγ1 signaling may function differently in various subsets of γδ T cells.

Inhibitory effect of Pterocarpus indicus Willd water extract on IgE/Ag-induced mast cell and atopic dermatitis-like mouse models.

Pterocarpus indicus Willd has been widely used as a traditional medicine to treat edema, cancer, and hyperlipidemia, but its antiallergic properties and underlying mechanisms have not yet been studied. The purpose of this study was to evaluate the antiallergic activity of Pterocarpus indicus Willd water extract (PIW) using activated mast cells and an atopic dermatitis (AD)-like mouse model. PIW decreased IgE/Ag-induced mast cell degranulation and the phosphorylation of Syk and downstream signaling molecules such as PLC-γ, Akt, Erk 1/2, JNK compared to stimulated mast cells. In DNCB-induced AD-like mice, PIW reduced IgE level in serum, as well as AD-associated scratching behavior and skin severity score. These results indicate that PIW inhibits the allergic response by reducing mast cell activation and may have clinical potential as an antiallergic agent for disorders such as AD.

Novel CD3-specific antibody induces immunosuppression via impaired phosphorylation of LAT and PLCγ1 following T-cell stimulation.

The activation of T cells is known to be accompanied by the temporary downmodulation of the TCR/CD3 complex on the cell surface. Here, we established a novel monoclonal antibody, Dow2, that temporarily induces downmodulation of the TCR/CD3 complex in mouse CD4(+) T cells without activating T cells. Dow2 recognized the determinant on CD3ε; however, differences were observed in the binding mode between Dow2 and the agonistic anti-CD3ε Ab, 145-2C11. An injection of Dow2 in vivo resulted in T-cell anergy, and prolonged the survival of cardiac allografts without a marked increase in cytokine release. The phosphorylated forms of the signaling proteins PLC-γ1 and LAT in Dow2-induced anergic T cells were markedly decreased upon stimulation. However, the levels of phosphorylated LAT and PLCγ1 in Dow2-induced anergic T cells could be rescued in the presence of the proteasome inhibitor MG-132. These results suggest that proteasome-mediated degradation is involved in hypophosphorylated LAT and PLCγ1 in Dow2-induced anergic T cells. The novel CD3-specific Ab, Dow2, may provide us with a unique tool for inducing immunosuppression.

NF-κB is crucial in proximal T-cell signaling for calcium influx and NFAT activation.

In the accepted model of T-cell activation, parallel signal-transduction pathways activate the transcription factors NF-κB, NFAT, and AP-1 to drive clonal expansion of T cells in response to Ag. Genome-wide transcriptional profiling following Ag-induced CD8(+) T-cell activation in C57BL/6 mouse T cells revealed that genes regulated by NFAT were also reduced in the absence of NF-κB p50 and cRel subunits. Importantly, p50(-/-) cRel(-/-) CD8(+) T cells had significantly diminished NFAT and AP-1 activation compared with WT or PKCθ(-/-) CD8(+) T cells. Attenuated NFAT activation after TCR engagement was associated with reduced calcium influx, PLCγ and Zap70 activation. Interestingly, pharmacological bypass of PLCγ-regulated pathways largely rescued p50(-/-) cRel(-/-) T-cell proliferative defects. These results indicate a crucial and unexpected requirement for NF-κB p50 and cRel subunits in proximal TCR signaling and calcium responses. They further suggest that key defects in T cells in the absence of NF-κB pathway components may be due to impaired proximal T-cell signaling.