Determination and clinical significance of anti-idiotypic antibody--in model experiments of thyroglobulin and TSH]

Anti-idiotypic (anti-ID) antibody in test serum was determined by the direct binding assay using 125I-anti-human thyroglobulin (hTg). Several positive cases were found in Graves' disease and thyroiditis chronica. Positive anti-ID antibodies could be classified into two types. Type 1 showed the positive anti-hTg antibody and high Tg levels by RIA using double antibody method. Type 2 showed the positive anti-hTg antibody but low Tg levels by RIA. The binding of 125I-hTg to anti-hTg antibody was displaced by anti-ID antibody in type 1, but was not anti-ID antibody in type 2. A case of coexistence of autoantibody to hTSH and auto-anti-ID antibody to anti-hTSH antibody was found. She showed normal thyroid function (T4, T3), but TSH level showed discrepancy by different assay methods. Both autoantibodies for hTSH and for anti-hTSH antibody were demonstrated by the reaction of patient's antibody with both 125I-hTSH and 125I-anti-hTSH (MoAb). These two autoantibodies belong to the polyclonal IgG. The autoantibody for hTSH recognized only the beta-subunit of hTSH. Neither stimulating type of TSH receptor antibody (TRAb) nor blocking type of TRAb interfered with the binding of patient's anti-ID to 125I-anti-hTSH. This binding reaction could be inhibited by the unlabeled hTSH. This anti-ID might represent the internal image of the non-biological active site of TSH molecule, because of absence of thyroid stimulating activity. These anti-ID antibodies may provide evidence supporting a network theory of the immune system.     

Kinetics of Thyroxine (T4) and Triiodothyronine (T3) Transport in the Isolated Rat Heart

The dynamics and kinetics of thyroid hormone transport in the isolated rat heart were examined using the modified unidirectional paired tracer dilution method. The uptake of (125)I-thyroxine ((125)I-T(4)) and (125)I-triiodothyronine ((125)I-T(3)) from the extracellular space into heart cells was measured relative to the extracellular space marker (3)H-mannitol. The thyroid hormone maximal uptake was 54.4 % for (125)I-T(4) and 52.15 % for (125)I-T(3). The thyroid hormone net uptake was 25.69 % for (125)I-T(4) and 25.49 % for (125)I-T(3). Backflux from the intracellular space was 53.17 % for (125)I-T(4) and 61.59 % for (125)I-T(3). In the presence of unlabelled thyroid hormones, (125)I-T(4) and (125)I-T(3) maximal uptakes were reduced from 10.1 to 59.74 % and from 34.6 to 65.3 %, respectively, depending on the concentration of the unlabelled hormone, suggesting a saturable mechanism of the thyroid hormone uptake by the heart cells, with K(m(T4))= 105.46 microM and the maximal rate of (125)I-thyroid hormone flux from the extracellular space to heart cells (V(max(T4))) = 177.84 nM min(-1) for (125)I-T(4) uptake, and K(m(T3)) = 80.0 microM and V(max(T3)) = 118.5 nM min(-1) for (125)I-T(3) uptake. Experimental Physiology (2001) 86.1, 13-18.     

甲状腺激素自身抗体的检测及其临床意义

为探讨患者血清甲状腺激素水平假性升高的原因及检测甲状腺激素自身抗体的方法，为临床正确判断甲状腺激素水平提供帮助，本研究进行了甲状腺激素自身抗体结合实验、自身抗体稀释度实验、自身抗体抑制曲线实验及自身抗体检定实验。结果显示：患者血清与^125I-T2或^125I-T4呈特异性结合，结合率分别为58．77％和49．05％，是正常人或三蒸水对照的8－13倍；得到满意的T3和T4自身抗体稀释度曲线和抑制曲线，并确定甲状腺自身抗体为IgG，与兔抗人T3和T4抗体出现在电泳同一位置。甲状腺激素水平假或高的主要原因是患者体内存在着内源性抗甲状腺激素抗体，检测甲状腺激素自身抗体有利于正确判断甲状腺激素水平，为临床诊断和治疗甲状腺疾病提供帮助。     

Nuclear binding of T3 and effects of QO2, Na-K-ATPase, and alpha-GPDH in liver and kidney

Thyroid status was altered by use of a low-iodine-perchlorate (PC) regimen and either reversal with NaI or injections of L-3,5,3'-triiodothyronine (T3). The PC regimen decreased renal and hepatic oxygen consumption (QO2), alpha-glycerophosphate dehydrogenase (alpha-GPDH), and Na+-K+-dependent adenosine triphosphatase (Na-K-ATPase) to comparable extents (25 vs. 23%, 26 vs. 39%, and 41 vs. 51%, respectively). Administration of T3 to hypothyroid rats elicited dose-dependent increases in hepatic and renal cortical QO2, ouabain-sensitive oxygen consumption (QO2(t)), alpha-GPDH, and Na-K-ATPase activities. The half-maximal increases in all of the response parameters in both kidney and liver were obtained at dosages of 6-32 micrograms T3/100 g body wt. The equivalences in the renal cortical vs. hepatic responses were indicated by correlation coefficients of approximately 0.97. Kidney and liver nuclei also showed similar high-affinity binding of 125I-T3-K1/2 = 29 vs. 18 micrograms T3/100 g body wt, and Nmax = 1.8 vs. 2.1 ng T3/mg DNA. The patterns of the responses plotted as a function of T3 occupancy of the high-affinity nuclear binding sites were indistinguishable in kidney and liver. These results imply similar modes of action of T3, probably initiated at the nuclear level, in both kidney and liver.     

Homodimeric forms of bombesin act as potent antagonists of bombesin on Swiss 3T3 cells.

Two Lys3-bombesin dimers were prepared by crosslinking epsilon-amino groups Lys3-bombesin with noncleavable (glutaraldehyde) and cleavable [dimethyl-3,3'-dithiobispropionimidate (DTBP)] crosslinkers. The dimers were purified by HPLC ion-exchange chromatography and were shown to have retained immunoreactivity with an anti-bombesin monoclonal antibody directed against the C-terminal binding region of bombesin. The glutaraldehyde cross-linked bombesin dimer specifically inhibited binding of 125I-GRP to its receptor on Swiss 3T3 cells. Bombesin, at 0.6-60 nM induced mitogenesis in quiescent Swiss 3T3 cells, whereas, incubation of cells with the glutaraldehyde bombesin dimer at concentrations up to 124 nM did not. In competition assays, the bombesin dimer exhibited a dose dependent inhibition of bombesin-induced mitogenic activity and intracellular Ca++ mobilization. The bombesin dimer was 100 to 1000-fold more potent than D-Phe12Leu14-bombesin and D-Phe12bombesin, respectively, in inhibiting bombesin-induced mitogenesis on quiescent Swiss 3T3 cells. Similarly, the DTBP-bombesin dimer was not mitogenic to Swiss 3T3 cells, however, cleavage of the disulfide crosslinker with DTT of cell bound DTBP dimer restored mitogenic activity. Finally, the glutaraldehyde bombesin dimer also inhibited growth of bombesin receptor positive H345 SCLC cells in vitro. These findings suggest that the dimeric forms of bombesin are potent antagonists of bombesin.     

Characterization and Expression of the Human T Cell Receptor-T3 Complex by Monoclonal Antibody F101.01

A murine monoclonal antibody (MoAb) F101.01 reacting with the T cell receptor (TCR)-T3 complex is presented. Immunohistological studies showed that F101.01 specifically stains T-zone lymphocytes in lymph nodes, tonsils, and splenic tissue. Two-colour immunofluorescence and flow cytometry demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20), and myeloid cells (CD13 and CD33) were excluded. The target antigen of F101.01 co-modulated with the CD3-defined antigen (T3) and the TCR recognized by the MoAb WT-31. CD3 antibody and WT-31 both blocked binding of F101.01. F101.01 precipitated the TCR-T3 complex from lysates of 125I-labelled peripheral blood mononuclear cells (PBMC) and HPB-ALL, when the lysate was prepared with a detergent (digitonin) that conserves the TCR-T3 complex. FACS analysis of T cells from a patient with a T cell immunodeficiency demonstrated that delta-TCS-1-CD3+CD4+ and delta-TCS-1-CD3+CD8+ cells were brightly F101.01+, whereas a large subpopulation of delta-TCS-1+CD3+CD4-CD8- cells were weakly F101.01+. We conclude that F101.01 recognizes a conformational epitope of the TCR-T3 complex and that it reacts with the alpha beta TCR-T3 and the gamma delta TCR-T3 complexes with different intensities.     

Human monoclonal thyroglobulin autoantibodies of high affinity. II. Interaction between thyroglobulin and thyroglobulin autoantibodies of different IgG subclasses.

The interaction of human thyroglobulin (Tg) autoantibodies of different IgG subclasses with Tg was investigated using four high affinity human monoclonal thyroglobulin (Tg) autoantibodies, secreted by human-mouse hybridomas, of subclasses IgG1 (kappa and lambda) and IgG2 (kappa and lambda) and an IgG4 kappa serum monoclonal Tg antibody. With exception of a low level of interference in binding between one IgG1 lambda Tg antibody and one IgG2 kappa Tg antibody (27% decrease), binding by human monoclonal Tg antibodies of one IgG subclass was unaffected by pre-incubation of 125-I Tg (or Tg on an ELISA plate) with a human monoclonal Tg antibody of a different IgG subclass. Furthermore, preincubation of Tg-coated ELISA plates with an IgG1 human monoclonal Tg antibody had little effect on binding to Tg by IgG2, IgG3 and IgG4 Tg antibodies present in the sera of 6 Hashimoto patients. Comparable observations were made using an IgG2 monoclonal Tg antibody and serum Tg antibodies of subclasses IgG1, IgG3 and IgG4. Binding of an IgG1 kappa Tg antibody was inhibited (> 80%) by pre-incubation of Tg with an IgG1 lambda Tg antibody derived by fusion of lymphocytes from the same Hashimoto patient. In contrast, pre-incubation of Tg with an IgG2 kappa Tg antibody had little effect on subsequent binding by an IgG2 lambda Tg antibody derived from lymphocytes of a different Hashimoto patient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of interleukin 2 receptiveness and proliferation in resting peripheral T cells by monoclonal anti-CD3 (T3) antibodies does not require the presence of macrophages

In this study, we sought to elucidate the sequence of events by which mitogenic monoclonal anti-CD3 antibodies (anti-CD3-MoAb) initiate T cell activation. In cultures of monocyte-depleted resting T cells, two anti-CD3-MoAb, OKT3 and anti-Leu 4, induced a state of interleukin 2 (IL-2) receptiveness which culminated in T lymphocyte proliferation when recombinant IL-2 was provided. Evidence that Fc-receptor mediation by monocytes did not contribute to this mitogenesis was supported by studies showing that polyclonal F(ab')2 anti-mouse IgG Fc antibody did not alter the magnitude of the IL-2 driven T cell proliferative response, and by the use of T cells from donors whose monocytes were unable to assist in the induction of anti-Leu 4 (IgG1 subclass) initiated proliferation. Anti-CD3-MoAb, in the absence of IL-2, induced IL-2 receptor expression on purified T cells, and anti-IL 2 receptor antibodies inhibited T cell proliferation in the presence of this growth factor. Furthermore, following modulation of the CD3 molecular complex in the presence of monocytes, depletion of accessory cells rendered the modulated T cells mitogenically dependent on exogenous IL-2. IL-2 itself did not suffice to promote T cell proliferation in the absence of anti-CD3-MoAb. These results indicate that the binding of monoclonal antibody to CD3 is capable of initiating, in an accessory cell-independent manner, premitotic alterations in T cells which can culminate in proliferation when exogenous IL-2 is provided.     

Target antigen of monoclonal reagent S5.7: comparison with T3 antigen

S5.7 recognizes a 20 kD cell surface protein which is present on T lymphocytes. S5.7 binds to a nonglycosylated protein, which can be labeled by cell-surface radioiodination and by a hydrophobic reagent [125I]-iodo-5-naphthyl-1-azide (INA). As the T-lymphocyte-specific T3 complex was found to contain a nonglycosylated 20 kD species, and since this 20 kD T3 form can be labeled preferentially by INA, a comparison between T3 and S5.7 was made. Isoelectric focusing experiments showed, however, that the two proteins are different. Moreover, the S5.7 monoclonal antibody does not block CML, is not mitogenic, reacts with immature cells of several hemopoietic lineages, and differs in that respect from anti-T3 monoclonal antibodies.     

The 70-kilodalton pertussis toxin-binding protein in Jurkat cells.

125I-ASD photoaffinity-labeling derivatives of pertussis toxin (125I-ASD-PT) or lipopolysaccharide (125I-ASD-LPS) labeled similar 70-kDa proteins in Jurkat cells, a cell line derived from human CD4+ T lymphocytes. Labeling of this 70-kDa protein by 125I-ASD-PT was inhibited by underivatized PT but not by underivatized LPS. However, an immunoglobulin M monoclonal antibody with specificity for the p73 LPS receptor in murine splenocytes (S. W. Bright, T.-Y. Chen, L. M. Flebbe, M.-G. Lei, and D. C. Morrison, J. Immunol. 145:1-7, 1990) inhibited 125I-ASD-PT labeling of the 70-kDa species in Jurkat cells. Our results suggested that PT may bind to the same 70-kDa protein as LPS does in Jurkat cells but that PT and LPS bind to different sites on this receptor candidate. 125I-ASD-PT photoaffinity labeling of the 70-kDa protein was also inhibited by underivatized glycoproteins to which PT has been shown to bind, and this inhibition correlated with the relative binding affinities of the glycoproteins for PT. 125I-ASD derivatives of two sialic acid-specific plant lectins, Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin, with oligosaccharide binding specificities similar to those of PT also labeled a 70-kDa protein in Jurkat cells. This suggests that the 70-kDa PT receptor candidate in Jurkat cells likely contains sialooligosaccharide sequences to which PT, M. amurensis leukoagglutinin, and S. nigra agglutinin bind. The cross-reacting epitope recognized by monoclonal antibody 5D3 in this 70-kDa species might overlap the PT- and LPS-binding sites.     

Human monoclonal thyroglobulin autoantibodies of high affinity. I. Production, characterisation and interaction with murine monoclonal thyroglobulin antibodies.

Four hybridomas secreting human thyroglobulin (Tg) autoantibodies of different IgG subclasses and light chain types (IgG1 lambda, IgG1 kappa, IgG2 lambda and IgG2 kappa) were obtained by direct fusion of Hashimoto thyroid lymphocytes with the mouse myeloma X63-Ag.653. The autoantibodies were specific for human Tg and the functional affinities were high (only 2.6-3.9 log10 pM Tg required to give 50% inhibition of binding in ELISA). Using thyroid lymphocytes, 4 lines secreting Tg autoantibodies were obtained from 11 fusions compared with 1 line from 32 fusions of Epstein Barr virus infected blood lymphocytes, which emphasises the importance of using lymphocytes derived from a tissue known to be enriched in thyroid autoantibody secreting precursor B cells. These 4 human Tg autoantibodies, as well as an IgG2 lambda Tg antibody previously derived from Hashimoto blood B cells and an IgG4 kappa monoclonal Tg antibody present in a Hashimoto serum, were used in attempts to probe the interaction between human Tg autoantibodies and the Tg molecule (2 polypeptides of 330 KD). The binding to 125-I Tg by 3/7 murine monoclonal antibodies was inhibited (36-78%) by an IgG2 lambda and an IgG4 kappa human monoclonal Tg autoantibody, indicating an overlap between the epitopes recognised by these 3 murine monoclonal Tg antibodies and 2 monoclonal human Tg autoantibodies. None of the human Tg autoantibodies (or the murine monoclonal Tg antibodies) bound to Tg denatured by reduction and alkylation. Although the number of observations is limited, our study demonstrates that high affinity human monoclonal Tg autoantibodies, like polyclonal serum Tg autoantibodies, recognise non-linear B cell epitopes on conformationally intact human Tg.     

Possible Autoantibody Binding to Sickle Erythrocytes

Excessive binding of possible autoantibodies to discrete subpopulations of circulating sickle erythrocytes has been documented. However, the mechanism by which putative autoantibody binding sites selectively develop on sickle cells is unknown. In the present study, autologous IgG binding has been quantified for low density sickle erythrocytes subjected to prolonged morphologic sickling under nitrogen in the absence of plasma (24 hours, 37°C), and to reoxygenation with subsequent incubations in varying dilutions of autologous plasma. Cell-bound IgG was measured using a nonequilibrium 125-iodinated protein-A-binding assay. Binding isotherms show that IgG binding was both concentration dependent and saturable. Sickle cells pretreated by deoxygenation exhibited approximately 2-fold increased saturation binding of autologous IgG as compared with oxygenated paired samples, suggesting that new autoantibody binding sites may have developed during prolonged sickling. Autologous IgG binding to sickle cells pretreated by deoxygenation was also inhibited 2-fold more by limiting quantities of deoxygenated autologous cells, as compared with inhibition by oxygenated sickle erythrocytes used for serum absorption. These findings indicate that the sickling-associated increase in IgG binding may represent an increase in specific autoantibody binding sites, and suggests that autoantibody binding sites are produced by permanent sickling-associated remodeling of the red cell surface.     

A new epitope of the T200 molecule family defined by the 3A35 monoclonal antibody and expressed by macrophages and activated T lymphocytes

A monoclonal antibody (mAb), 3A35, produced against mouse macrophages (M phi) was found to react against certain activated T cells. This mAb, a rat IgM, resulted from a cell fusion between a mouse plasmacytoma and rat lymphocytes immunized against mouse M phi. It bound more avidly to activated than to resident M phi. It did not react against B cells and resting T lymphocytes but recognized certain dividing T cells like EL4 lymphoma, concanavalin A-activated and interleukin 2-expanded spleen cells, and helper T cell hybridomas. By contrast, other T lymphocyte-derived cell lines such as YAC-1 and CTLL2 were unreactive. No clear relationship was found between the binding of 3A35 to cells and the expression of L3T4 and Lyt-2 antigens. The specific stimulation of T cell clones with antigen rapidly induced a strong reactivity with 3A35 mAb which declined thereafter to a low (helper clones) or non-reactivity (cytotoxic clones) after 10 days of culture. Immunoprecipitation experiments, performed with M phi derived from bone marrow cell cultures, surface iodinated with 125I or metabolically labeled with [35S]methionine, showed that 3A35 bound to a 200-kDa molecule, shifting to 175 kDa under reducing conditions. In peritoneal M phi activated in vivo, in addition to the 175-kDa band, new bands migrating at 140, 120 and 85 kDa were identified by 3A35 and could be absorbed on a commercial anti-T200 mAb bound to Sepharose beads. After strengthening the cell binding of 3A35 to EL4 lymphoma cells by a cross-linking agent, only a 85-kDa molecule was immunoprecipitated. Thus, 3A35 identifies a new epitope of the T200 molecule family which is expressed on M phi and activated T cells.     

Detection of cross-reacting murine I-J like determinants on a human subset of T8+ antigen binding, presenting and contrasuppressor cells

Murine I-J gene products have been found in T suppressor cells (SC) and factors, macrophages and contrasuppressor cells (CSC). However, a human counterpart of the murine I-J has not been reported. As there is strong evidence that some murine anti-Ia antisera cross-react with human Ia antigens, the possibility was tested that mouse anti-I-J antibodies might cross-react with corresponding human class II determinants. Indeed, this revealed that three anti-I-J monoclonal antibodies (MoAb) and two antisera tested react with human mononuclear cells and that a significantly greater proportion of T8+ than T4+ cells or monocytes (Mo) react with the I-J antibodies. This was corroborated by autoradiography with significant inhibition of 125I-SA (streptococcal antigen) binding to T8+ cells but not to Mo by the MoAb or antisera to murine I-J. Functional reconstitution experiments of T4+ helper cells with the SA binding and presenting T8+ Vicia villosa adherent cells (VV-AC) and assessment of specific antibody forming cells to SA suggest that the antigen presenting function of this T8+ subset can be significantly inhibited by killing with the MoAb to I-J and complement. Furthermore, the subset of T8+ VV-AC also functions as CSC, for killing with MoAb to I-J and complement significantly inhibited the contrasuppressor function. This is consistent with the presence of I-J gene products in murine CSC. However, similar treatment of T8+ VV-NAC (non-adherent cells) or monocytes (Mo) failed to affect the suppressor or accessory helper function of these cells, respectively. Phenotypic characterization, inhibition of 125I-SA binding and reconstitution experiments for helper and suppressor functions, suggest that a subset of T8+ antigen binding, presenting and CSC may express determinants cross-reacting with murine I-J molecules.     

Triggering of the lethal hit in human cytotoxic T lymphocytes: a functional role for a 103-kDa T cell-specific activation antigen

A monoclonal antibody (CB.1) is described that defines a new triggering signal for human cytotoxic T lymphocytes (CTL). The antibody precipitates a 103-kDa surface antigen from activated normal human T cells. The antigen is undetectable or present in only low amounts on resting T lymphocytes but its expression increases strongly after activation and proliferation on T4+ and T8+ T lymphocytes. Binding of antibody CB.1 to CTL results in triggering of the lethal hit. This induction of cytotoxicity is dependent on cross-linking of CTL and an Fc receptor-bearing target cell with CB.1 and requires Ca2+ like antigen-specific triggering. CB.1-induced triggering can be specifically inhibited by binding of antibodies to the T8 or T4 molecules on T8+ or T4+ CTL.     

The relationship between autoantibodies to triiodothyronine (T3) and thyroglobulin (Tg) in the dog.

The relationship between T3 autoantibodies (T3AA) and thyroglobulin autoantibodies (TgAA) in dogs was investigated by determining the inhibitory effect of triiodothyronine (T3), thyroxine (T4) and thyroglobulin (Tg) on T3AA and TgAA binding activity and by determining the pattern of occurrence of the two activities in canine serum samples. Strong similarity in binding characteristics between the two activities, as one might expect if T3AA activity were merely a cross-reactivity of TgAA, was not observed. Canine T3AA activity exhibited a cross-reactivity to purified canine Tg that was intermediate between that of T3 and T4, indicating an antigenic relationship to an epitope of Tg. Average affinity constants of canine T3AA (N = 11) for T3, Tg and T4 were 1.76 x 10(10) M-1, 2.29 x 10(9) M-1, and 1.02 x 10(8) M-1, respectively. Canine TgAA activity, however, did not cross-react significantly with T3 or T4. Canine TgAA (N = 21) binding to canine Tg was not inhibited by T4 or T3 at concentrations up to 2 x 10(-4) M. Each of 23 canine serum samples containing T3AA also exhibited TgAA activity, although there was poor correlation between the magnitudes of the two activities. Neither T3AA nor TgAA activity was observed in serum samples from 16 euthyroid dogs; however, 46.7% of the samples from 15 hypothyroid dogs had detectable TgAA activity. T3AA is so rare that is was not observed in this small population of samples from hypothyroid dogs. The [125I] T3 binding in serum from hypothyroid dogs was elevated compared to that in euthyroid dogs, but was considerably lower than in samples generally designated as containing T3AA. These results suggest that T3AA found in occasional canine serum samples are due to the presence of autoantibodies recognizing a T3 containing epitope of Tg that is different from the epitopes involved in eliciting the predominant population of canine Tg autoantibodies.     

Cross-linking of the T cell receptor complex with the subset-specific differentiation antigen stimulates interleukin 2 receptor expression in human CD4 and CD8 T cells

Nonpolymorphic interactions between the T cell differentiation antigens CD4 or CD8 and major histocompatibility complex (MHC)-encoded molecules have been postulated to participate in antigen recognition of MHC-restricted T cells. This would imply simultaneous binding of CD4/8 and of the T cell receptor complex (Ti/CD3) to MHC molecules on the stimulator or target cell. In this report experimental evidence is provided that simultaneous binding by antibodies of Ti/CD3 and of CD4 or CD8 leads to the expression of interleukin 2 (IL 2) receptors in resting human T cells and to their subsequent proliferation in the presence of recombinant IL 2 (rIL 2). This could be shown by using a novel anti-CD3 monoclonal antibody (BMA 030) which alone only marginally stimulates highly purified human T cells even when applied in cross-linked form. However, human T cell subpopulations could be stimulated to grow in the presence of rIL 2 when BMA 030 was fixed to a solid support in combination with antibodies to either CD4 or CD8. In limiting dilution experiments, the frequencies of CD4 and CD8 T cells activated by the antibody combinations were similar to those activated by phytohemagglutinin in the presence of irradiated adherent cells. No stimulation was achieved if both or one antibody was applied in soluble form. In contrast, soluble antibodies inhibited activation by solid-phase antibodies. Taken together, cross-linking of Ti/CD3 with CD4/8 seems to be essential for T cell activation in cases of ligands that bind but do not activate T cells on their own--a situation that may reflect the interaction of T cell receptors with MHC-encoded molecules in association with antigen.     

Activation of human T lymphocytes II. Involvement of the T3 antigen in polyclonal T cell activation by mitogenic lectins and oxidation

The effect of monoclonal antibodies against the T cell surface antigens T3 and T4 on accessory cell-dependent mitogen-induced proliferation of human antigen-specific T4⁺ T lymphocyte clones and purified peripheral blood T cells was studied. To avoid the Fc receptor-dependent mitogenic effects of the OKT3 antibodies, monocytes were replaced by Epstein-Barr virus-transformed B lymphoblastoid cells as accessory cells. Monoclonal antibody OKT3 but not OKT4 inhibited the response of both T lymphocyte clones and purified T cells to mitogenic lectins and oxidation. The inhibition was not due to nonspecific effects of the monoclonal antibodies since it affected only the initial triggering but not the proliferation of activated T cells and it could be overcome by higher concentrations of lectin indicating a competition between OKT3 antibody and lectin. Furthermore, OKT3 antibody at the same concentration that was inhibitory in this system was mitogenic in the presence of Fc receptor-bearing monocytes. Surface modulation of T3 but not of T4 antigens led to unresponsiveness to a mitogen pulse given directly after modulation. These findings suggest that antigen-specific and mitogen-induced T cell triggering are due to interaction with the same receptors of the T lymphocyte.     

Anti-CD81 activates LFA-1 on T cells and promotes T cell-B cell collaboration

CD81 is expressed on human T cells at all stages of development. CD81 is physically associated with CD4 and CD8 and antibodies against CD81 generate signals which influence thymocyte adhesion and proliferation. Here we evaluate the function of CD81 on mature T cells. We employ a system in which B cells present superantigen to autologous T cells and find that anti-CD81 promotes T cell-B cell collaboration. Anti-CD81 induces T cell-B cell adhesion of peripheral blood lymphocytes which is partially mediated by LFA-1. CD81 engagement promotes LFA-1-dependent T cell activation, IL-2 production and proliferation. The antibody 5A6 was uniquely potent in exerting these effects compared to another antibody to CD81 or to antibodies that react with other tetraspanins expressed on T cells, anti-CD53 or anti-CD82. CD81-derived signals rapidly induce high-avidity LFA-1 as measured by cell binding to recombinant ICAM-3-coated fluorescent microspheres or by cell adhesion to ICAM-3-coated plastic. 5A6 activation of LFA-1 does not expose the high-affinity conformation epitope recognized by monoclonal antibody 24.     

Ligandin and Z protein in binding of thyroid hormones by the liver.

Sephadex G-100 chromatography of rat liver supernatant after addition of [125I]T3 revealed four peaks of protein-bound radioactivity in the void volume, albumin, ligandin, and Z-containing regions, respectively. The peaks were identified by cochromatography of BSP and [125I]T3 and immonodiffusion with antiratligandin IgG and antirat Z IgG. Binding of [125I]T4 to rat liver supernatant occurred in void volume, albumin, and Z regions only. Studies in vivo reveal a pattern of [125I]T3 binding to rat liver supernatant fractions quantitatively different from that observed in vitro. [125I]T4 binding to liver supernatant fractions in vivo occurred in all four peaks. BSP or bilirubin added to liver supernatant decreased T3 and T4 binding by each fraction. Flavaspidic acid inhibited binding of T3 and T4 to albumin, ligandin, and Z protein. Phenobarbital pretreatment of rats increased binding of T3 by ligandin and of T4 by albumin-containing fractions. Circular dichroism studies with purified rat liver ligandin suggest that T3 and T4 bind competitively to the same site as does bilirubin; the association constants of T3 and T4 for ligandin are 10(6) and 10(5) M-1, respectively. T4 was bound only by purified ligandin and not by ligandin in liver supernatant. To determine whether unconjugated bilirubin interferes with hepatic uptake of T3, [125I]T3 was administered to icteric homozygous and phenotypically normal heterozygous Gunn rats. Hepatic uptake and supernatant binding [125I]T3 were significantly reduced in homozygous Gunn rats. Hepatic uptake of [125I]T3 was also reduced in vivo by infusion of BSP with or without flavaspidic acid. BSP infusion abolished [125I]T3 binding to ligandin; BSP and flavaspidic acid abolished binding to ligandin and Z. These observations suggest that ligandin and Z protein are thyroid hormone binding proteins in rat liver cytosol and may influence the net flux of iodothyronies from plasma into the liver.