Pair Of mAbs Research Articles

Production and characterization of monoclonal antibodies against pregnancy-associated plasma protein A.

Pregnancy-associated plasma protein A (PAPP-A) was found to be a good first trimester maternal serum marker, together with free beta-human chorionic gonadotrophin (HCG) subunits, for the biochemical screening of fetal trisomy 21 (Down's syndrome). We have raised monoclonal antibodies (mAbs) against PAPP-A purified from human pregnancy serum. The different antibodies were characterized biochemically by Western blot analysis and in terms of specificity (reaction with non-pregnant and male serum). Their performance in Down's syndrome screening was assessed in comparison with an existing enzyme-linked immunosorbent assay method after labelling of the different mAbs with biotin or horseradish peroxidase. A pair of mAbs was eventually chosen for a double-antibody sandwich protocol. The selected combination was found to have a significantly increased specificity (P = 0.0116) over the method using (purified) polyclonal antibodies, together with slightly increased sensitivity. In our limited number of Down's syndrome pregnancy samples (n = 17) and controls (n = 18), the medians as well as the multiples of the median values (for the affected cases) were comparable between the two methods described.

Characterisation of monoclonal antibodies to haemocyte subpopulations of tiger shrimp (Penaeus monodon): immunochemical differentiation of three major haemocyte types

In order to study immune cellular effectors in tiger shrimp (Penaeus monodon), monoclonal antibodies (mAbs) raised from haemocytes could be a potential tool for separating and identifying haemocyte subpopulations. In this study, four mAbs – Z5E10 (IgG2a), Z6A5 (IgG1), Z6A6 (IgG1), and Z6H8 (IgG2b) – were produced by immunising balb/c mouse with a high-density granular cell (GC) suspension prepared from tiger shrimp through negative selection with concanavalin A-linked beads. Haemocyte reactivities were determined by means of immunoenzyme staining, and the molecular masses of antigens were analysed by Western blotting. Z5E10mAb showed reactivity with both GCs and semigranular cells (SGCs) and recognised a 29kDa antigen. Z6A6 specifically bound to SGCs while recognising a 163kDa antigen. The other two mAbs (Z6A5 and Z6H8) separately reacted with SGCs and were able to recognise two antigens which were larger than 205kDa under non-reducing conditions but could not recognise them under reducing conditions. Data from a periodate oxidation assay revealed that Z6A6 antibody was specific for one glycoprotein, while the antigens recognised by the other three mAbs consisted of protein molecules. According to these results, it was concluded that the Z5E10 antigen and the Z6A6 antigen were different not only from each other, but also from those recognised by the other two mAbs. Furthermore, additivity test results showed that the additivity index value for the mAb pairs Z6H8-Z6A6 (29%) was higher than that of Z6A5-A6A6 (0%). It is suggested that Z6A5 and Z6H8mAbs could be specific for non-identical epitopes. Following the negative selection with mAb-linked beads, an increase in hyaline cell density in haemocyte suspension was observed which suggests that the epitopes recognised by the four mAbs were located on the surface of haemocytes and that these mAbs may be useful when they are employed to label and separate haemocyte subpopulations for further study of haemocyte functions.

Two-site immunoassays for osteoclastic tartrate-resistant acid phosphatase based on characterization of six monoclonal antibodies.

Tartrate-resistant acid phosphatase (TRAP), an enzyme expressed in bone-resorbing osteoclasts, is secreted into the circulation during bone resorption. We used six monoclonal antibodies (MAbs) to optimize direct two-site fluoroimmunoassays for determining serum TRAP concentrations. Four of the MABs, 1F1, 2H1, 4E6, and 5C1, were raised against recombinant human TRAP, and the other two, O1A and J1B, against human bone TRAP. 2H1, J1B, and O1A appeared to be highly specific for TRAP. 1F1 and 4E6 were poor in recognizing bone TRAP and were not useful in the assay. 5C1, while having a good affinity for the bone enzyme, was not specific. Serum TRAP is relatively stable, because 7 days of storage of serum samples at 4 degreesC and -20 degreesC or five thawing-freezing cycles, did not change the TRAP concentration detected using the two-site assays. All studied assays detected an increase in serum TRAP concentrations of postmenopausal women compared with premenopausal women, the difference being highest with MAB pairs 2H1-5C1 and O1A-J1B. These results suggest that serum TRAP may be a useful bone resorption marker, and the MAB pairs 2H1-5C1 and O1A-J1B may be useful in determining the bone resorption rate.

Association of p59fyn with the T Lymphocyte Costimulatory Receptor CD2: BINDING OF THE Fyn Src HOMOLOGY (SH) 3 DOMAIN IS REGULATED BY THE Fyn SH2 DOMAIN

Human CD2 is a 50-55-kDa cell surface receptor specifically expressed on the surface of T lymphocytes and NK cells. Stimulation of human peripheral blood T cells with mitogenic pairs of anti-CD2 monoclonal antibodies (mAbs) is sufficient to induce interleukin-2 production and T cell proliferation in the absence of an antigen-specific signal through the T cell receptor. CD2 has been shown previously to associate physically with the Src family protein-tyrosine kinases p56(lck) and p59(fyn). We now report that stimulation of T cells with mitogenic pairs of anti-CD2 mAbs enhanced the association of the Fyn polypeptide with the CD2 complex, whereas stimulation with single anti-CD2 mAb had minimal effect. Using glutathione S-transferase (GST) fusion proteins, we found that CD2 bound to the Src homology (SH) 3 domain of Fyn. Interestingly, the CD2-Fyn association was negatively regulated by the Fyn SH2 domain; CD2 bound poorly to GST fusion proteins expressing both the SH2 and SH3 domains of Fyn. However, the inhibitory effect of the Fyn SH2 domain on binding of the Fyn SH3 domain to CD2 was relieved by peptides containing a phosphorylated YEEI sequence that bound directly to the Fyn SH2 domain. In addition, we found that the ability of the Fyn SH2 domain to precipitate tyrosine-phosphorylated proteins, including the CD3zeta chain, was enhanced after T cell stimulation with mitogenic pairs of CD2 mAbs. Finally, overexpression of a mutated Fyn molecule, in which the ability of the Fyn SH2 domain to bind phosphotyrosine-containing proteins was abrogated, inhibited CD2-induced transcriptional activation of the nuclear factor of activated T cells (NFAT), suggesting a functional involvement of the Fyn SH2 domain in CD2-induced T cell signaling. We thus propose that stimulation through the CD2 receptor leads to the tyrosine phosphorylation of intracellular proteins, including CD3zeta itself, which in turn bind to the Fyn-SH2 domain, allowing the direct association of the Fyn SH3 domain with CD2 and the initiation of downstream signaling events.

Potent Apoptotic Signaling and Subsequent Unresponsiveness Induced by a Single CD2 mAb (BTI-322) in Activated Human Peripheral T Cells

Manipulation of CD2 molecules with CD2 mAb pairs has been shown to deliver apoptotic signals to activated mature T cells. We show that BTI-322, a CD2 mAb directed at a peculiar epitope of CD2, can trigger on its own the apoptotic death of IL-2-activated peripheral T cells and of OKT3-stimulated T cells, contrasting in this respect with a series of other mouse or rat CD2 mAb. F(ab')2 fragments were as potent as the whole Ab. BTI-322-induced apoptosis proceeded in a few hours and was independent of the Fas/Fas ligand system. Less than 5 ng/ml of BTI-322, added at the beginning of culture, were able to eliminate within 4 days most CD3+ cells from OKT3- and IL-2-stimulated lymphocytes, the only cells remaining being CD16+CD2- NK cells. T cell proliferative responses induced by a mitogenic CD2 mAb pair or by PHA-P (which mainly binds to CD2) were not inhibited by BTI-322. In this case, the apoptotic effect was successfully counteracted by simultaneous enhancement of T cell divisions. Thus, the killing effect of BTI-322 was most effective when T cells were exclusively stimulated through the CD3/TCR complex. Apoptosis of the responding T cells may explain why T cells recovered from a primary MLC performed in the presence of BTI-322 responded to third party cells but not to the primary stimulatory cells. These data constitute the rational basis for the use of BTI-322 for inducing tolerance in human allotransplantation.

The transmembrane region of CD2-associated signal-transducing proteins is crucial for the outcome of CD2-mediated T-cell activation.

Signalling through the CD2 molecule was shown previously to employ similar signalling molecules as the T-cell receptor (TCR). Here, we show that CD2-mediated signalling is strongly influenced by the expressed transmembrane region of the employed signal-transducing molecule. We used TCR-negative cells expressing chimeric fusion proteins that consist of human interleukin-2 (IL-2) receptor alpha-chain-derived sequences (hCD25) fused to mouse-specific zeta-chain segments (hCD25-zeta). One set of TCR-negative cell lines expressed the hCD25-derived extracellular part fused to mouse-specific transmembrane and cytoplasmic zeta-protein sequences ('TZZ'). The second type of cell lines expressed the hCD25-derived extracellular and transmembrane portions fused to the mouse-specific zeta-chain cytoplasmic segment ('TTZ'). After cross-linking the hCD25-zeta molecules with specific monoclonal antibodies (mAb), all TCR-negative cell lines produced similar amounts of IL-2. Cross-linking with stimulating pairs of CD2-specific mAb, however, led to IL-2 production only in cell lines expressing the zeta-chain-specific transmembrane segment. Co-cross-linking of CD25 and CD2 molecules resulted in an effective stimulation of both TZZ- and TTZ-expressing cell lines. Moreover, TTZ- and TZZ-expressing cell lines differed in their pattern of tyrosine-phosphorylated proteins after stimulation with hCD25-specific mAb. Thus, although CD2 and TCR molecules share signalling components and pathways, the fine tuning of CD2 co-receptor function appears to be regulated in part by transmembrane regions of signal-transducing molecules like the TCR-associated zeta-chain.

A new set of monoclonal antibodies against human Fc gamma RII (CD32) and Fc gamma RIII (CD16): characterization and use in various assays.

Four mouse anti-human Fc gamma RII (CD32) (6C4, 2B2, 3D3, 93.4) (IgG1, kappa) and one anti-human Fc gamma RIII (CD16) (7.5.4) IgG1, kappa) MAbs were raised. An in vitro switch variant, 7.5.4Sw50 (IgG2b, kappa), was also derived from the 7.5.4 MAb. 6C4, 2B2, and 3D3 MAbs bind both Fc gamma RIIa and Fc gamma RIIb isoforms. Two of them (6C4 and 2B2 MAbs) allow a complete blockade of the binding of immune complexes to Fc gamma RII. All three MAbs immunoprecipitate the receptor and bind both its glycosylated and nonglycosylated forms. The fourth anti Fc gamma RII MAb, 93.4, directed against the intracellular region of Fc gamma RIIa1/2, allows its detection by Western blotting only when it is not phosphorylated. The 7.5.4 MAb binds both Fc gamma RIIIa and Fc gamma RIIIb, can be used in Western blotting and does not inhibit aggregated IgG binding. ELISA using IV.3 (anti-Fc gamma RIIa1/2)/6C4 and 3G8 (anti-Fc gamma RIIIa/b)/7.5.4Sw50 MAb pairs make it possible to detect soluble Fc gamma RIIa1/2 and Fc gamma RIII, with a sensitivity of 200 pg/mL and 1 ng/mL, respectively. Surface plasmon resonance analyses indicated that the KD of two of the three anti-Fc gamma RII and of the anti-Fc gamma RIII are in the same order of magnitude (6C4: 0.78 nM, 2B2: 0.28 nM, 7.5.4: 0.47 nM). The anti-Fc gamma RII 3D3 MAb exhibits an off-rate constant higher than the 6C4 and 2B2 MAbs and a KD of 2.19 nM.

Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex

Fourteen monoclonal antibodies (mAbs) against human cardiac troponin I (cTnI) were generated by commonly used experimental techniques. All these antibodies, as well as antibody 414 (HyTest), were specific for human cTnI. Fifteen antibodies thus obtained were tested in a sandwich cTnI immunofluorescence assay (altogether 196 combinations). Ten pairs giving the highest sensitivity were selected for further investigation. The effect of TnI-TnC complex formation on antibody interaction with antigen was analyzed. The formation of TnI-TnC complex results in a significant decrease of the interaction of mAbs with TnI for seven of 10 analyzed pairs of antibodies. Using two pairs of cTnI-specific mAbs, one that recognized only free cTnI but not cTnI complexed with cTnC, and another that could be used for measurement of total cTnI (free cTnI and cTnI in complex with cTnC), we demonstrated that the main part of cTnI in serum collected from acute myocardial infarction patients is presented in the complex from. We concluded that effective and reliable immunological detection of TnI is possible only when antibodies used for assay development recognize both free TnI and TnI complexed with other troponin components.

5531943 Method of making a carbon/metal composite

Further studies were carried out on the monoclonal antibodies (mAbs) from the First International Swine CD Workshop which react with γδ Null T-lymphocytes, defined by the binding of mAb w020141 (MAC320) as Swine Workshop Cluster number SWC6. Studies were also carried out on several other mAbs from the same workshop which identify other CD antigens, but whose binding is not restricted exclusively to γδ Null T-lymphocytes. The first group consists of 11 mAbs (w021, w022, w059–w065, w105 and w117) and the second group of 18 mAbs (w008, w026, w056, w067–w071, w080, w091–w094, w110, w111, w118, w119 and w121). All mAbs were characterised by binding to peripheral blood lymphocytes (PBL) from normal, sham-thymectomized (STx) and thymectomized (Tx) pigs of the Babraham SLAbb line and by their overlap, using two-colour immunofluorescence with biotinylated mAb MAC320 (w020141, which identifies all γδ Null cell T-lymphocytes and the Null cell subpopulation identified by MAC319 (w021). The Null cell-specific mAbs were also used in inhibition studies of MAC319 and MAC320 binding and by staining PBL with pairs of mAbs together with either MAC319 or MAC320. Based on these data we suggest a putative relationship of the Null cell subsets defined by these mAbs with each other.

5597074 Packing tray of semiconductor devices and a method for manufacturing the same: Ko Seung S Seoul, Korea

Further studies were carried out on the monoclonal antibodies (mAbs) from the First International Swine CD Workshop which react with γδ Null T-lymphocytes, defined by the binding of mAb w020141 (MAC320) as Swine Workshop Cluster number SWC6. Studies were also carried out on several other mAbs from the same workshop which identify other CD antigens, but whose binding is not restricted exclusively to γδ Null T-lymphocytes. The first group consists of 11 mAbs (w021, w022, w059–w065, w105 and w117) and the second group of 18 mAbs (w008, w026, w056, w067–w071, w080, w091–w094, w110, w111, w118, w119 and w121). All mAbs were characterised by binding to peripheral blood lymphocytes (PBL) from normal, sham-thymectomized (STx) and thymectomized (Tx) pigs of the Babraham SLAbb line and by their overlap, using two-colour immunofluorescence with biotinylated mAb MAC320 (w020141, which identifies all γδ Null cell T-lymphocytes and the Null cell subpopulation identified by MAC319 (w021). The Null cell-specific mAbs were also used in inhibition studies of MAC319 and MAC320 binding and by staining PBL with pairs of mAbs together with either MAC319 or MAC320. Based on these data we suggest a putative relationship of the Null cell subsets defined by these mAbs with each other.

Neoepitope immunoassay: an assay for human interleukin 1 beta based on an antibody induced conformational change.

A secondary monoclonal antibody (mAb2) was generated by immunization with immune complexes of human IL-1 beta and a primary monoclonal (mAb1). mAb2 bound to a neoepitope on the IL-1 beta/mAb1-complex with a dissociation constant (Kd) of 26 pM but not to uncomplexed IL-1 beta. As assessed by the binding of labeled IL-1 beta and neutralization of bioactivity, mAb2 enhanced the affinity of IL-1 beta to mAb1; Kd-values were 108 pM in absence and 5.4 pM in presence of mAb2. By analyzing a series of mutants of IL-1 where surface loops had been exchanged with the corresponding loops of human IL-1 receptor antagonist protein, a critical region responsible for mAb2 binding was localized to the C-terminal region. In addition to mAb1/IL-1 beta-complexes, mAb2 bound pro-IL-1 beta/mAb1 complexes as well as pro-IL-1 beta suggesting that mAb2 recognized a conformation of IL-1 beta resembling that of pro-IL-1 beta. Using this pair of mAbs, chemiluminescent and enzyme linked assays with detection limits of 2 pg/ml hIL-1 beta have been established.

Leukosialin (CD43)-major histocompatibility class I molecule interactions involved in spontaneous T cell conjugate formation.

Resting T cells spontaneously adhere in a selective manner to potent accessory cells, such as dendritic cells (DC) and lymphoblastoid B blasts (LCL). Here we demonstrate that leukosialin (CD43) and major histocompatibility complex class I molecules (MHC-I) might play a critical role in this process. T cell conjugate formation with monocyte-derived DC (md-DC) and LCL could be strongly inhibited by either preincubating T cells with Fab fragments of CD43 monoclonal antibody (mAb) 6F5 or by preincubating md-DC or LCL with MHC-I mAb W6/32. Intact CD43 mAb 6F5, in contrast to monovalent Fab fragments, enhanced T cell adhesiveness by transactivating CD2 binding to CD58 molecules. Interestingly, induction of this proadhesive signal via CD43 with intact 6F5 mAb was found to revert mAb W6/32-mediated inhibition of T cell conjugate formation. These observations indicated that CD43 cross-linkage mimics and monovalent mAb 6F5 inhibits interaction of T cell CD43 with a stimulatory ligand on opposing cells, presumably MHC-I. For the demonstration of direct physical interaction between CD43 on T cells and MHC-I-coated beads it was necessary, however, to ligate CD2 on T cells with a stimulatory pair of CD2 mAbs (VIT13 plus TS2/18). This suggests that CD2 ligation crosswise upregulates CD43 binding avidity for MHC-I and that both adhesion molecule pairs (CD43/MHC-I and CD2/CD58) act in concert to induce and mediate T cell conjugate formation with certain cell types.

Enhancement in antigen binding by a combination of synergy and antibody capture.

The effects of orientating pairs of synergistic monoclonal antibodies (mAb) on binding of human chorionic gonadotropin (hCG) was studied by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Antibody synergy towards hCG required two functionally intact antibodies located adjacent to each other and with different epitope specificities. We investigated whether immobilization procedures avoiding protein denaturation, increasing proper orientation and promoting higher molecular flexibility of the synergistic mAb resulted in significantly enhanced antigen, binding. Synergistic mAb pairs captured through their Fc-region by protein G or a polyclonal serum against the Fc-part of mouse IgG could be used at 10-fold lower coating concentrations to achieve maximal binding of the analyte as compared with the same mAb pairs coated directly onto polystyrene. The synergistic effect observed with protein A used as capture varied greatly with the subclasses of the two synergistic antibodies employed. Scatchard analysis revealed that the number of functionally synergistic antibody sites participating in the binding of hCG for one mAb pair was about 10 times higher for the protein G-captured as compared with the directly coated synergistic pair. Biotinylated synergistic mAb pairs, coated directly or captured by streptavidin, did not display any enhanced antigen binding when tested in SPR or ELISA. With SPR, synergy was only observed when the synergistic mAb had been captured through their Fc-region. Using protein G or a polyclonal rabbit anti-IgG1 serum as capture reagents in SPR, synergistic triple mAb combinations against hCG were demonstrated.

Relative location of epitopes involved in synergistic antibody binding using human chorionic gonadotropin as a model.

We systematically screened a large panel of well-characterized monoclonal antibodies (mAb) directed towards various epitopes on human chorionic gonadotropin (hCG) for synergistic binding of 125I-hCG when they were adsorbed to a solid phase. The epitope locations involved in synergy were then related to the crystal structure of hCG and discussed in accordance with available data on the hCG epitopes. Enhanced binding of hCG was specific for certain pairs of mAb and was reflected in a 3-50-fold increased apparent functional affinity constant for hCG. Surface plasmon resonance revealed that when the mAb were captured by a polyclonal anti-IgG1 coupled to the Biacore chip, the off rates for hCG were significantly slower with synergistic mAb combinations than for the corresponding single mAb or nonsynergistic pairs of mAb, whereas the on rates did not differ appreciably. Each of the two antibodies involved in synergistic binding of hCG (more than 3-fold compared to additive binding of the two mAb) always belonged to a different epitope cluster in a separate antigenic domain on hCG. Synergistic epitope combinations on holo-hCG were located in similar structural planes. Combinations of mAb directed towards the epitope clusters alpha 2/beta 3/5, alpha 2/hCG beta CTP (C-terminal peptide) and beta 3/5/hCG beta CTP showed the strongest enhancement, with binding more than 10-fold greater than the sum of 125I-hCG bound to the individual mAb, followed by pairs of mAb directed towards the epitope groups beta 1/beta 3/5, c 1/2/beta 3/5, beta 1/alpha 2, and alpha 2/alpha 3/5 (3-9-fold). The greater frequency of synergy obtained with the linear epitopes of the hCG beta CTP can be ascribed to their greater molecular flexibility relative to the constrained discontinuous epitopes on hCG alpha and core-hCG beta (residues 1-112). In general, these studies provide a method for rapid screening of synergistic antibody pairs which also helps to identify non-overlapping epitopes that are accessible in similar structural planes. In turn, this facilitates the design of high-affinity bispecific antibodies targetted to a single antigen molecule.

Structure-function relationships of human cholesteryl ester transfer protein: analysis using monoclonal antibodies.

Cholesteryl ester transfer protein (CETP), a 476 amino acid glycoprotein, mediates cholesteryl ester (CE), triglyceride, and phospholipid transfer among plasma lipoproteins. A monoclonal antibody (mAb), TP2, specific for an epitope within the last 26 amino acids of CETP has been shown to block all CETP-mediated lipid transfer, apparently by limiting access to lipid-binding sites in the carboxy terminal of CETP. A new panel of 16 anti-human CETP mAbs has now been used to further probe the structure-function relationships of CETP. Of the new mAbs, 9 partially inhibit CETP-mediated CE transfer (24-43%) from HDL to LDL. The corresponding epitopes were mapped within the CETP primary structure by the reactivity of the mAbs with CETP variants having deletions or amino acid substitutions. Of the 9 new, neutralizing mAbs, 6 are specific for epitopes situated between residues 410-450 and two others for epitopes between residues 184-260 and 332-366, respectively. The epitope of one neutralizing mAbs could not be mapped. Therefore, binding of mAbs to epitopes situated in four non-overlapping regions within CETP primary structure that are separated by as many as 280 residues can neutralize CETP-mediated CE transfer. Epitopes of mAbs that do not influence CE transfer activity map to the regions 184-260, 261-331, and 367-409, respectively. When pairs of mAbs were tested for their abilities to mutually compete for binding to immobilized CETP, competition was observed for mAbs specific for epitopes that are distant in CETP primary structure. The cross-competition patterns demonstrate that the carboxy terminal 60% of CETP adopts a compact structure. Together with previous mutagenesis studies, the data suggests that a carboxy terminal neutral lipid binding domain may be in close proximity to a lipoprotein binding region within native CETP.

Prolonged allograft survival by the inhibition of costimulatory CD2 signals but not by modulation of CD48 (CD2 ligand) in the rat.

The CD2 receptor is an important costimulatory molecule in T cell activation. Its ligand CD48 in rodents is supposed to be a homologue of human CD58, because of its similarities in structure and distribution. We evaluated the immunosuppressive activity of CD2/CD48-directed therapy in vitro and in vivo for the efficacy in prolonging rat heart allograft survival in a high responder transplant model. CD2-directed monoclonal antibody (mAb) therapy significantly prolonged median survival time to 45 days (P < 0.001). Suppression was mediated by down-modulation of CD2 below 20% on lymph node cells without considerable cell depletion. In contrast, CD48 mAb could not prolong graft survival. Rejection occurred in the presence of complete CD48 modulation and, therefore, despite disruption of the CD2-CD48 interaction. CD48 mAb failed to inhibit lymphocyte activation via a mitogenic pair of CD2 mAbs and inhibited mixed lymphocyte reaction (MLR) only by an unspecific mechanism. In conclusion, our results suggest a negative regulatory signal transduction by inhibitory CD2 mAbs and argue against a pivotal role of mere disruption of the CD2-CD48 interaction in CD2-mediated immunosuppression.

CD2 singnaling in T cells involves tyrosine phosphorylation and activation of the Tec family kinase, EMT/ITK/TSK

Ligation of the CD2 cell surface glycoprotein expressed on T lymphocytes and NK cells induces protein tyrosine phosphorylation and activation of the Src kinases, LCK and FYN. We show here that in Jurkat T leukemia cells and in peripheral blood T cells, CD2 stimulation also leads to tyrosine phosphorylation and activation of the Tec family kinase, EMT/ITK/TSK. Activation of EMT by CD2 was induced by mitogenic pairs of CD2 mAb, certain single CD2 mAb followed by secondary antibody cross-linking, and CD58-bearing sheep red blood cells. With the use of different Jurkat cell mutants it was demonstrated that CD2-mediated activation of EMT required expression of LCK, but not require surface expression of the CD3 zeta chain. Receptor-mediated activation of LCK does not in itself lead to activation of this Tec kinase since induction of LCK by ligation of CD4 or CD5 did not result in activation of EMT. The activation of EMT during CD2 signaling suggests an important role for this kinase in CD2 co-stimulation of T cell responses.

The behaviour of monoclonal antibodies in the First International Pig CD Workshop reacting with γδ/Null T lymphocytes in the blood of [formula omitted] line pigs

Further studies were carried out on the monoclonal antibodies (mAbs) from the First International Swine CD Workshop which react with γδ Null T-lymphocytes, defined by the binding of mAb w020 141 (MAC320) as Swine Workshop Cluster number SWC6. Studies were also carried out on several other mAbs from the same workshop which identify other CD antigens, but whose binding is not restricted exclusively to γδ Null T-lymphocytes. The first group consists of 11 mAbs (w021, w022, w059–w065, w105 and w117) and the second group of 18 mAbs (w008, w026, w056, w067–w071, w080, w091–w094, w110, w111, w118, w119 and w121). All mAbs were characterised by binding to peripheral blood lymphocytes (PBL) from normal, sham-thymectomized (STx) and thymectomized (Tx) pigs of the Babraham SLAb b line and by their overlap, using two-colour immunofluorescence with biotinylated mAb MAC320 ( w020 141 , which identifies all γδ Null cell T-lymphocytes and the Null cell subpopulation identified by MAC319 (w021). The Null cell-specific mAbs were also used in inhibition studies of MAC319 and MAC320 binding and by staining PBL with pairs of mAbs together with either MAC319 or MAC320. Based on these data we suggest a putative relationship of the Null cell subsets defined by these mAbs with each other.

Monocyte-Independent T Cell Activation by Simultaneous Binding of Three CD2 Monoclonal Antibodies (D66 + T11.1 + GT2)

Mitogenic pairs of CD2 mAb typically transduce activation/proliferation signals within T cells. However, complementary signal(s) provided by accessory cells are required to induce T cell proliferation. We show here that a particular combination of three CD2 mAb, D66 + GT2 + T11.1, leads to the proliferation of highly purified human T lymphocytes, without other complementary signal(s). The CD2 triplet was able to induce CD4+ and, to a lesser extent, CD8+ cells to proliferate. Interestingly, the so-called "naive" T cells (CD45RA+) were strongly stimulated, but more immature cells, such as thymocytes, were not. The proliferative response induced by the CD2 triplet was entirely mediated by the IL-2 autocrine pathway, as shown by the complete inhibition with anti-IL-2 Ab. T cells stimulated with the CD2 triplet were also able to secrete TNFα. We found no evidence for an unusual secretion of cytokines that might explain the lack of requirement of complementary signal(s). As high as it was, the proliferation induced by the CD2 mAb triplet could be further increased by the addition of IL-1, and this proliferative fraction could be inhibited by antibodies against TNFα. The CD2 mAb triplet increased [Ca2+1, while mitogenic CD2 mAb pairs needed the presence of a cross-linking agent. Thus, our data show that T cells can be activated to fully proliferate by this particular CD2 pathway, in the absence of accessory signal(s).

Comitogenic effects of very late activation antigens on CD3-stimulated human thymocytes. Involvement of various tyrosine kinase pathways.

Thymocytes display several integrins that are involved in cell-extracellular matrix interactions and differentiation processes. We have examined the role of very late activation Ag (VLA) on human thymocyte stimulation. VLA-4, VLA-5, and VLA-6 activated with either mAbs or their natural ligands (fibronectin, laminin, and vascular cell adhesion molecule-1) are able to transduce costimulatory signals in thymocytes activated via the CD3 pathway, i.e., enhancement of thymocyte proliferation, CD25 and CD69 expression, and IL-2 secretion. In contrast, activation of thymocytes with a mitogenic pair of CD2 mAb was not modified by VLA molecules. Cross-linking of both beta 1- and alpha 5-chains induced tyrosine phosphorylation of several proteins, whereas the cross-linking of the alpha 4- and alpha 6-chains did not. Moreover, a different pattern of tyrosine phosphorylation was observed when thymocytes were activated via either beta 1- or alpha 5-chains. These results suggest that VLA molecules activate tyrosine kinase pathways in thymocytes, and that different pathways would be implicated during thymocyte interactions with extracellular matrix or accessory cells, which are likely to play a role in thymocyte differentiation.