Production and characterization of a novel monoclonal antibody against phosphorylated T cell receptor zeta chain.

One of the earliest events following T cell receptor (TCR) triggering is the activation of the protein kinase Lck and induction of tyrosine phosphorylation of zeta, the major signal transduction subunit of the T cell receptor complex. Here we report the generation and characterization of a monoclonal antibody specific for human phosphozeta. The antibody was produced by immunizing mice with a truncated recombinant form of human zeta together with the Lck enzyme. The C415.9A antibody recognizes recombinant as well as cellular phosphozeta but is unreactive with unphosphorylated zeta or other tyrosine phosphorylated proteins. Using this antibody, we have demonstrated aberrant TCR-zeta tyrosine phosphorylation in Jurkat T cell transduction mutants. Therefore, this antibody can be used to elucidate T cell signal transduction mechanisms by analyzing and monitoring tyrosine phosphorylation of zeta in vitro and in vivo directly. Furthermore, this antibody could find application in the analysis of abnormal T cell signaling in autoimmune disease, cancer, and immunodeficiency disorders.

Induction of mouse beta integrin expression following transfection with human alpha 4 chain.

We report here an analysis of the expression and function of the alpha chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the beta chain in these processes. L cells were transfected with human alpha 4 cDNA or alpha 4 and human beta 1 cDNA. Unexpectedly, human alpha 4 cDNA, when transfected alone, could induce de novo surface expression of host beta 7 and increased expression of host beta 1. Induction of mouse beta 7 and beta 1 surface expression was not due to de novo gene activation, but instead represented alpha 4/beta intracellular subunit association and transport to the cell surface. Transfection with human beta 1 prevented surface expression of mouse beta integrins. Whereas human alpha 4 and human beta 1 subunits associated very tightly in anti-alpha 4 immunoprecipitates, human alpha 4 and mouse beta subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for alpha 4 beta 7 and alpha 4 beta 1, was very poor, whereas human alpha 4/human beta 1 receptors bound strongly to VCAM. One alpha 4 transfectant, which exhibited a tight human alpha 4/mouse beta 1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that alpha 4 subunits have specific affinity for beta 7 and beta 1 integrins and require beta subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the alpha 4 and beta subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the beta subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression.

Aspartate 698 within a novel cation binding motif in alpha 4 integrin is required for cell adhesion.

The interactions of alpha 4 beta 1 integrin with vascular cell adhesion molecule (VCAM) and fibronectin play important roles in many physiological and pathological processes. To understand the mechanism of alpha 4 beta 1 integrin-mediated cell adhesion, we made mutant alpha 4 constructs. Three aspartic acid (Asp) residues in alpha 4, Asp-489, Asp-698, and Asp-811, were replaced with glutamic acids (Glu). The wild-type and mutant alpha 4 constructs were transfected into K562 cells, and stable transfectants with similar levels of alpha 4 surface expression were established. The Asp-->Glu substitutions did not affect alpha 4 beta 1 association or heterodimer formation as demonstrated by immunoprecipitation analysis. However, the glutamate substitutions at Asp-489 and Asp-698 severely impaired cell adhesion to VCAM and fibronectin, whereas the substitution at Asp-811 had no detectable effect on cell adhesion. In contrast to these results, isolated alpha 4 beta 1, containing the D489E or D698E substitution, was able to bind to VCAM, suggesting that these two residues are not critical for ligand recognition. In searching for a mechanism to explain inhibition of adhesion by Asp-489 and Asp-698 mutations, we found that the sequences flanking Asp-698 resemble the DxxxxxD-S-Sx divalent cation/ligand binding motif in beta integrins and the I-domains of alpha integrins. This suggests that Asp-698 in the alpha 4 integrin, which does not possess an I-domain, may also be involved in cation binding and may be part of a sequence functionally similar to that found in the I-domains of other alpha integrins.

Detection of bovine trichomoniasis with a specific DNA probe and PCR amplification system.

Trichomoniasis is a widespread, economically important venereal disease of cattle which causes infertility and abortion. Effective control of trichomoniasis has been impeded by the insensitivity of traditional diagnostic procedures, which require the isolation and cultivation of the parasite, Tritrichomonas foetus, from infected cattle. We developed a 0.85-kb T. foetus DNA probe by identifying conserved sequences in DNAs from T. foetus that were isolated from cattle in California, Idaho, Nevada, and Costa Rica. The probe hybridized specifically to DNAs of T. foetus isolates from different geographic areas but not to DNA preparations of Trichomonas vaginalis, bovine cells, or a variety of bacteria from cattle. The probe detected DNA from a minimum of 10(5) T. foetus organisms. To improve sensitivity, a partial sequence of the probe was used to identify oligonucleotide primers (TF1 and TF2) which could be used to amplify a 162-bp product from T. foetus DNAs by PCR. A chemiluminescent internal T. foetus sequence probe was hybridized to Southern blots of the amplification product. This system detected as few as one T. foetus organism in culture media or 10 parasites in samples containing bovine preputial smegma. Analysis of 52 clinical samples showed that 47 (90.4%) of the 52 samples were correctly identified, with no false-positive reactions. In comparison, the traditional cultivation method detected 44 (84.6%) of the 52 samples from T. foetus-infected and uninfected bulls. These results indicate that the PCR-based amplification system could be a useful alternative method for the diagnosis of bovine trichomoniasis.

Role of third N-terminal domain of VCAM-1.

The interaction between VLA-4 and VCAM-1 has been implicated in the recruitment, adhesion, and activation of mononuclear leukocytes in chronic inflammatory conditions and autoimmune disease. The seven domain extracellular portion of VCAM-1, sVCAM1-7, and the first three and two N-terminal domains of VCAM-1, sVCAM1-3 and sVCAM1-2, respectively, were expressed in baculovirus and purified. Using these purified soluble forms of VCAM-1 and cellular transfectants expressing various cell bound forms of VCAM-1, we show that the major binding site for VLA-4 is located within the first two domains of VCAM-1 and that the third domain of VCAM-1 appears to be required for functional integrity of the VLA-4 binding site.

Tissue-specific and cell surface expression of human major histocompatibility complex class I heavy (HLA-B7) and light (beta 2-microglobulin) chain genes in transgenic mice.

We introduced the human genes HLA-B7 and B2M encoding the heavy (HLA-B7) and light [beta 2-microglobulin (beta 2m)] chains of a human major histocompatibility complex class I antigen into separate lines of transgenic mice. The tissue-specific pattern of HLA-B7 RNA expression was similar to that of endogenous class I H-2 genes, although the HLA-B7 gene was about 10-fold underexpressed in liver. Identical patterns of RNA expression were detected whether the HLA-B7 gene contained 12 or 0.66 kilobase(s) (kb) of 5' flanking sequence. The level of expression was copy number dependent and as efficient as that of H-2 genes; gamma interferon enhanced HLA-B7 RNA expression in parallel to that of H-2. In addition to the mechanism(s) responsible for gamma interferon-enhanced expression, there must be at least one other tissue-specific mechanism controlling the constitutive levels of class I RNA. Tissue-specific human beta 2m RNA expression was similar to that of mouse beta 2m, including high-level expression in liver. Cell surface HLA-B7 increased 10- to 17-fold on T cells and on a subset of thymocytes from HLA-B7/B2M doubly transgenic mice compared to HLA-B7 singly transgenic mice. The pattern of expression of HLA-B7 on thymocytes resembled that of H-2K as opposed to H-2D. These results confirm that coexpression of both human chains is required for efficient surface expression and that HLA-B7 may share a regulatory mechanism with H-2K, which distinguishes it from H-2D.

Prostaglandin E2 depresses antigen-presenting cell function of peritoneal macrophages.

Eicosanoids play a prominent role in trauma. Such mediators of inflammation negatively influence cell-mediated immunity (CMI). There is, however, no information available on the effect of eicosanoids on a critical event in CMI, i.e., antigen-presenting (AP) cell function of macrophages (M luminal diameter), a cellular process responsible for the activation of T and B lymphocytes. The aim of this study, therefore, was to examine the effect of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) on AP cell function of the peritoneal M luminal diameter. To study this, a T-helper-cell clone, D10.G4.1 was employed. This cell clone proliferates in the presence of Iak (Class II glycoprotein, MAC product) bearing M luminal diameter and specific antigen (conalbumin A) thus directly reflecting the AP capability of the M luminal diameter. Peritoneal M luminal diameter were harvested from B10.BR mice (H2k) and their AP was tested in vitro by incubating varying numbers of M luminal diameter with 2 X 10(4) D10.G4.1 cells/well and conalbumin (400 micrograms/ml) in the presence and absence of different concentrations of PGE2 or TXB2. Cultures were incubated for 72 hr, pulsed with [3H]-thymidine, and harvested. At concentrations of 10, 30, and 100 nM of PGE2, D10.G4.1 proliferations were 38, 35, and 20% of control, respectively (P less than 0.05 compared to control). TXB2 added at the above-mentioned concentrations did not suppress the proliferative response of D10. Thus, PGE2 but not TXB2 has a potent immunosuppressive effect on AP of peritoneal M luminal diameter.(ABSTRACT TRUNCATED AT 250 WORDS)

An external stimulus that mimics Mls locus responses.

The response to a novel set of T cell mitogens has been analysed and compared to the response to Mls locus differences. These polyclonal T cell activators, staphylococcal enterotoxins A and B, stimulate T cells in a way that requires an antigen-presenting cell bearing class II MHC products and involves the CD4:T cell receptor complex. However, the specificity of MHC recognition by the T cell receptor is lost in this response. Thus, these mitogens produce a response with characteristics similar to that induced by Mls locus differences. These mitogens can be used to analyse the immunobiology of this response, and may help in understanding and identifying the Mls locus product as well.

Depressed antigen presentation function and membrane interleukin-1 activity of peritoneal macrophages after laparotomy.

Although major tissue trauma produces profound depression of cell-mediated immunity, it is not known whether surgical trauma (i.e., midline laparotomy) has any adverse effect on the antigen presentation function and membrane interleukin-1 (IL-1) activity of peritoneal macrophages. To study this, C3H/HEJ (endotoxin-tolerant) mice were anesthetized. An approximately 1-inch midline abdominal incision was made, followed by abdominal closure. On days 1, 3, 5, and 7, peritoneal macrophages were harvested by means of peritoneal lavage, and antigen presentation capability was tested by incubating various numbers of peritoneal macrophages with 2 X 10(4) D10.G4.1 cells per well in the presence of conalbumin (400 micrograms/ml). The T helper cell clone (D.10.G4.1) proliferates on recognition of conalbumin in the context of Iak and also proliferates in the presence of membrane-bound IL-1 plus concanavalin A. To measure membrane IL-1 expression in peritoneal macrophages, Concanavalin A (10 micrograms/ml) was substituted for conalbumin. Cultures were incubated for 72 hours, pulsed with tritiated thymidine, and harvested. Peritoneal macrophages from laparotomized mice induced significantly less T helper cell proliferation on days 1 and 3 in the antigen presentation assay (37% and 30%, respectively; p less than 0.05) and in the membrane IL-1 assay (14% and 10%, respectively; p less than 0.05) as compared with the control. This difference was not detectable on day 5. More effective antigen presentation capability (167% of control; p less than 0.05) was seen on day 7. Thus laparotomy by itself produces marked depression of both antigen presentation function and membrane IL-1 activity of peritoneal macrophages, which may enhance susceptibility to intra-abdominal sepsis.

Differential effects of antibodies to Lyt-2 and L3T4 on cytolysis by cloned, Ia-restricted T cells expressing both proteins.

Cloned T cell lines specific for the antigen ovalbumin (OVA) in the context of self I-A or I-E class II MHC-encoded molecules were found to express equivalent levels of the Lyt-2 and L3T4 surface molecules by FACS analysis. Functionally, these cloned T cell lines will kill OVA-pulsed, class II MHC-bearing B lymphoma cells. This system allowed us to examine the relative contribution of the Lyt-2 and L3T4 molecules to recognition of antigen in the context of self class II MHC molecules. We find that anti-L3T4 is 25- to 100-fold more potent at inhibiting cytotoxicity by these cloned lines than is anti-Lyt-2, where potency is calculated as the ratio of the concentration needed for inhibition divided by the concentration needed for binding. Both antibodies can completely inhibit cytotoxicity. These results suggest that the accessory molecules Lyt-2 and L3T4 can play two different roles in antigen:MHC recognition. In class II MHC recognition, L3T4 plays a dominant role, highly sensitive to inhibition by anti-L3T4. By contrast, Lyt-2 plays a minor yet important role in the cell interaction, perhaps by adhesion strengthening. Previous analysis in several laboratories supports the concept that the ligand for L3T4 is class II MHC, whereas the ligand for Lyt-2 is class I MHC. The present results are consistent with the hypothesis that these molecules are actually part of the T cell antigen:MHC recognition complex, and play an important role in the class of MHC molecule recognized by a T cell alpha:beta heterodimeric receptor. As such, they are not accessory proteins, but a direct part of the recognition apparatus, and the term accessory protein may apply only in those cases in which the specificity of the T cell receptor is for a different class of MHC than that normally associated with the L3T4 or Lyt-2 molecule.

Autocrine growth inhibition of a cloned line of helper T cells.

The growth of T lymphocytes is dependent on the T-cell growth factor interleukin 2 (IL-2), which causes T cells bearing high-affinity receptors for IL-2 to proliferate. Most cloned helper-T-cell lines can be shown to both produce and respond to IL-2; thus, growth of such cells is by an autocrine mechanism. We report that the failure of the cloned murine T-cell line D10.G4.1 to respond to its own IL-2 results from the secretion, by the same cells, of a potent inhibitor of the IL-2-driven T-cell proliferative response. This inhibition can be overcome by increasing the number of IL-2 receptors expressed by the target cell. In the cloned T-cell line producing the inhibitory substance, this increase in IL-2 receptors is driven by the monokine interleukin-1. We propose that this inhibitor of IL-2 responses may play a role in preventing "bystander" activation of T cells by IL-2 released in vivo and could be a potent pharmacologic agent.

Cell interactions in the immune system: the role of self recognition in the targeting of nonspecific effector molecules by helper T cells.

Helper T cells are activated by cross-linking of their receptors by antigen:Ia complexes on the surface of antigen-presenting cells and B cells. As a result of this cross-linking, the helper T cell releases several lymphokines that in turn affect the Ia-bearing cell with which the helper T cell is in contact. This interaction is cognate when the effect on the target cell is examined, but it operates by a mechanism that is neither antigen specific nor MHC restricted. Whether the cognate nature of this interaction reflects solely the intimate contact of the T cell with the Ia antigen-bearing cell or whether it reflects a receptor-directed focal release of lymphokines remains to be determined. The molecular basis for functional diversity in helper T cells will have to be determined by examining the factors that regulate lymphokine gene expression in such cells, a process that appears to act at several levels.

Monoclonal antibodies specific for Ia glycoproteins raised by immunization with activated T cells: possible role of T cellbound Ia antigens as targets of immunoregulatory T cells.

Two monoclonal antibodies, Y-3P and Y-8P, specific for conventional mouse Ia glycoprotein antigens are described. Both were raised by repeated immunization of primed mice with activated T cells over a period of 1 yr, and both detect a new and broad public Ia specificity. Both of the antibodies react with I-A subregion-controlled A alpha:A beta complexes of all mouse strains apart from the responder strain (H-2d), as well as the equivalent of A alpha:A beta complexes in rats carrying seven independent haplotypes. These antibodies have great utility as almost universal Ia reagents. On the basis of these results, we propose that Ia antigens presented to the immune system bound to activated T cells are immunogenic, and may induce the production of Ia antibodies of novel and broad specificity. In addition, we propose that such bound Ia glycoproteins could be a target for immunoregulatory T cells, and could account for the specificity of suppression of graft-vs-host reactions and Ia-restricted helper T cells observed by others in F1 animals injected with parental T cells.

The expression of I-Ed molecules in F1 hybrid mice detected with antigen-specific, I-Ed-restricted cloned T-cell lines.

The expression of polymorphic determinants on I-E molecules is largely dependent on allelic variation in the E beta chain. We have previously analyzed the expression of Ek beta and Eb beta chains in F1 hybrid mice by a combination of techniques, and have shown that functional variation detected by the responsiveness of cloned T-cell lines specific for these molecules correlates well with serological determination of E beta expression. In the present study, we have extended our analysis to Ed beta expression in F1 hybrid mice. We show that Ed beta is relatively poorly expressed in three F1 combinations: H-2d X H-2b, H-2d X H-2s, and H-2d X H-2u. The former two crosses express E alpha chains from the H-2d parent only; when recombinant strains carrying Eb beta or Es beta and an active E alpha gene are used, Ed beta expression is significantly increased. On the other hand, H-2u mice synthesize E alpha chains; the poor expression of Ed beta chains in this F1 hybrid apparently reflects the strong preferential association of Eu beta chains with all E alpha molecules thus far analyzed. These results confirm that E beta chains compete for binding to E alpha chains and that preferential association of different allelic forms of E beta chains with E alpha chains is a generalized phenomenon. They also illustrate the importance of the rate of biosynthesis of Ia chains for cell-surface expression.

Efficiency of antigen presentation differs in mice differing at the Mls locus.

In the mouse, potent primary in vitro proliferation of T cells can be induced by allelic variants of cell-surface glycoproteins, Ia antigens, the genes for which are located in the I region of the major histocompatibility complex (MHC) on chromosome 17. The only other potent primary proliferative response known is induced by mixing MHC-identical lymphocytes from strains that differ at the locus termed Mls (ref. 1) (for mixed lymphocyte stimulating), which has been mapped to chromosome 1. While it is relatively easy to raise antibodies against Ia antigens, and thus determine both their chemical nature and their role in T-cell stimulation, the nature of the product of the Mls locus has remained obscure. It has been proposed that the Mls locus product is a minor antigen recognized in association with self-Ia antigens, a translocated Ia-like element, or a mitogenic molecule found on the surface of antigen presenting cells (APC). Here, we demonstrate that APCs from mice carrying stimulatory Mls locus alleles present antigen more efficiently to cloned, antigen-specific, Ia-restricted T cells than APCs from mice carrying nonstimulating Mls locus alleles. We propose that the Mls locus does not encode a unique cell-surface antigen at all; we suggest instead that the T-cell proliferative response induced by Mls-locus disparate cells reflects recognition of self-Ia molecules on APCs. If our interpretation is correct, it provides further evidence both for the quantitative nature of self tolerance and for the existence of a distinct recognition site for self-Ia molecules on antigen-specific T lymphocytes.

Differential expression of Ia glycoprotein complexes in F1 hybrid mice detected with alloreactive cloned T cell lines.

T cell lines alloreactive to Aeb:E alpha Ia antigen complexes have been prepared and used to determine the relative amount of Aeb:E alpha expressed by stimulator cells in homozygous recombinant and F1 mice expressing these complexes. We find that homozygous cells stimulate most strongly and therefore probably express the highest density of Aeb:E alpha. Among heterozygotes, H-2bxd F1 mice preferentially express Aeb:E alpha d complexes, H-2bxa F1 mice express relatively fewer Aeb:E alpha k complexes, and H-2bxu F1 mice express preferentially Aeu:E alpha u complexes. This differential association of Aeb chains with E alpha chains thus influences the biologic activity of these Ia antigens in this and other functional assays. The functional data are supported by biochemical analysis of Aeb:E alpha complex expression. These findings suggest that a reanalysis of HLA-DR associations with human diseases should be undertaken in which both HLA-DR alleles are included, with the prediction that certain combinations would show greater susceptibility, whereas others would show less susceptibility than predicted from the susceptibility associated with presence of a single allele at HLA-DR.