Expression of non-MHC-restricted T cell antigen-binding molecules by thymic lymphocytes

Heterologous antisera which recognize non-major histocompatibility complex (MHC)-restricted T cell antigen-binding molecules (TABM) were used to characterize the expression and structure of TABM on thymic lymphocytes. Approximately 70% of thymocytes express membrane molecules bound by anti-TABM antibodies (mTABM). Antibody activity for thymocyte TABM could be removed by adsorption to splenic T cells, but not by adsorption to splenic B cells. Similarly, adsorption of the antiserum to thymocytes or splenic T cells removed antibody activity to a purified TABM whereas adsorption with B cells had no effect. Radioiodinated thymic and splenic T cell mTABM were resolved by 2D-polyacrylamide gel electrophoresis and when reduced, both populations of mTABM migrated primarily as Mr 23,000 proteins with an isoelectric point range of 6.8-7.8. Multimers of this protein were also observed at Mr 85-97,000 and 130-150,000 on both thymocytes and splenic T cells. These data indicate that MHC-unrestricted antigen-binding molecules are expressed by a majority of thymocytes and these thymic TABM are structurally and antigenically similar to mTABM on peripheral cells. This suggests an ontogenic relationship between thymic TABM and peripheral TABM.     

Ontogeny and expression of non-MHC-restricted T cell antigen-binding molecules by thymocytes and peripheral T cell subsets

The ontogeny of T cells which express major histocompatibility complex (MHC)-unrestricted T cell antigen-binding molecules (TABM) on the cell membrane was investigated. We used a rabbit anti-mouse TABM antiserum to investigate the expression of TABM by subsets of adult thymocytes, peripheral T cells, and thymocytes during gestation. TABM are expressed by CD4+, CD8-, CD4+, CD8+ thymocytes and single-positive thymocytes. During gestation, TABM are expressed as early as Day 16, and at birth the expression of TABM on thymocytes has reached adult levels. Data are also presented which suggest that the expression of membrane TABM (mTABM) on peripheral T cells can be upregulated during T cell activation. The results suggest that TABM are expressed by different T cell subsets and that TABM+ cells may utilize the same intrathymic developmental pathway as that of T cells which express the alpha/beta T cell receptor.     

Wnt family proteins are secreted and associated with the cell surface.

Members of the Wnt gene family are proposed to function in both normal development and differentiation as well as in mammary tumorigenesis. To understand the function of Wnt proteins in these two processes, we present here a biochemical characterization of seven Wnt family members. For these studies, AtT-20 cells, a neuroendocrine cell line previously shown to efficiently process and secrete Wnt-1, was transfected with expression vectors encoding Wnt family members. All of the newly characterized Wnt proteins are glycosylated, secreted proteins that are tightly associated with the cell surface or extracellular matrix. We have also identified native Wnt proteins in retinoic acid-treated P19 embryonal carcinoma cells, and they exhibit the same biochemical characteristics as the recombinant proteins. These data suggest that Wnt family members function in cell to cell signaling in a fashion similar to Wnt-1.     

Cell cycle-dependent expression of antigen-binding and I-J-bearing molecules on suppressor T cell hybridomas

The I-J and antigen-binding chains with constant region determinant (Ct) that compose an antigen-specific suppressor T cell factor were found on the surface of suppressor T cell hybridomas, serologically and morphologically demonstrated by a fluorescence-activated cell sorter (FACS) and immunoelectron microscopic analyses. Moreover, the surface expression of the I-J and Ct-bearing chains fluctuating with the same kinetics depended entirely upon the cell cycle. The maximum expression of these two chains was observed in the early stage of the M phase, and the minimum in the S phase. Similarly, the magnitude of the suppressor activity was maximal in the late stage of the M phase, and was minimal in the S phase. The results therefore demonstrated that there exists good correlation between the cell surface expression of the I-J and Ct-bearing chains and the magnitude of the suppressor activity produced. The antigen recognition units on suppressor T cell hybridomas have serologically and morphologically been characterized by using radiolabeled antigens or monoclonal antibodies against the I-J or Ct on the antigen-binding molecule. Cell-binding assay and radioautographic analysis demonstrated that the suppressor T cell hybridoma possesses the capacity to bind native antigen in an antigen-specific fashion as does the hybridoma-derived, antigen-specific suppressor factor composed of the I-J and the Ct-bearing chains, indicating that the recognition unit on the cell surface is composed of a structure similar to the factor.     

Mast cells enhance T cell activation: importance of mast cell costimulatory molecules and secreted TNF

We recently reported that mast cells stimulated via FcepsilonRI aggregation can enhance T cell activation by a TNF-dependent mechanism. However, the molecular mechanisms responsible for such IgE-, Ag- (Ag-), and mast cell-dependent enhancement of T cell activation remain unknown. In this study we showed that mouse bone marrow-derived cultured mast cells express various costimulatory molecules, including members of the B7 family (ICOS ligand (ICOSL), PD-L1, and PD-L2) and the TNF/TNFR families (OX40 ligand (OX40L), CD153, Fas, 4-1BB, and glucocorticoid-induced TNFR). ICOSL, PD-L1, PD-L2, and OX40L also are expressed on APCs such as dendritic cells and can modulate T cell function. We found that IgE- and Ag-dependent mast cell enhancement of T cell activation required secreted TNF; that TNF can increase the surface expression of OX40, ICOS, PD-1, and other costimulatory molecules on CD3(+) T cells; and that a neutralizing Ab to OX40L, but not neutralizing Abs to ICOSL or PD-L1, significantly reduced IgE/Ag-dependent mast cell-mediated enhancement of T cell activation. These results indicate that the secretion of soluble TNF and direct cell-cell interactions between mast cell OX40L and T cell OX40 contribute to the ability of IgE- and Ag-stimulated mouse mast cells to enhance T cell activation.     

Serologic heterogeneity in I-J determinants associated with functionally distinct T cell regulatory factors

Information transfer among regulatory T cell subsets is mediated by biologically active T cell factors. Many of these factors are comprised of two molecules: one that binds antigen, and another that is I-J+ and determines the self recognition capability of the factor (I-J molecule). In the in vitro response to sheep red blood cells, we used three functionally distinct I-J+ factors to study the relationship between polymorphic I-J determinants and the biological activity of these factors. Our study shows that several monoclonal I-J antibodies react with I-J molecules associated with T suppressor-inducer factor (TsiF) and T suppressor-effector factor (TseF), but not with T contrasuppressor inducer factor (TcsiF). In contrast, a different set of monoclonal I-J reagents reacts with TcsiF but not TsiF or TseF. Finally, some monoclonal I-J antibodies distinguish between I-J molecules associated with TsiF and TseF. Thus anti-I-J reagents differentially react with I-J determinants on regulatory factors, and this differential pattern of reactivity correlates with the functional activity of the factors. The possible relationship between I-J heterogeneity and the biological function of I-J molecules in regulation is discussed.     

Preterm labor and chorioamnionitis are associated with neonatal T cell activation

Background

Preterm parturition is characterized by innate immune activation and increased proinflammatory cytokine levels. This well established association leads us to hypothesize that preterm delivery is also associated with neonatal T lymphocyte activation and maturation.

Methodology/Principal Findings

Cord blood samples were obtained following term, preterm, and deliveries complicated by clinical chorioamnionitis. Activation marker expression was quantitated by flow cytometric analysis. Infants born following preterm delivery demonstrated enhanced CD4⁺ T lymphocyte activation, as determined by CD25 (Term 9.72% vs. Preterm 17.67%, p = 0.0001), HLA-DR (Term 0.91% vs. Preterm 1.92%, p = 0.0012), and CD69 expression (Term 0.38% vs. Preterm 1.20%, p = 0.0003). Neonates delivered following clinical chorioamnionitis also demonstrated increased T cell activation. Preterm neonates had an increased frequency of CD45RO⁺ T cells.

Conclusion/Significance

Preterm parturition is associated with neonatal CD4⁺ T cell activation, and an increased frequency of CD45RO⁺ T cells. These findings support the concept that activation of the fetal adaptive immune system in utero is closely associated with preterm labor.     

Quantitation of T cell antigen-binding molecules (TABM) in the sera of nonimmunized, immunized, and desensitized mice

Heterologous antibodies to non-MHC-restricted T cell antigen-binding molecules (TABM) were used in an ELISA to detect and quantitate TABM levels in the sera of nonimmune mice and in mice immunized and/or desensitized to methylated bovine serum albumin (MBSA). TABM were detected at low levels in sera from nonimmune and immune mice. Amounts of TABM rose 300-fold in MBSA-immune mice within 12 hr of a desensitizing injection of MBSA, and rapidly returned to pre-desensitization levels. Serum TABM were purified by precipitation with 50% (NH4)2SO4 and chromatography in carboxymethylcellulose. The purified TABM were Mr 30,000 to 37,000 reduced, 72,000 nonreduced, and had a pI range of 5.5 to 7.1. A portion of the elevated TABM in desensitized mice bound MBSA specifically, whereas TABM from the sera of nonimmune/nondesensitized mice did not bind MBSA. The timing of the appearance of TABM early in desensitization suggests the possibility that they may play a role in the subsequent appearance of suppressor cells in later stages of this phenomenon. In addition, the results presented here raise the possibility that fluctuations in TABM levels might prove to be a sensitive indicator of immunoregulatory status in general.     

The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck

The CD4 and CD8 T cell antigens are thought to transduce an independent signal during the process of T cell activation. We report our evaluation of the possible involvement of the lymphocyte-specific tyrosine kinase p56lck in these transduction pathways. Our data demonstrate that p56lck is specifically modulated with either CD4 or CD8 following antibody-mediated cross-linking of these molecules and that a large fraction of the total cellular lck protein can be coimmunoprecipitated with these surface glycoproteins. These results suggest that p56lck is functionally and physically associated with CD4/CD8 in normal murine T lymphocytes and support the concept that an independent signal is transduced by the interaction of these surface molecules with major histocompatibility complex determinants.     

T cell regulation of B cell activation: antigen-specific and antigen-nonspecific suppressor pathways are mediated by distinct T cell subpopulations

The present studies were carried out to characterize the cellular events involved in the induction and function of carrier-specific Ts cells, which selectively regulate the generation of IgG responses by Lyb-5- B cells. It was demonstrated that this regulation is in fact mediated by two distinct suppressor pathways. In one pathway, carrier-primed Lyt-1 + 2 - Ts cells are specifically activated by in vitro reexposure to the priming antigen. After this specific activation, these Lyt-1 + 2 - Ts cells are able to suppress IgG responses in an antigen-nonspecific manner. This suppression requires the participation of unprimed Lyt-1 - 2 + T cells, and is effective in both the early and the late phases of antibody responses. A second suppressor pathway requires the antigen-specific activation of primed Lyt-1 - 2 + Ts cells. Suppression of antibody responses by activated Lyt-1 - 2 + Ts cells is highly carrier specific, in contrast to the nonspecific effector function of Lyt-1 + 2 - Ts cells, appears to act without requirement for additional T cell populations; and is effective only early in the course of the antibody response. Thus, it appears that two Ts cell populations may function through distinct mechanisms to regulate the generation of IgG Lyb-5- B cell responses.     

Demonstration of the third antigenically distinct outer membrane lipoprotein (OmlA) in Actinobacillus pleuropneumoniae serotype 7

The gene encoding an outer membrane lipoprotein (OmlA) of Actinobacillus pleuropneumoniae strain WF83 (serotype 7 reference strain), designated omlA₇, was sequenced. The amino acid sequence of OmlA₇ showed 64.5 and 71.6% identity to that of OmlA from serotypes 1 (OmlA₁) and 5 (OmlA₅), respectively. The first 134 amino acids of OmlA₇ were identical to those of OmlA₅. A Southern blot analysis revealed the presence of a gene highly homologous to the omlA₇ in the reference strains of serotypes 3, 4, 6, and 7. A Western blot analysis using a specific antiserum against a recombinant OmlA₇ detected expression of the homologous proteins in the serotypes 4, 6, and 7 reference strains and a serotype 3 field strain, but not in a serotype 3 reference strain. The data demonstrate the third antigenically distinct OmlA is expressed in A. pleuropneumoniae.     

T cell infiltration is associated with kidney injury in patients with anti-glomerular basement membrane disease

Cell-mediated autoimmunity, particularly that involving autoreactive T cells, participates in mediating anti-glomerular basement membrane(GBM) disease. However, direct kidney injury mediated by renal infiltrated T cells has not been clearly elucidated in humans. The T cell profile(CD3, CD4, CD8, IL-17, and foxp3) and macrophage(CD68) were examined by immunohistochemistry on renal biopsy tissues from 13 patients with anti-GBM disease. The correlation between cell infiltration and clinical data was also analyzed. We found that the distribution of T cell infiltration was predominant in the peri-glomerular and interstitial areas. CD3~+ T cell infiltratrion around the glomeruli with cellular crescent formations was significantly higher than that around the glomeruli with mild mesangial proliferation. CD8~+ T cells significantly accumulated around the glomeruli with cellular crescents without Ig G deposits compared to those with Ig G deposits. The prevalence of infiltrating CD8~+ T cells was correlated with the percentage of ruptured Bowman's capsules. In conclusion, cellular immunity may play a crucial role in the inflammatory kidney injury in anti-GBM patients. The periglomerular infiltration of T cells, especially CD8~+ T cells, may participate in the pathogenic mechanism of glomerular damage.     

Three different erythrocyte surface molecules are required for spontaneous T cell rosette formation

We have obtained three anti-sheep erythrocyte (E) monoclonal antibodies (MAb) which block rosettes with human T cells. They also block rosettes with E from all the species we have tested, including rosettes with autologous E. They define three different minor components on the E surface: MAb N217 precipitates a single 42-kilodalton (kDa) chain and MAb N4 a single 14-kDa chain, and MAb N23 immunoprecipitates under nonreducing conditions a single band at approximately 220 kDa, which is resolved under reducing and alkylating conditions, in two bands migrating at approximately 110 kDa and approximately 220 kDa. Thus MAb N23 is likely to react with a complex made of covalently linked 100-kDa chains associated in a noncovalent fashion with approximately 220 kDa chains. In addition, we have observed a puzzling phenomenon, i.e., that binding the MAb N23 first increases, to a large extent, the amount of N4 epitopes which can be subsequently detected on E. This effect was not observed when N217 MAb or antiglycophorin (either monoclonal or polyclonal) antisera were first bound on E. Therefore the N23 and N4 molecules must interact on the E surface. This finding discloses the complex interactions between the T cell and E surface component that must occur in the process of rosette formation, including that with autologous E. These observations are of interest in view of the recent evidence that the CD2 molecule is involved both in adhesion and activation aspects of T cell functioning.     

Differential loss of T cell signaling molecules in metastatic melanoma patients' T lymphocyte subsets expressing distinct TCR variable regions

In this study we tested the hypothesis that loss of T cell signaling molecules in metastatic melanoma patients' T cells may affect differently T cell subsets characterized by distinct TCR variable regions. By a two-color immunofluorescence technique, expression of zeta-chain, lck, and ZAP-70 was evaluated in CD3+ T cells and in three representative T cell subsets expressing TCRAV2, TCRBV2, or TCRBV18. Partial loss of lck and ZAP-70 was found in CD3+ T cells from PBL of most melanoma patients, but not of healthy donors. The extent of zeta-chain, lck, and ZAP-70 loss depended on the TCRV region expressed by the T cells, and this association was maintained or increased during progression of disease. Coculture of patients' or donors' T cell with melanoma cells, or with their supernatants, but not with normal fibroblasts or their supernatants, down-modulated expression of zeta-chain, lck, and ZAP-70 in a TCRV region-dependent way. Immunodepletion of soluble HLA class I molecules present in tumor supernatants, but not of soluble ICAM-1, blocked the suppressive effect on T cell signaling molecule expression. T cell activation with mAbs to a single TCRV region and to CD28 led to significant and TCRV region-specific re-induction of zeta-chain expression. These findings indicate that extent of TCR signaling molecules loss in T lymphocytes from metastatic melanoma patients depends on the TCRV region and suggest that tumor-derived HLA class I molecules may contribute to induce such alterations.     

An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM)

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.     

Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.     

Serum and liver cytosolic growth-hormone-binding proteins are antigenically identical with liver membrane 'receptor' types 1 and 2.

Studies with a panel of monoclonal antibodies (MAbs) reactive towards the presumptive rabbit liver growth-hormone (GH) receptor show that the rabbit serum GH-binding proteins share seven antigenic determinants (three at the hormone-binding site and four located elsewhere) with the liver cytosolic GH-binding proteins and the putative GH 'receptors' associated with the hepatocyte membrane. The rabbit serum binding proteins have an affinity for GH similar to the membrane GH receptors [for human GH, Ka = 2.45 (+/- 0.15) X 10(9) M-1 (mean +/- S.E.M., n = 8)] and high capacity relative to membrane 'GH receptors'. Analogues of the postulated membrane 'receptor' subtypes 1 and 2 exist in the serum, but not subtype 3, which is also absent from liver cytosol. The serum and cytosolic binding proteins have identical cation-dependence properties; hGH binding is Ca2+-dependent, whereas oGH binding is Ca2+-independent. Affinity labelling of hGH-affinity-purified serum binding proteins with 125I-hGH demonstrated a major GH-binding subunit, of Mr 55,000, identical with the major component purified from membranes. In view of their high affinity and capacity, the serum binding proteins could control availability of GH to membrane receptors. It is suggested that the cytosolic binding proteins may be newly synthesized serum binding proteins. The existence of a close relationship between subsets of membrane-associated GH-binding sites, the serum GH-binding proteins and cytosolic GH-binding proteins dictates a reappraisal of earlier ligand-binding studies, which did not distinguish between binding-site subsets in the liver.     

T cell glycolipid-enriched membrane domains are constitutively assembled as membrane patches that translocate to immune synapses

In T cells, glycolipid-enriched membrane (GEM) domains, or lipid rafts, are assembled into immune synapses in response to Ag presentation. However, the properties of T cell GEM domains in the absence of stimulatory signals, such as their size and distribution in the plasma membrane, are less clear. To address this question, we used confocal microscopy to measure GEM domains in unstimulated T cells expressing a GEM-targeted green fluorescent protein molecule. Our experiments showed that the GEM domains were assembled into membrane patches that were micrometers in size, as evidenced by a specific enrichment of GEM-associated molecules and resistance of the patches to extraction by Triton X-100. However, treatment of cells with latrunculin B disrupted the patching of the GEM domains and their resistance to Triton X-100. Similarly, the patches were coenriched with F-actin, and actin occurred in the detergent-resistant GEM fraction of T cells. Live-cell imaging showed that the patches were mobile and underwent translocation in the plasma membrane to immune synapses in stimulated T cells. Targeting of GEM domains to immune synapses was found to be actin-dependent, and required phosphatidylinositol 3-kinase activity and myosin motor proteins. We conclude from our results that T cell GEM domains are constitutively assembled by the actin cytoskeleton into micrometer-sized membrane patches, and that GEM domains and the GEM-enriched patches can function as a vehicle for targeting molecules to immune synapses.