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CD147/Basigin/EMMPRIN is overexpressed in several cancerous tissues and it has been shown to induce matrix metalloproteinases (MMPs) whose expression is associated with cancer metastasis. Thus, targeting CD147 with monoclonal antibodies (mAbs) potentially has therapeutic applications in cancer immunotherapy. Here, we report the use of anti-CD147 mAbs targeting domain 1 of CD147, namely M6-1D4 (IgM), M6-1F3 (IgM), M6-2F9 (IgM) and M6-1E9 (IgG2a), against several human cancer cell lines. Strikingly, IgM but not IgG mAbs against CD147, especially clone M6-1D4, induced acute cellular swelling, and this phenomenon appeared to be specifically found with hepatocellular carcinoma (HCC) cells. Furthermore, molecular investigation upon treating HepG2 cells with M6-1D4 showed unfolded protein response (UPR) activation, autophagosome accumulation, and cell cycle arrest, but without classic apoptosis related features. More interestingly, prolonged M6-1D4 treatment (24 h) resulted in irreversible oncosis leading to necroptosis. Furthermore, treatment with a mixed lineage kinase domain-like psuedokinase (MLKL) inhibitor and partial knockout of MLKL resulted in reduced sensitivity to necroptosis in M6-1D4-treated HepG2 cells. Surprisingly however, the observed necroptotic signaling axis appeared to be non-canonical as it was independent of receptor-interacting serine/threonine-protein kinase (RIPK) phosphorylation. In addition, no cytotoxic effect on human dermal fibroblast (HDF) was observed after incubation with M6-1D4. Taken together, this study provides clues to target CD147 in HCC using mAbs, as well as sheds new light on a novel strategy to kill cancerous cells by the induction of necroptosis.

Human CD63 has been reported to play a role either as an inhibitor or as a co-stimulator of T- cell responses, although the mechanism of this is unclear. In this study, an anti-human CD63 monoclonal antibody (mAb) COS3A was used to monitor the role of CD63 in T-cell activation. MAb COS3A could inhibit CD3-mediated T-cell proliferation and CD25 expression in peripheral blood mononuclear cells (PBMCs), used as a study model, but the suppressive effect was not observed when purified T-cells were used instead of PBMCs. The inhibitory phenomenon was associated with downregulation of IL-2 and IFN-γ by T-cells, but upregulation of IL-10 by monocytes. Neutralizing IL-10 with anti-IL-10 mAb improved the T-cell response, indicating the role of IL-10 in T-cell suppression. In this study, monocytes were demonstrated to play a role in impeding T-cell activation by the anti-CD63 mAb COS3A. This is the first evidence that anti-CD63 mAb induces IL-10 secretion by monocytes, which later play a role in T-cell hypo-responsiveness.

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.

α0-Thalassemia is an inherited hematological disorder caused by the deletion of α-globin genes. The Southeast Asian deletion (--SEA) is the most common type of α0-thalassemia observed in Southeast Asian countries. Regarding WHO health policy, an effective α0-thalassemia screening strategy is needed to control new severe α-thalassemia cases. In this study, a monoclonal antibody panel was used to develop immunochromatographic (IC) strip tests for detecting the Hb Bart's and ζ-globin chain. Among 195 samples, all α0-thalassemia traits (78 α0-thalassemia (--SEA) and 4 α0-thalassemia (--THAI)) had low MCV or MCH values. The sensitivity, specificity, PPV and NPV of the IC strip tests for ζ-globin and Hb Bart's for screening α0-thalassemia (--SEA) within the low MCV or MCH samples were 100%, 65.2%, 90.7%, 100% and 96.2%, 47.8%, 86.6%, 78.6%, respectively. All 4 α0-thalassemia (--THAI) traits were negative for ζ-globin chains but positive for Hb Bart's using the IC strip tests. These results led to a α0-thalassemia screening being proposed in which blood samples are first evaluated by MCV, MCH and Hb typing. Samples with high MCV and MCH values are excluded for the presence of the α0-thalassemia gene. Samples with low MCV or MCH values are assayed using the developed IC strip tests, where only samples testing positive are further assayed for α0-thalassemia by PCR. Patients with Hb H, EA Bart's or EF Bart's diseases do not need to use this IC strip assay. Thus, in this study, a simple and cost effective α0-thalassemia point of care test was developed.

Background Couples who carry α-thalassemia-1 deletion are at 25% risk of having a fetus with hemoglobin Bart’s hydrops fetalis. Southeast Asian deletion (−-(SEA)) is the most common type of α-thalassemia 1 among Southeast Asian populations. Thus, identification of the (−-(SEA)) α-thalassemia 1 carrier is necessary for controlling severe α-thalassemia in Southeast Asian countries. Results Using our generated anti ζ-globin chain monoclonal antibodies (mAbs) clones PL2 and PL3, a simple immunostick test for detecting ζ-globin chain presence in whole blood lysates was developed. The procedure of the developed immunostick test was as follows. The immunostick paddles were coated with 50 μg/mL of mAb PL2 as capture mAb, or other control antibodies. The coated immunostick was dipped into cocktail containing tested hemolysate at dilution of 1:500, 0.25 μg/mL biotin-labeled mAb PL3 and horseradish peroxidase-conjugated streptavidin at dilution of 1:1000. The immunostick was then dipped in precipitating substrate and the presence of ζ-globin chain in the tested sample was observed by the naked eye. Upon validation of the developed immunostick test with various types of thalassemia and normal subjects, 100% sensitivity and 82% specificity for detection of the (−-(SEA)) α-thalassemia-1 carriers were achieved. The mAb pre-coated immunostick can be stored at room temperature for at least 20 weeks. Conclusion In this study, a novel simple immunostick test for the screening of (−-(SEA)) α-thalassemia 1 carriers was presented. The developed immunostick test, within a single test, contains both positive and negative internal procedural controls.

CD99 has been reported to be involved in T cell regulation. CD99 ligand involvement in the regulation of T cell activation has been postulated. In this study, recombinant CD99 proteins were produced and used as a tool for determining the role of CD99 and its ligand interaction. Recombinant CD99 proteins induced the upregulation of IL-6 and TNF-α expression, but not IFN-γ, in anti-CD3 monoclonal antibody activated T cells. The cytokine alteration was not observed in unstimulated T cells indicating the cytokine upregulation required the signal from T cell activation. The upregulation of IL-6 and TNF-α was, in addition, observed in CD3- mononuclear cell population including monocytes and NK cells. The recombinant CD99 proteins, however, did not affect either CD25, CD69 or MHC class II expression or T cell proliferation, upon T cell activation. The CD99 ligands were demonstrated to be expressed on monocytes, NK cells and dendritic cells, but not on B and T cells. Our results indicated the presence of CD99 ligands on leukocyte surface. Interaction between CD99 and its ligands involves the regulation of cytokine production.

T cells play a crucial role in orchestrating body immune responses. T cell hyperfunction, however, leads to inflammation and induction of autoimmune diseases. Understanding of T cell regulation mechanisms and successful modulation of T cell responses is beneficial in treatment of disease associated to T cell hyperresponsiveness. Our previous study indicated that monoclonal antibody (mAb) P-3E10, a mAb to Na, K ATPase β3 subunit, inhibited anti-CD3-induced PBMC proliferation. In the current study, we further investigated the mechanism of mAb P-3E10 in the induction of T cell hypofunction. We demonstrated that mAb P-3E10 decreased T cell proliferation and Th1, Th2 and Th17 cytokine production. Monocytes were the cells playing a key role in mediation of mAb P-3E10 induced T cell hypofunction. The inhibition of T cell activation by mAb P-3E10 required cell contact between monocytes and T cells. The mAb P-3E10 induced the down-expression level of MHC class II and CD86 and increased IL-6, IL-10 and TNF-α production of monocytes. We concluded that ligation of the Na, K ATPase β3 subunit on monocytes by mAb P-3E10 arbitrated T cell hypofunction. This mAb might be a promising novel immunotherapeutic antibody for the treatment of hyperresponsive T cell associated diseases.

A silicon nitride Ion Sensitive Field Effect Transistor (ISFET) based immunosensor was developed as a low-cost and label-free electrical detection for the detection of antigen 85 complex B (Ag85B). The sensing membrane of the ISFET was modified with 3-aminopropyltriethoxysilane (APTES) followed by glutaraldehyde (GA), yielding an aldehyde-terminated surface. This group is available for immobilization of a monoclonal antibody against a recombinant Ag85B protein (anti-Ag85B antibody). The optimal concentration for anti-Ag85B antibody immobilization onto the modified ISFET was 100 μg ml-1. This optimal condition provided the maximal binding capability and minimal non-specific background signal. The binding event between the recombinant Ag85B antigen and anti-Ag85B antibody on the ISFET surface is presented by monitoring the gate potential change at a constant drain current. The dose response for the recombinant Ag85B protein showed a linear response between 0.12 and 1 μg ml-1 without significant interference from other recombinant proteins. The analytical imprecision (CV%) and accuracy of this Ag85B protein biosensor were 9.73-10.99% and 95.29%, respectively. In addition, an irrelevant antibody and other recombinant proteins were employed as a negative control to demonstrate the non-specific interaction of the antigen and antibody. The success of this immunosensor system for Ag85B protein detection facilitates the construction of a promising device which can shorten the turnaround time for the diagnosis of tuberculosis compared to a standard culture method. Furthermore, this device could also be applied for real-time growth monitoring of Mycobacterium tuberculosis in a mycobacterial culture system.

Background Human trefoil factor (TFF) peptides consist of three members: TFF1, TFF2 and TFF3. TFF3 is the most abundant TFF peptide in saliva. TFF3 homodimer was suggested to be involved in apoptosis inhibition and malignancy. Determination of TFF3 homodimer expression profiles in saliva may lead to new information about oral biology and diseases. The objective of this study was to generate monoclonal antibodies (mAbs) against TFF3 and apply the produced mAbs for the establishment of ELISA for quantification of dimeric TFF3 in saliva. Results With our modified hybridoma technique, three hybridoma clones producing anti-TFF3 mAbs having IgG isotype were generated. The mAbs were specific for TFF3 with no cross-reactivity to other TFFs. Using the generated mAbs, a modified-sandwich ELISA with high sensitivity for the quantification of dimeric TFF3 in saliva was developed. Using this ELISA, the amount of dimeric TFF3 in saliva could be measured. Conclusions A modified-sandwich ELISA for the quantification of TFF3 dimeric form was established. The established ELISA will be a valuable tool for facilitating the investigation of the physiological roles and the diagnostic values of TFF3 in oral diseases. The concept of this modified-sandwich ELISA may be applied for the determination of other homodimeric peptides of interest.

Couples in which both partners carry the α-thalassemia-1 trait have a 25% risk of hemoglobin Bart's hydrops fetalis in each pregnancy. Identification of α-thalassemia-1 trait is, therefore, necessary in order to control this severe form of α-thalassemia. We have generated monoclonal antibodies specific to the ζ-globin chain without cross reaction with other globin chains. A simple and sensitive ELISA was developed by using poly-l-lysine to increase the protein binding to the ELISA plate. The developed poly-l-lysine pre-coated ELISA has a very high sensitivity (100%) and specificity (97%) for detection of carriers of α-thalassemia-1 with Southeast Asian-type deletion.

In this study, we describe the application of a molecular biology technique for the production of mouse polyclonal antibodies (pAbs) specific to human cell surface molecules. Production of the pAb specific to the human CD99 surface molecule was used as the study model. The retroviral expression system was employed to generate human CD99 expressing mouse myeloma cells. After cell sorting and single cell cloning, a myeloma clone which stably expressed high levels of human CD99 on its surface was established. The human CD99 expressing mouse myeloma cells were then used as the immunogen for immunization of BALB/c mice. As endogenous proteins of mouse myeloma cells possess self-non-immunogenicity for BALB/c mice, after immunization, only the expressed human CD99 molecules induce antibody response. After three immunizations, high titers of mouse anti-CD99 pAbs were successfully produced. The produced pAb specifically reacted to both recombinant human CD99 and native CD99 molecules expressed on human blood cells. The established technology is simple and valuable for the production of pAbs specific to human CD99 membrane proteins which can be used for characterization of the CD99 molecule.

Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A2, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart's hydrops fetalis in which no α-globin chain is produced.

Background: Monoclonal antibodies (mAbs) have become essential tools in life science research and in medicine, because of their extreme specificity. Several mAbs against leukocyte surface molecules have been generated in our laboratory. From these, mAb COS3A was selected for biochemical and functional analysis. Objective: To identify and characterize the molecule recognized by mAb COS3A. Methods: Cellular distribution was analyzed by immunofluorescence staining and flow cytometry. Biochemical characterization of the molecular target of COS3A was approached by immunoprecipitation, Western blotting and amino acid sequencing using LC-MS. N-glycosidase F treatment of COS3A-precipitated protein and culture of U937 cells in the presence of tunicamycin before cell lysate preparation were used to study the glycosylation state of proteins. Phagocytosis was examined by flow cytometry. Results: MAb COS3A bound specifically to a molecule expressed on the surface of various human hematopoietic cells and cell lines but not on red blood cells. The antigen had a molecular weight of 30-70 kDa, which was reduced to 25 kDa by elimination of N-linked glycan. LC-MS data and immunoprecipitation indicated that mAb COS3A bound specifically to CD63 molecule. Remarkably, functional analysis demonstrated that mAb COS3A dramatically reduced granulocyte phagocytosis. Conclusions: The mAb COS3A recognized the CD63 molecule and strongly diminished phagocytosis of bacteria by granulocytes, suggesting that CD63 may play a crucial role in the initial step of phagocytosis. MAb COS3A is therefore suitable for both biochemical and functional studies of CD63, and may be used for further study of the mechanism of phagocytosis and also in therapeutic approaches. DOI 10.12932/AP0735

Beta3 subunit is described as one of the Na, K ATPase subunits. Recently, we generated a monoclonal antibody (mAb), termed P-3E10. This mAb was shown to react with the Na, K ATPase beta3 subunit or CD298. By immunofluorescence analysis using mAb P-3E10, it was found that all peripheral blood leukocytes express Na, K ATPase beta3. The presence of beta3 subunit on leukocytes is not in a quantitative polymorphic manner. Upon phytohemagglutinin or phorbol myristate acetate activation, the expression level of the Na, K ATPase beta3 subunit on activated peripheral blood mononuclear cells was not altered in comparison with those of unstimulated cells. Red blood cells (RBCs) of healthy donors showed negative reactivity with mAb P-3E10. However, more than 80% of thalassemic RBCs showed positive reactivity. By immunoprecipitation, moreover, a protein band of 55-65 kDa was precipitated from normal RBC membrane using mAb P-3E10. These results evidenced that the beta3 subunit of Na, K ATPase is expressed on RBC membrane but the epitope recognized by mAb P-3E10 is hidden in normal RBCs. Furthermore, we showed the association of beta3 subunit and alpha subunit of Na, K ATPase. This information is important for further understanding of the functional roles of this molecule.

A CD4 monoclonal antibody which reacts with CD4 + lymphocytes but does not react significantly with monocytes was generated and used to develop a simple method for CD4 count. In a comparison with standard flow cytometry, high correlation was obtained. The developed method can be an alternative to flow cytometry for resource-limited countries.

This study was carried out to understand the effect of the recombinant multivalent extracellular matrix metalloproteinase inducer (EMMPRIN) extracellular domain, designated as rmEMMPRINex, on the apoptotic cell death of human leukemia U937 cells. Expression of monocarboxylate transporter 1 (MCT1) and caspase-9 in U937 treated with rmEMMPRINex was investigated in this study. Levels of membrane MCT1 and intracellular procaspase-9 were decreased in rmEMMPRINex-treated cells in comparison to controls. However, the expression of activated caspase-9 was undetectable. rmEMMPRINex also induced DNA fragmentation and apoptosis in U937 cells. Taken together, we concluded that interaction of rmEMMPRINex with U937 cells leads to inhibition of MCT1 membrane expression, intracellular activation of procaspase-9, followed by DNA fragmentation and apoptosis. This may contribute to the conceptual development of novel cancer drugs in the future.

Background: Several molecules are known to be involved in T-cell activation via the TCR/CD3 complex and while the mechanisms of late T cell signaling have been well characterized, the very early events are still not fully understood. Objective: The aim of this study was to identify yet unknown molecules associated with the TCR/CD3 complex . Results: To identify new molecules associated with the TCR/CD3 complex, a monoclonal antibody termed MT3 was produced by immunoprecipitated beads immunization. C olocalization of the MT3 mAb recognizing molecules with the TCR/CD3 complexes was verified by confocal microscopic analysis. The surface antigen recognized by MT3 antibody was expressed on a subpopulation of CD3 + T cells, and on both CD4 + and CD8 + lymphocytes. The antigen was also expressed on naive CD4 + T cells and on a subset of memory CD4 + T cells. In contrast, in the CD8 population, the majority of MT3 + cells were found in the naive population. The MT3 mAb recognizing molecules were also expressed on red blood cells but only in particular subjects. Similar to peripheral blood leukocytes, MT3 mAb recognizing molecules are exclusively expressed on T cell lines. Conclusions: Based on the cellular distribution patterns and confocal microscopic analysis, the MT3 mAb recognizing molecule that we investigated is proposed to be a TCR/CD3 associated molecule and might be involved in the antigen recognition of T cells.

Objective To examine CD99 expression and its functional role in ICAM-1 induction in human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGECs) by activating cells with anti-CD99 monoclonal antibody, MT99/3. Background Engagement of CD99 with agonistic antibodies has been shown to regulate immune responses, cell adhesion and migration, and cell death in several studies. Particularly, this engagement results in transendothelial migration of leukocytes mediated by intercellular adhesion molecule-1 (ICAM-1) induction in endothelial cells. Methods Total mRNA and protein were isolated from HGFs and HGECs for analyses of CD99 and ICAM-1 expression. Surface expression of CD99 and ICAM-1 was analysed by flow cytometry, and the detection of soluble ICAM-1 was assayed by immunoprecipitation and ELISA. Results CD99 surface expression was constitutive on HGFs to a greater extent than that on HGECs. CD99 ligation with MT99/3 induced ICAM-1 mRNA expression in HGFs, but not in HGECs. Interestingly, CD99 ligation led to an increased level of soluble ICAM-1 detected in culture supernatant, whereas interleukin-1β (IL-1β) treatment induced expression of membrane-bound ICAM-1. Furthermore, ICAM-1 induction by CD99 engagement was demonstrated to involve the activation of the p50 subunit of nuclear factor-kappaB (NF-κB), extracellular signal-regulated kinase, and p46 c-Jun N-terminal kinase that differed from that by IL-1β treatment. Conclusion Our study has shown the involvement of CD99 ligation in the up-regulation of ICAM-1 expression and its secretion in gingival fibroblasts, which may be essential for better understanding of the pathogenesis of periodontal disease.

Monoclonal antibodies (MAb) specific to the protein of interest can be achieved following the classical hybridoma technique. However, obtaining a desired MAb is not always straightforward. The intrinsic quality of immunogen is one of the critical success factors. In this study, three sources of immunogens were compared for CD4 MAb production. CD4 proteins were isolated by immunoprecipitation and the CD4 immunoprecipitated (CD4-IP) beads were used as an immunogen. Recombinant CD4 protein-biotin carboxyl carrier protein (BCCP) fusion proteins (CD4-BCCP) were produced in Escherichia coli, isolated by streptavidin-coated beads, and the CD4-BCCP beads were used as an immunogen. CD4 expressing COS (CD4-COS) cells were generated, enriched by immunosorting, and used as an immunogen. After three immunizations, anti-CD4 antibodies could be observed in all immunized mice. The CD4 MAbs that were generated from CD4-IP bead and CD4-COS cell immunizations reacted with both CD4 expressed on transfected COS cells and lymphocytes. These MAbs could be used for immunoprecipitation of CD4 molecules from lymphocyte lysate and for enumerating CD4+ lymphocytes by flow cytometry. In contrast, the MAb generated from CD4-BCCP bead immunization reacted only with recombinant CD4-BCCP proteins but not with native CD4 expressed on CD4+ lymphocytes. Our results indicate that the proposed methods can facilitate the production of desired MAbs where the purified protein antigens are not available or difficult to prepare, but either the encoding cDNA or specific MAb is available.

Objective To quantitatively measure the increased expression of Akt2 and its phosphorylated form (p-Akt) in oral cancer cell lines and investigate the post-translational mechanism for Akt2 and p-Akt overexpression. Methods Three oral cancer cell lines and three cell lines of primary human oral keratinocytes (HOKs) were cultured and the degrees of Akt2 and p-Akt expression was evaluated by immunoblot analysis and flow cytometry. Each cell line was incubated with cycloheximide, an inhibitor of new protein synthesis, for various times to quantitatively determine the remaining expression levels of Akt2 and p-Akt by flow cytometry. The localization of Akt2 and p-Akt was assessed by immunofluorescence. Results The levels of Akt2 and p-Akt proteins were significantly higher in cancer cell lines than those in HOKs (P < 0.05). When the new protein synthesis was blocked by cycloheximide treatment, the degradation rate of Akt2 and p-Akt in oral cancer cells was significantly lower than that in HOKs (P < 0.05). Both Akt2 and p-Akt were more intensely stained in the cytoplasm of cancer cells, whereas HOKs expressed Akt2 and p-Akt only minimally. Conclusion Both Akt2 and p-Akt were overexpressed in oral cancer cells, which may be partly explained by a reduced rate of protein degradation in order to maintain high cytosolic levels.

CD147 is highly expressed in activated lymphocytes and cancer cells. CD147 plays many important roles in cellular functions. We have previously reported that binding of multivalent CD147 extracellular domain displayed on phage particles (multivalent CD147Ex phages) induced apoptosis in a monocytic cell line, U937, by triggering caspase-3 activation. Activation of caspase-8, caspase-9 and MCT-1 was investigated in this study. Levels of intracellular procaspase-9 and membrane MCT-1 were noticeably decreased in multivalent CD147Ex phage-induced U937 cells in comparison to controls. However, activation of caspase-8 was not obvious. Multivalent CD147Ex phages also induced DNA fragmentation and growth arrest of U937 cells. Taken together with previous studies, we hypothesize that the CD147 extracellular domain on phage particles may bind to its ligand(s) on the cell surface of U937 leading to inhibition of MCT-1 cell surface expression, intracellular activation of caspase-9, followed by caspase-3 activation, DNA fragmentation and growth arrest. We identified a mechanism of CD147 in triggering its ligand(s) on U937 cells leading to U937 apoptotic cell death. This may contribute to the conceptual development of novel cancer drugs based on CD147 polymers. Citation Format: Nutjeera Intasai, Supansa Pata, Khajornsak Tragoolpua, Chatchai Tayapiwatana. Binding of multivalent CD147 extracellular domain on U937 cells induces apoptosis via activation of caspase-9 and downregulation of MCT-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5433. doi:10.1158/1538-7445.AM2014-5433

The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described “heavy rafts” or non-raft membrane. Our data show that only Csk targeted into “classical” raft but not to “heavy raft” or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.