[Analysis of T cell subsets with monoclonal antibodies].
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Antibodies, Monoclonal↗
Biomedical subjects
C Morimoto
Publications and source records attributed to C Morimoto.
Structure and function of Cas-L, a 105-kD Crk-associated substrate-related protein that is involved in beta 1 integrin-mediated signaling in lymphocytes.
Integrin/ligand binding evokes tyrosine phosphorylation of various proteins. We reported previously that a 105 kD protein (pp105) was tyrosine phosphorylated by the engagement of beta 1 integrins in T lymphocytes. We show here that pp105 is a novel p130Cas (Crk-associated substrate)-related protein. Deduced amino acid sequence revealed that pp105 contains conserved motifs with p130Cas, and both pp105 and p130Cas bind to focal adhesion kinase (pp125FAK) and Crk. However, pp105 has a clearly distinct structure from p130Cas, and pp105 is preferentially expressed in lymphocytes, whereas p130Cas is expressed in adherent cells. With these findings, we designate pp105 as Cas-L, lymphocyte-type Cas. Furthermore, we demonstrate that integrin/ligand binding results in the recruitment of Crk, Nck, and SHPTP2 to pp105. These findings further define the roles of pp105/Cas-L and pp125FAK in the integrin-mediated signaling pathways.
Adaptor Proteins, Signal Transducing↗
Requirement of Fas(CD95), CD45, and CD11a/CD18 in monocyte-dependent apoptosis of human T cells.
Our previous studies demonstrated that upon activation, monocytes (Mo) were able to sensitize peripheral blood T (PBT) cells to apoptosis induced by treatment with PMA. However, it is unknown what gene products provide the death signal to the sensitized PBT cells and how activated Mo enable PBT cells to become susceptible to apoptosis. Here, we show that PBT cells, but not Mo, express functional Fas ligand upon treatment with PMA. Moreover, this Mo-dependent T cell apoptosis could be blocked by a Fas-Ig fusion protein, as well as by a nonlytic mAb against Fas molecule. These results strongly suggest involvement of Fas-Fas ligand interaction in the death of PBT cells. Unlike Fas-induced apoptosis, however, Mo-dependent T cell death was completely inhibited by overexpression of the Bcl-2 protein, and PMA alone was sufficient to trigger apoptosis in T cells when Mo were included in culture. Furthermore, anti-CD11a, anti-CD18, or anti-CD45/CD45RA mAbs; could prevent PBT cells from death triggered by PMA plus Mo, suggesting that these Ags participate in the apoptotic process. The participation of CD45RA in the death of PBT cells was further demonstrated by the observation that the J45.01 cell line, a CD45-deficient variant of Jurkat cells, did not undergo apoptosis by this Mo-dependent mechanism. When transfected with cDNA encoding CD45RA, J45.01 cells acquired apoptotic response to PMA stimulation in the presence of Mo to a similar, but lesser, degree than normal Jurkat cells.
Antigens, CD↗
Src kinase plays an essential role in integrin-mediated tyrosine phosphorylation of Crk-associated substrate p130Cas.
A novel signaling molecule p130Cas has been shown to undergo tyrosine phosphorylation in response to integrin-mediated cell adhesion. In this study, we have attempted to identify kinases that mediate Cas phosphorylation in integrin signaling by examining various mutant cell lines that do not express either p125FAK, c-Scr, c-Fyn or c-Abl. We found that deficiency of c-Src but not of other kinases completely abrogated integrin-mediated Cas phosphorylation. Importantly, paxillin phosphorylation was not compromised in each mutant cell line examined. These results suggest that c-Src primarily mediates adhesion-dependent Cas phosphorylation. As for paxillin phosphorylation, there may exist substantial redundancy amongst multiple kinases. Finally, adhesion-induced Cas phosphorylation resulted in its association with c-Crk adapter protein via the Crk-SH2 domain. Thus, Cas plays a role in the transmission of integrin-initiated signals through tyrosine phosphorylation and subsequent binding to c-Crk.
3T3 Cells↗
Serum high molecular weight dipeptidyl peptidase IV (CD26) is similar to a novel antigen DPPT-L released from activated T cells.
This study demonstrates that the 175-kDa form of dipeptidyl peptidase IV (DPPIV) found in normal human serum is identical with a similarly-sized Ag, DPPT-L, found to be rapidly expressed on the surface of activated T cells. As activation progresses, the expression of DPPT-L reaches a peak on day 3, after which expression falls, whereas expression of the 105-kDa CD26/DPPIV detected by the mAb 1F7 increases, as does the ability to bind adenosine deaminase. The loss of DPPT-L from the surface of activated T cells correlates exactly with the appearance of DPPT-L and DPPIV activity in serum-free tissue culture medium. The release of DPPIV was generally greater from CD4+ cells than from CD8+ T cells, and within the CD4+ subset, the CD45RO+ subset was the major source, which correlated with surface expression before culture. We show that the DPPIV released from activated T cells is antigenically, biochemically, and enzymatically similar to DPPIV circulating in the serum and is distinct from the DPPIV activity of 105-kDa CD26. The T cell-released DPPIV is able to function as a costimulating molecule for the response to the recall Ag, tetanus toxoid, at levels similar to those at which recombinant soluble CD26 and serum DPPIV exhibit costimulatory function, suggesting that the released DPPIV may serve an important immunoregulatory function in vivo, both locally and within the systemic circulation.
Animals↗
Stimulation of tyrosine phosphorylation after ligation of beta7 and beta1 integrins on human B cells.
B lymphocytes express several members of the integrin family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. In addition to beta1 integrins, predominantly alpha4 beta1, mature B cells also express alpha4 beta7, which is a receptor for vascular cell adhesion molecule-1 and fibronectin, and is also involved in the homing of B cells to mucosal sites through binding to a third ligand, mucosal address in cell adhesion molecule-1. Here we describe that crosslinking of alpha4 beta7 integrins on B cell lines and normal tonsillar B cells, induces tyrosine phosphorylation of multiple substrates of 105-130 kD, indicating that beta7 integrin plays a role as signaling molecule in B cells. This pattern of phosphorylated proteins was very similar to that induced following ligation of alpha4 beta1. Interestingly, ligation of alpha5 beta1 or alpha6 beta1 also stimulated the 105-125 kD group of phosphorylated proteins, whereas ligation of beta2 integrins did not. The focal adhesion tyrosine kinase p125FAK was identified as one of these substrates. Beta1 or beta7 mediated tyrosine phosphorylations were markedly decreased when the microfilament assembly was inhibited by cytochalasin B. These results suggest that intracellular signals initiated by different integrins in B cells may converge, to similar cytoskeleton-dependent tyrosine phosphorylated proteins.
Antibodies, Monoclonal↗
Characterization of adenosine deaminase binding to human CD26 on T cells and its biologic role in immune response.
CD26, a T cell activation Ag, also known as dipeptidyl peptidase IV, is directly associated with adenosine deaminase (ADA) on the surface of T cells and T cell lines. In the present study, we examined both the binding of ADA and CD26 and the functional consequences of this interaction. We found that ADA was associated with CD26 on T cell lines lacking either ADA or dipeptidyl peptidase IV enzymatic activity, indicating that the association between dipeptidyl peptidase IV and ADA did not require enzymatic activity. Moreover, using immunoelectron microscopy, we demonstrated that CD26 and ADA co-localized on the cell surface, but not inside cells, suggesting that CD26 did not transport ADA to the surface. In keeping with this observation, we showed that human CD26-transfected murine pre-B cell lines lacking human ADA acquired ADA from an extracellular source. More importantly, adenosine in the absence of cell surface ADA inhibited T cell proliferation and IL-2 production induced by various stimuli. On the other hand, cells expressing ADA and CD26 on the surface were much more resistant to the inhibitory effect of adenosine. These data suggest that ADA on the cell surface is involved in an important immunoregulatory mechanism by which released ADA binds to cell surface CD26, and this complex is capable of reducing the local concentration of adenosine.
Adenosine↗
Influence of CD26 and integrins on the antigen sensitivity of human memory T cells.
The antigen sensitivity of class II MHC restricted human CD4 T-cell clones is demonstrated to increase gradually with time after restimulation. This is manifested in a requirement of less antigen in culture, as well as decreased numbers of peptide-MHC complexes per APC for T-cell activation, and in an increased resistance to inhibition by class II MHC blockade. The increase in antigen sensitivity is accompanied by increased cell-surface expression of CD26, LFA-1, and VLA-1, whereas the expression of TCR and a series of other cell-surface molecules remains unchanged. Using appropriate monoclonal antibodies, we have shown that CD26 and LFA-1 contribute directly to the increased antigen sensitivity of "late-stage" T-cell clones. The late-memory T-cell phenotype established in this study is shown to occur also among T cells activated in vivo. We suggest that increasing the antigen sensitivity via antigen-nonspecific molecules is a physiologic mechanism for maintaining T-cell memory in face of decreasing antigen concentration, and for ensuring preferential activation of memory T cells upon repeated encounter with antigen.
Antigen Presentation↗
Beta-Adrenergic receptors in rat fat cells and their relationship with lipolysis.
Norepinephrine stimulated lipolysis in rat fat cells while (-)-alprenolol completely inhibited this lipolysis. (-)-Alprenolol competed for (-)-[3H] dihydroalprenolol (DHA) binding sites on rat fat cells. The specific (-)-[3H]DHA binding sites identified by competition with (-)-alprenolol were found to be transferred to the solubilized supernatant during preparation of endogenous lipid droplets from the fat cells. Although the lipid droplets did not exhibit specific (-)-[3H]DHA binding, norepinephrine induced lipolysis in a cell-free system consisting of the lipid droplets and hormone-sensitive lipase (HSL). Norepinephrine-induced lipolysis in the cell-free system was inhibited by propranolol and (-)-alprenolol, but not by phenoxybenzamine. The lipolytic action of norepinephrine and the anti-lipolytic actions of propranolol and (-)-alprenolol disappeared after sonication of the lipid droplets in the cell-free system. These results suggest that the adrenergic receptor concerned with lipolysis in fat cells may not be a specific (-)-[3H]DHA binding site, but may be closely related to the lipid droplets.
Adipocytes↗
CD27/CD70 interaction directly induces natural killer cell killing activity.
The CD27 molecule is expressed on a portion of natural killer (NK) cells as well as T and B cells. To provide the functional capacity of CD27 molecule on NK cells, we here highly purified CD3- CD56+ NK cells by flow cytometry (purity > 98%), and investigated their NK cell activity via CD27/CD70 interaction using a CD70-transfectant by a 4 h 51Cr-release assay. The enhancement of NK activity by purified NK cells in the presence of interleukin-2 (IL-2) or interleukin-12 (IL-12) against CD70/Nalm-6 was not recognized as compared to against mock/Nalm-6. However, after a coculture with the fixed CD70/300-19, the purified NK cells increased the NK cell activity against K562, the value being 10 to 20% higher than coculture with the mock/300-19 in the presence of IL-2 or IL-12. The enhancement of NK activity was blocked by the addition of anti-CD70 monoclonal antibody (mAb). In addition, conjugation of NK cells to the target was increased by coculture with the CD70/300-19 without increased expression of adhesion molecules. In the parallel experiment, there was no increase in the killing capacity of NK cells. These results strongly show that CD27/CD70 interaction directly enhances NK activity in the presence of IL-2 or IL-12 by increasing the effector and target conjugate formation, indicating that CD27/CD70 interaction plays an important role in the cytotoxic function of NK cells.
Antigens, CD↗
Selective immunomodulation: utilization of CD29/VLA molecules.
Accumulating evidence suggests that the VLA/CD29 molecule plays an important role in T-cell costimulation, and CD4+CD29/VLA+ memory T cells play a key role in induction of CD8 killer effector T cells which are considered to be a major population involved in graft rejection. To target limited elements of the T-lymphocyte population, we have described the preparation of a bispecific antibody-toxin conjugate designed to target CD4+CD29+ memory T cells. We also showed that the solid-phase crosslinking of VLA-4 by the antibody against this molecule or by its ligand, the CS-1 region of fibronectin, stimulates tyrosine phosphorylation of 140, 120-105, 80-70, 60-55, 50 and 45 kilodalton proteins. In addition, we identified the pp140 protein as PLC gamma, pp120 protein as pp125FAK, pp70 and pp50 proteins as paxillin, and pp60-55 proteins as pp59fyn and pp56lck, and pp45 as MAP kinase, respectively. Moreover, we demonstrated that pp125FAK is directly associated with paxillin. The paxillin binding domain of pp125FAK is homologous to the paxillin binding domain of vinculin. Mutations in the conserved amino acid residues between pp125FAK and vinculin result in the loss of paxillin-binding activity. Because VLA/CD29 is preferentially expressed on CD4 memory T cells, the above described system will be used to develop a novel drug design for providing selective immunosuppression useful for organ transplantation.
Adjuvants, Immunologic↗
Increased population of high fluorescence 1F7 (CD26) antigen on T cells in synovial fluid of patients with rheumatoid arthritis.
OBJECTIVE: To investigate the activation of T cells in peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA). METHODS: The expression of CD26 (Ta1 and 1F7) antigen on T cells was analyzed in 7 women with RA and 7 healthy control subjects by immunofluorescence. RESULTS: The percentage of CD3+ CD26+ cells was significantly higher in PB of patients with RA compared with healthy subjects. The IF7+ cell population was divided into high (1F7+high cells) and low fluorescence populations (1F7+low cells), based on 1F7 antigen density. The percentage of 1F7+high cells in SF of RA was markedly increased compared with PB of patients and healthy subjects. However, RA SF contained lower percentages of whole 1F7+ cells compared with PB. CONCLUSION: Our results indicate that SF of patients with RA contains activated T cells, and suggest that T cells with high levels of CD26 antigen may preferentially migrate into the rheumatoid synovium to induce inflammation and tissue destruction.
Adult↗
Role of CD26 for CD4 memory T cell function and activation.
CD26 is a 110 kDa T cell activation antigen and has been shown to have DPPIV enzyme activity which cleaves amino-terminal dipeptides with either L-proline or L-alanine at the penultimate position. Recent studies showed that CD26 plays an integral role in T cell activation. A partial explanation of the mechanism of CD26 mediated T cell signaling appears to be its association with CD45 tyrosine phosphatase, which may be importance in T cell activation and signal transduction. In addition, we showed that CD26 is a receptor for adenosine deaminase (ADA). Moreover, ADA on the cell surface is involved in an important immunoregulatory mechanism by which released ADA binds to cell surface CD26 and this complex is capable of reducing the local concentration of adenosine. Thus, CD26 is a multifunctional molecule controlling many key aspects of lymphocyte function.
Base Sequence↗
Beta 1 integrin-mediated signaling in human T cells.
Beta 1 integrin/ligand binding evokes tyrosine phosphorylation of various proteins. In our previous studies, we have shown that the engagement of beta integrin molecules induced tyrosine phosphorylation of 140, 120, 110-105, 80-70, 60-55 and 45 KD proteins in peripheral T lymphocytes. Several tyrosine-phosphorylated proteins have been identified such as PLC gamma (pp140), pp125FAK(pp120), Paxillin(pp70), p59fyn/p56lck(pp60-55) and MAPkinase (pp45). However, a 105 KD tyrosine-phosphorylated protein(pp105) has not been identified. Recently, we demonstrated that pp105 is identified as a novel p130Cas related protein. pp105 is preferentially expressed in lymphoid cells, while p130Cas is expressed in adherent cells. With these findings, we designated pp105 as Cas-L, "lymphocyte type Cas." Cas-L is directly associated with FAk-C-terminal region in an integrin stimulation-dependent manner, and tyrosine phosphorylated Cas-L binds to the SH2 domains of Crk, Nck and SHPTP2. These findings reveal a novel architecture of beta 1 integrin-mediated protein tyrosine phosphorylation and further suggest the involvement of Crk, Nck and SHPTP2 in the downstream of beta 1 integrin-mediated signaling pathway.
Humans↗
CD27-CD70 interactions regulate B-cell activation by T cells.
CD27, a member of the tumor necrosis factor (TNF) receptor family, binds to its ligand CD70, a member of the TNF family, and subsequently induces T-cell costimulation and B-cell activation. CD27 is expressed on resting T and B cells, whereas CD70 is expressed on activated T and B cells. Utilizing transfected murine pre-B-cell lines expressing human CD27 or CD70, we have examined the effect of such transfectant cells on human B-cell IgG production and B-cell proliferation. We show that the addition of CD27-transfected cells to a T-cell-dependent, pokeweed mitogen-driven B-cell IgG synthesis system resulted in marked inhibition of IgG production, whereas the addition of CD70-transfected cells enhanced IgG production. The inhibition and enhancement of pokeweed mitogen-driven IgG production by CD27 and CD70 transfectants were abrogated by pretreatment with anti-CD27 and anti-CD70 monoclonal antibodies, respectively. In contrast, little or no inhibition of IgG production and B-cell proliferation was noted with CD27-transfected cells or either anti-CD27 or CD70 monoclonal antibody in a T-cell-independent Staphylococcus aureus/interleukin 2-driven B-cell activation system. In this same system CD70-transfected cells enhanced B-cell IgG production and B-cell proliferation, and this enhancement could be gradually abrogated by addition of increasing numbers of CD27-transfected cells. These results clearly demonstrate that interactions among subsets of T cells expressing CD27 and CD70 play a key role in regulating B-cell activation and immunoglobulin synthesis.
Antigens, CD↗
Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK.
Focal adhesion kinase (pp125FAK) is localized to focal adhesions and tyrosine phosphorylated by the engagement of beta 1 integrins. However, it is unclear how pp125FAK is linked to integrin molecules. We demonstrate that pp125FAK is directly associated with paxillin, a 68-kD cytoskeleton protein. The COOH-terminal domain of pp125FAK spanning FAK residues 919-1042 is sufficient for paxillin binding and has vinculin-homologous amino acids, which are essential for paxillin binding. Microinjection and subsequent immunohistochemical analysis reveal that glutathione S-transferase-FAK fusion proteins, which bind to paxillin, localize to focal adhesions, whereas fusion proteins with no paxillin-binding activity do not localize to focal adhesions. These findings strongly suggest that pp125FAK is localized to focal adhesions by the direct association with paxillin.
Amino Acid Sequence↗
Role of the VLA-4 molecule in T cell costimulation. Identification of the tyrosine phosphorylation pattern induced by the ligation of VLA-4.
The very late activated Ag (VLA) molecules not only mediate T cell adhesions, but also provide costimulation in a TCR/CD3-dependent manner. However, little is known about the signals mediated by the ligation of VLA molecules. Previous work from our laboratory identified a 105-kDa protein that is predominantly phosphorylated on tyrosine residue upon engagement of VLA-4 in a human T lymphoblastic cell line, H9, and in peripheral T cells. In the present study, we have shown that the A and B epitope of VLA-4 plays a key role in VLA-4-mediated T cell costimulation. Moreover, we have demonstrated that the solid phase cross-linking of VLA-4 using Ab (against A and B) or the CS-1 region of fibronectin, stimulated tyrosine phosphorylation of 140-, 120-, 80- to 70-, 60- to 55-, 50-, and 45-kDa proteins in addition to the 105-kDa protein. In contrast, Ab ligation of the C epitope of VLA-4 mainly induced tyrosine phosphorylation of pp105, weakly induced other protein tyrosine phosphorylation, and additionally induced only minimal T cell costimulation. Using immunoblotting, we have identified some of the tyrosine-phosphorylated proteins to be phospholipase C gamma (pp140), pp125 focal adhesion kinase (pp120), paxillin (pp70 and pp50), p59fyn/p56lck (pp60-55), and mitogen-activated protein kinase (pp45). Since solid phase cross-linking of VLA-4 by B2 epitope-specific Ab induced T cell costimulation most strongly via the CD3 pathway, our results suggested that the above tyrosine-phosphorylated proteins may play an important role in VLA-4-mediated T cell costimulatory signaling events.
Antibodies, Monoclonal↗
Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130Cas, a Src homology 3-containing molecule having multiple Src homology 2-binding motifs.
p130Cas (Cas) has been recently identified as a 130-kDa protein that is highly phosphorylated on tyrosine residues and is stably associated with p47v-crk (v-Crk) and p60v-src (v-Src) oncogene products in cells transformed by the respective genes. Cas is a novel signaling molecule having a single Src homology (SH) 3 domain and a cluster of multiple SH2-binding motifs. While the tight association of Cas with v-Crk and v-Src is strongly suggestive of a significant role in regulating cellular transformation, the function of Cas in normal untransformed cells is totally unknown. We report here that cell adhesion to fibronectin rapidly promotes tyrosine phosphorylation of Cas in human and rat fibroblast cell lines. The response was equally induced by cell adhesion to plates coated with vitronectin, laminin, and collagen but not by cell attachment to nonspecific substrate poly-L-lysine. The kinetic profile of Cas phosphorylation was almost identical with that of tyrosine phosphorylation of focal adhesion kinase pp125FAK (Fak), which is well known to be activated subsequent to integrin-mediated cell adhesion. Adhesion-dependent Cas phosphorylation was completely inhibited by treating cells with cytochalasin D, an agent that disrupts polymerization of actin stress fibers. These results suggest that tyrosine phosphorylation of Cas is stimulated by normal cell adhesion in close association with Fak phosphorylation and the formation of actin stress fibers. In v-Src- or v-Crk-transformed cells, however, the tyrosine phosphorylation of Cas is markedly increased in an adhesion-independent manner that is insensitive to treatment with cytochalasin D. Thus, Cas plays a role in signaling pathways mediated by cell adhesion as well as by transformation. We propose that Cas may amplify and propagate integrin-mediated signals by interacting with SH2-containing molecule(s).
Animals↗