Anti-CD69 autoantibodies cross-react with low density lipoprotein receptor-related protein 2 in systemic autoimmune diseases

We investigated whether autoantibodies to CD69, one of the earliest markers of lymphocyte activation, exist in the sera of patients with systemic autoimmune disease. Serum samples were obtained from patients with rheumatoid arthritis (RA), systemic lupus erythematosus, and Behcet's disease, and were tested for the presence of anti-CD69 autoantibodies by ELISA and Western blotting using rCD69 fusion proteins. IgG-type autoantibodies to CD69 were detected in the sera of 38.3% of the RA patients, 14.5% of the systemic lupus erythematosus patients, and 4.0% of the patients with Behcet's disease. Among those with RA, the anti-CD69 autoantibody-positive patients had a higher serum level of rheumatoid factors and a more accelerated erythrocyte sedimentation rate than the anti-CD69 autoantibody-negative patients. Further, the predominant epitope on the CD69 molecule to which most of the anti-CD69 autoantibody-positive serum samples exclusively reacted, was mapped at the C terminus of CD69. Of interest, this epitope is homologous to a stretch of amino acids in the protein sequence of low-density lipoprotein receptor-related protein 2 (LRP2), which is a receptor for multiple ligands including beta-very low density lipoprotein and is also an autoantigen responsible for Heymann nephritis in rats. The anti-CD69 autoantibody cross-reacted to LRP2 through the homologous amino acid sequence. To our knowledge, this is the first evidence of the existence of anti-CD69 autoantibodies. This autoantibody may modulate the function of CD69- and LRP2-expressing cells.     

Ligation of the T cell receptor complex results in phosphorylation of Smad2 in T lymphocytes

TGF-beta modulates immune responses by regulating T cell function. The Smad family of proteins has been recently shown to transduce signals for the TGF-beta superfamily and Smad2 mediates TGF-beta signaling. Here, we showed that TGF-beta phosphorylated Smad2 and induced interaction between Smad2 and Smad4 in primary T cells and the Jurkat T cell line. Interestingly, ligation of the T cell receptor (TCR)/CD3 complex with anti-CD3 mAb also phosphorylated Smad2, but failed to induce interaction between Smad2 and Smad4 in the Jurkat T cell line. Phosphorylation of Smad2 via the TCR/CD3 complex was not abrogated by treatment with neutralizing antibody against TGF-beta. Furthermore, PD98059, a MEK inhibitor, suppressed Smad2 phosphorylation by stimulation with anti-CD3 mAb in Jurkat T cell line. These findings indicated that not only TGF-beta but also stimulation via the TCR/CD3 complex phosphorylated Smad2 through mitogen-activated protein (MAP) kinase cascades, suggesting that Smad2 may function in both TGF-beta- and TCR/CD3 complex-mediated signaling pathways in T cells.     

An HIV-1-infected T cell clone defective in IL-2 production and Ca2+ mobilization after CD3 stimulation

A chronically HIV-1-infected T cell clone (J1.1) derived from Jurkat cells was developed that possesses defects in CD3 signaling. This clone was phenotypically determined to be CD4- and express a reduced surface density of CD3 as compared with a pool of uninfected Jurkat clones. Although J1.1 could be induced with TNF-alpha to produce HIV-1 particles, stimulation via the CD3 (T3-Ti) complex, using mAb cross-linking, had no effect on viral production. Further investigation revealed that J1.1 secreted approximately 20-fold less IL-2 than did uninfected Jurkat cells after anti-CD3 treatment. In addition, a separate defect in Ca2+ mobilization was noted in the HIV-1-infected J1.1 line when compared with uninfected Jurkat cells after anti-CD3 cross-linking. The cell line described offers a new model in which to study the mechanisms of several defects directly imposed by HIV-1 on CD3+ cells.     

Signaling via the inositol phospholipid pathway by T cell antigen receptor is limited by receptor number.

Engagement of the TCR initiates at least two transmembrane signaling pathways, the phosphatidylinositol pathway and a tyrosine kinase pathway. The T cell leukemic line Jurkat was used to study the relationship between the number of occupied TCR on the cell surface and the TCR-mediated activation of phosphatidylinositol-specific phospholipase C. We characterized a series of Ti beta-chain transfectants of the Jurkat mutant J.RT3-T3.5, in which surface expression of the TCR is limited by expression of the TCR beta-chain. Calibrated flow cytometry was used to determine the number of binding sites for anti-CD3 mAb on the surface of these cells, which was less than 1.2 x 10(3) to 1.2 x 10(4) sites/cell. In the presence of lithium chloride, the accumulation of inositol phosphates (InsP) in these cell lines in response to saturating concentrations of anti-CD3 mAb was proportional to the calculated surface TCR number. This result was consistent with dose-response studies using anti-CD3 mAb in Jurkat cells, in which ligand concentration, rather than number of binding sites, was limiting. Increase in intracellular free calcium concentration was a sensitive indicator of TCR engagement and correlated with the level of TCR expression, but less closely than did InsP levels. Induction of the early lymphocyte activation marker CD69 by anti-CD3 mAb also correlated with surface expression of TCR. In order to test whether limitation of this signaling pathway by TCR number may be relevant to signal transduction in the wild-type cell, we compared PLC activity in Jurkat cells during soluble anti-CD3 mAb-induced internalization of the TCR and also in response to immobilized mAb. The net accumulation of InsP per min decreased linearly with TCR number during the rapid phase of TCR internalization, confirming the limiting role of TCR number in this system. When internalization was prevented by immobilization of the stimulus, there was no decrease in the net accumulation of InsP per minute over time. In a Jurkat cell line transfected with the heterologous human muscarinic receptor, subtype 1, the InsP response to a muscarinic agonist was unaffected by TCR internalization, indicating that the distal phosphatidylinositol pathway was not affected by prolonged stimulation of the TCR. We conclude that transmembrane signaling through the TCR may be regulated by the number of surface TCR-ligand complexes. This observation has implications for transmembrane signaling in both mature T cells and thymocytes.     

Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation.

Antiphosphotyrosine immunoblots were used to characterize tyrosine phosphorylated proteins after stimulation of the human TCR. Increased tyrosine phosphorylation was evident on at least 12 substrates within 2 min after ligation of the TCR with mAb. Analysis of the time course for increased tyrosine phosphorylation revealed distinct patterns. Increased phosphorylation of 135-kDa and 100-kDa substrates was evident within 5 s, whereas increased phosphorylation of the TCR-zeta-chain required several minutes after treatment with anti-CD3 mAb. This rapid cellular tyrosine phosphorylation occurred independent of the cell cycle, as it occurred after stimulation of resting T cells, T cell blasts, and the Jurkat T cell leukemia line. When the TCR complex was cross-linked together with the CD4 receptor by heteroconjugate anti-CD3/CD4 mAb, an increased magnitude of tyrosine phosphorylation occurred, although no new substrates could be detected. The increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates was specific in that anti-HLA class I, anti-CD6, anti-CD7, and anti-CD28 antibodies did not cause increased tyrosine phosphorylation. Anti-CD4 stimulation of resting T cells did not cause increased tyrosine phosphorylation of pp100 and pp135, suggesting that the CD4-associated kinase, lck, does not account for the tyrosine phosphorylation observed after TCR stimulation. Similarly, pharmacologic treatment of cells with phorbol ester and calcium ionophore did not cause increased tyrosine phosphorylation of these substrates, indicating that activation of protein kinase C or phospholipase C does not account for these early increases in tyrosine phosphorylation. The time of onset of pp100 phosphorylation, and the magnitude of phosphorylation correlated with the magnitude of calcium mobilization when cells were stimulated with different forms of TCR stimulation. When cells were labeled with [3H]myoinositol and analyzed after stimulation by anti-CD3 mAb, increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates preceded the activation of phospholipase C, as measured by the appearance of inositol 1,4,5-trisphosphate. This occurred in both T cell blasts and in the Jurkat T cell line. Thus, these findings show that increased tyrosine phosphorylation is the earliest yet detected signal observed after ligation of the TCR complex, and furthermore suggest that tyrosine phosphorylation might link the TCR to the phosphatidylinositolbisphosphate hydrolysis signaling pathway.     

Expression of CD5 regulates responsiveness to IL-1

The role of the CD5 surface molecule in T cell responsiveness to IL-1 was examined. A CD5-mutant Jurkat cell line was generated from a CD5+ parent cell line. This CD5- mutant subclone was infected with a defective retrovirus containing the CD5 cDNA and/or the neo gene encoding G418 resistance. The CD5+ wild type Jurkat produced IL-2 in response to anti-CD3 mAb, OKT3, cross-linked to a solid surface. IL-2 production was enhanced by co-culture with IL-1 or anti-CD5 Mab. Neither the CD5- mutant nor the CD5- G418-resistant infectant responded to anti-CD5 mAb or to IL-1. Responsiveness to IL-1 was restored by cell surface expression of CD5 in the CD5+ infectant. Both the CD5+ wild type Jurkat and the CD5+ infectant responded equivalently to purified IL-1, IL-1 alpha and rIL-1 beta. Optimal concentrations of IL-1 and anti-CD5 mAb had an additive effect on the enhancement of IL-2 production stimulated with cross-linked anti-CD3 mAb suggesting that IL-1 and CD5 act through distinct, complementary pathways to augment T cell activation. The correlation of CD5 expression and specific binding of rIL-1 beta was examined in these cell lines. Both the specific binding (at 4 degrees C) and subsequent internalization (at 37 degrees C) of 125I labeled rIL-1 beta was equivalent in the CD5+ infectant and the CD5+ wild type Jurkat cell, whereas specific binding of 125I-labeled rIL-1 beta was markedly decreased in the CD5-G418-resistant infectant. These observations strongly suggest that cell surface expression of CD5 regulates binding of and responsiveness to IL-1.     

The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

S100A8 and S100A9, two Ca²⁺-binding proteins of the S100 family, are secreted as a heterodimeric complex (S100A8/A9) from neutrophils and monocytes/macrophages. Serum and synovial fluid levels of S100A8, S100A9, and S100A8/A9 were all higher in patients with rheumatoid arthritis (RA) than in patients with osteoarthritis (OA), with the S100A8/A9 heterodimer being prevalent. By two-color immunofluorescence labeling, S100A8/A9 antigens were found to be expressed mainly by infiltrating CD68⁺ macrophages in RA synovial tissue (ST). Isolated ST cells from patients with RA spontaneously released larger amounts of S100A8/A9 protein than did the cells from patients with OA. S100A8/A9 complexes, as well as S100A9 homodimers, stimulated the production of proinflammatory cytokines, such as tumor necrosis factor alpha, by purified monocytes and in vitro-differentiated macrophages. S100A8/A9-mediated cytokine production was suppressed significantly by p38 mitogen-activated protein kinase (MAPK) inhibitors and almost completely by nuclear factor kappa B (NF-κB) inhibitors. NF-κB activation was induced in S100A8/A9-stimulated monocytes, but this activity was not inhibited by p38 MAPK inhibitors. These results indicate that the S100A8/A9 heterodimer, secreted extracellularly from activated tissue macrophages, may amplify proinflammatory cytokine responses through activation of NF-κB and p38 MAPK pathways in RA.     

Expression of prohibitins on the surface of activated T cells

Prohibitins (prohibitin-1 and -2) comprise a family of highly conserved proteins that are mainly localized to mitochondria. Recent studies showed that prohibitins are up-regulated upon T cell activation and play an essential role in maintaining mitochondrial homeostasis. In the present study, we found that a considerable proportion of prohibitin-1 and -2 induced in response to T cell activation was expressed on the surface of activated T cells. When mouse and human T cells were stimulated with PMA and ionomycin, prohibitins expressed on the cell surface were increased significantly, peaking at 48 h after stimulation. Stimulation of mouse T cells with anti-CD3 and anti-CD28 antibodies also remarkably induced the cell surface expression of prohibitins. Their expression on the cell surface was also detected in T cell leukemia cells such as Jurkat cells. In Jurkat cells, prohibitin-1 and -2 were co-localized with CD3 on the cell surface, and anti-CD3 antibody-induced signaling, the MAP kinase cascade, was inhibited on treatment with protein A magnetic beads co-conjugated with anti-CD3 antibody and anti-prohibitin-1 or anti-prohibitin-2 antibody. These results suggest that prohibitins expressed on the surface of activated T cells are involved in the T cell receptor-mediated signaling cascade.     

The myeloid expressed EF-hand proteins display a diverse pattern of lipid raft association

EF-hand proteins are known to translocate to membranes, suggesting that they are involved in signaling events located in the cell membrane. Many proteins involved in signaling events associate cholesterol rich membrane domains, so called lipid rafts, which serve as platforms for controlled protein-protein interaction. Here, we demonstrate that the myeloid expressed EF-hand proteins can be distinguished into three classes with respect to their membrane association. Grancalcin, a myeloid expressed penta EF-hand protein, is constitutively located in lipid rafts. S100A9 (MRP14) and S100A8 (MRP8) are translocated into detergent resistant lipid structures only after calcium activation of the neutrophils. However, the S100A9/A8 membrane association is cholesterol and sphingolipid independent. On the other hand, the association of S100A12 (EN-RAGE) and S100A6 (calcyclin) with membranes is detergent sensitive. These diverse affinities to lipid structures of the myeloid expressed EF-hand proteins most likely reflect their different functions in neutrophils.     

Zap-70-independent Ca(2+) mobilization and Erk activation in Jurkat T cells in response to T-cell antigen receptor ligation

The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) and increased the intracellular Ca(2+) concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCgamma1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.     

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-κB ligand (RANKL) expression in rheumatoid arthritis

Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4(+) T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4(+) T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4(+) T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4(+) T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4(+) T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.     

A novel role for p21-activated protein kinase 2 in T cell activation

To identify novel components of the TCR signaling pathway, a large-scale retroviral-based functional screen was performed using CD69 expression as a marker for T cell activation. In addition to known regulators, two truncated forms of p21-activated kinase 2 (PAK2), PAK2DeltaL(1-224) and PAK2DeltaS(1-113), both lacking the kinase domain, were isolated in the T cell screen. The PAK2 truncation, PAK2DeltaL, blocked Ag receptor-induced NFAT activation and TCR-mediated calcium flux in Jurkat T cells. However, it had minimal effect on PMA/ionomycin-induced CD69 up-regulation in Jurkat cells, on anti-IgM-mediated CD69 up-regulation in B cells, or on the migratory responses of resting T cells to chemoattractants. We show that PAK2 kinase activity is increased in response to TCR stimulation. Furthermore, a full-length kinase-inactive form of PAK2 blocked both TCR-induced CD69 up-regulation and NFAT activity in Jurkat cells, demonstrating that kinase activity is required for PAK2 function downstream of the TCR. We also generated a GFP-fused PAK2 truncation lacking the Cdc42/Rac interactive binding region domain, GFP-PAK2(83-149). We show that this construct binds directly to the kinase domain of PAK2 and inhibits anti-TCR-stimulated T cell activation. Finally, we demonstrate that, in primary T cells, dominant-negative PAK2 prevented anti-CD3/CD28-induced IL-2 production, and TCR-induced CD40 ligand expression, both key functions of activated T cells. Taken together, these results suggest a novel role for PAK2 as a positive regulator of T cell activation.     

L-selectin serves as an E-selectin ligand on cultured human T lymphoblasts

Previous studies reported that L-selectin (CD62L) on human peripheral blood neutrophils serves as an E-selectin ligand. This study shows that CD62L acquired E-selectin-binding activity following phorbol ester (PMA) treatment of the Jurkat T cell line and anti-CD3/IL-2-driven proliferation of human T lymphocytes in vitro. The recombinant porcine E-selectin/human Ig chimera P11.4 showed neuraminidase-sensitive and calcium-dependent attachment to PMA-stimulated human Jurkat T cells in a flow cytometry assay. The anti-CD62L mAb (DREG 56) blocked this binding interaction by approximately 60% and P11.4 precipitated CD62L from detergent lysates of PMA-activated Jurkat cells. In contrast, P11.4 precipitated minimal amounts of CD62L from detergent lysates of nonactivated human PBL. As reported previously, P-selectin glycoprotein ligand 1 and a distinct 130-kDa glycoprotein were the major species in these precipitates. However, T cell activation on plate-immobilized anti-CD3 and growth in low-dose IL-2 increased the percentage of CD62L molecules with E-selectin-binding activity. After two cycles of activation and culture, approximately 60-70% of the CD62L was precipitated with the P11.4 chimera. These cultured T lymphoblasts rolled avidly on both E-selectin and P-selectin at physiologic levels of linear shear stress. The DREG 56 Ab partially blocked rolling on the E-selectin substrate, whereas no effect was seen on P-selectin. Thus, CD62L on human cultured T lymphoblasts is one of several glycoproteins that interacts directly with E-selectin and contributes to rolling under flow.     

Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance.

The role of transmembrane 4 superfamily (TM4SF) proteins during muscle cell fusion has not been investigated previously. Here we show that the appearance of TM4SF protein, CD9, and the formation of CD9-beta1 integrin complexes were both regulated in coordination with murine C2C12 myoblast cell differentiation. Also, anti-CD9 and anti-CD81 monoclonal antibodies substantially inhibited and delayed conversion of C2C12 cells to elongated myotubes, without affecting muscle-specific protein expression. Studies of the human myoblast-derived RD sarcoma cell line further demonstrated that TM4SF proteins have a role during muscle cell fusion. Ectopic expression of CD9 caused a four- to eightfold increase in RD cell syncytia formation, whereas anti-CD9 and anti-CD81 antibodies markedly delayed RD syncytia formation. Finally, anti-CD9 and anti-CD81 monoclonal antibodies triggered apoptotic degeneration of C2C12 cell myotubes after they were formed. In summary, TM4SF proteins such as CD9 and CD81 appear to promote muscle cell fusion and support myotube maintenance.     

Lck Mediates Signal Transmission from CD59 to the TCR/CD3 Pathway in Jurkat T Cells

The glycosylphosphatidylinositol (GPI)-anchored molecule CD59 has been implicated in the modulation of T cell responses, but the underlying molecular mechanism of CD59 influencing T cell signaling remained unclear. Here we analyzed Jurkat T cells stimulated via anti-CD3ε- or anti-CD59-coated surfaces, using time-resolved single-cell Ca²⁺ imaging as a read-out for stimulation. This analysis revealed a heterogeneous Ca²⁺ response of the cell population in a stimulus-dependent manner. Further analysis of T cell receptor (TCR)/CD3 deficient or overexpressing cells showed that CD59-mediated signaling is strongly dependent on TCR/CD3 surface expression. In protein co-patterning and fluorescence recovery after photobleaching experiments no direct physical interaction was observed between CD59 and CD3 at the plasma membrane upon anti-CD59 stimulation. However, siRNA-mediated protein knock-downs of downstream signaling molecules revealed that the Src family kinase Lck and the adaptor molecule linker of activated T cells (LAT) are essential for both signaling pathways. Furthermore, flow cytometry measurements showed that knock-down of Lck accelerates CD3 re-expression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3ζ completely abolished CD59-triggered Ca²⁺ signaling, while signaling was still functional upon direct TCR/CD3 stimulation. Altogether, we demonstrate that Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells, and propose that CD59 may act via Lck to modulate T cell responses.     

The CD2 ligand LFA-3 activates T cells but depends on the expression and function of the antigen receptor

The T cell Ag receptor (CD3/Ti) and the sheep E receptor (CD2) expressed on the surface of human T cells are both capable of initiating intracellular signals necessary for T cell activation. CD3/Ti interacts with Ag to initiate cellular immune responses. Although the exact function of CD2 is unknown, lymphocyte function-associated Ag 3 (LFA-3), a 55- to 70-kDa receptor expressed on a broad spectrum of hemopoietic and nonhemopoietic cells, has recently been shown to be its natural ligand. We show here that although purified multimeric LFA-3 is not capable of initiating transmembrane signaling events on its own, the combination of LFA-3 and the anti-CD2 mAb CD2.1 induces intracellular calcium increases, phosphatidylinositol second messenger generation and lymphokine secretion in the T cell leukemic line Jurkat. In order to study the signaling requirements of CD2, we compared the ability of CD2 mAb and LFA-3 to initiate activation signals in Jurkat and in three Jurkat-derived mutants. A CD3-CD2+ mutant failed to increase calcium or exhibit phosphatidylinositol hydrolysis to either the combination of agonist CD2 mAb 9-1 and 9.6 or LFA-3 and CD2.1. Reconstitution of the Ag receptor by transfection of the Ti-beta-chain restored the expression of the CD3/Ti complex and the ability to respond to either combination of CD2 ligands. However, no response to CD2 ligands was detected in a CD3+CD2+ mutant selected for signaling defects to CD3/Ti ligands. Complementation of the CD3/Ti signaling defect by cell fusion also restored competency to respond to CD2 agonists. These results demonstrate that LFA-3 under appropriate conditions can activate T cells via the CD2 complex and that this activation requires not only the cell surface expression of the CD3/Ti complex but also a functional Ag receptor pathway.     

Role of protein kinase C in signal attenuation following T cell receptor engagement.

T lymphocyte activation through stimulation of the T cell receptor complex and co-stimulatory receptors is associated with acute tyrosine phosphorylation of intracellular proteins, which in turn mediate downstream signaling events that regulate interleukin-2 expression and cell proliferation. The extent of protein tyrosine phosphorylation is rapidly attenuated after only 1-2 min of stimulation as a means of tightly controlling the initial signaling response. Here we show that this attenuation of tyrosine phosphorylation of Shc, CrkL, and the proto-oncogene Cbl is mimicked by treatment of mouse T lymphocytes or cultured Jurkat cells with phorbol 12-myristate 13-acetate. This effect is blocked by the specific protein kinase C inhibitor GF109203X, but not by PD98059, an inhibitor of MEK1/2 kinase. Activation of protein kinase C by phorbol ester also causes rapid (t(1)/(2) = 2 min) dissociation of both CrkL and p85/phosphoinositide 3-kinase from Cbl concomitant with Cbl tyrosine dephosphorylation. More important, GF109203X treatment of Jurkat cells prior to T cell receptor stimulation by anti-CD3/CD4 antibodies results in an enhanced (2-fold) peak of Cbl phosphorylation compared with that observed in control cells. Furthermore, the rate of attenuation of both Cbl tyrosine phosphorylation and its association with CrkL following stimulation with anti-CD3/CD4 antibodies is much slower in Jurkat cells treated with GF109203X. Taken together, these data provide strong evidence that one or more isoforms of phorbol ester-responsive protein kinase C play a key role in a feedback mechanism that attenuates tyrosine phosphorylation of proteins and reverses formation of signaling complexes in response to T cell receptor activation.     

Heparan sulfate modification of the transmembrane receptor CD47 is necessary for inhibition of T cell receptor signaling by thrombospondin-1

Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent M(r) > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent M(r) 230,000) and CD47 (apparent M(r) > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser(64) and Ser(79). Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser(64).     

MiR-522-3p inhibits proliferation and activation by regulating the expression of SLC31A1 in T cells

We investigated the role of miR-522-3p in thymoma-associated myasthenia gravis (TAMG), and the mechanism of action in T cells. The miR-522-3p expression in normal serum, non-thymoma MG patient serum and TAMG patient serum and tissues was detected by quantitative real-time PCR (qRT-PCR), respectively. We assessed miR-522-3p expression in Jurkat cells and human CD4⁺ T cells after activation by anti-CD3 and anti-CD28 using qRT-PCR. The viability, proliferation, cycle distribution and the levels of CD25, CD69, interleukin-2 (IL-2) and IL-10 in transfected Jurkat cells were detected by Cell counting kit-8, 5-ethynyl-2′-deoxyuridine (EdU), flow cytometry, qRT-PCR, respectively. Targeting relationships of miR-522-3p and SLC31A1 were predicted and validated by bioinformatics analysis and dual-luciferase reporter. The viability, proliferation, cycle distribution and the levels of SLC31A1, CD25, CD69, IL-2 and IL-10 in transfected Jurkat cells were detected by above methods and western blot. The miR-522-3p expression was declined in TAMG and activated T cells. MiR-522-3p inhibitor promoted cell viability, EdU positive cells, cycle progression, and the level of CD25, CD69, IL-2 and IL-10 in Jurkat cells, while the effect of miR-522-3p mimic was the opposite. SLC31A1 was targeted by miR-522-3p, and miR-522-3p inhibited SLC31A1 expression. Overexpressed SLC31A1 reversed the inhibitory effects of miR-522-3p mimic on cell viability, EdU positive cell, cycle progression, and the levels of IL-2 and IL-10 in transfected Jurkat cells. MiR-522-3p expression was down-regulated in TAMG, and miR-522-3p inhibited proliferation and activation by regulating SLC31A1 expression in T cells.