Biomedical subjects

M L Blue

Publications and source records attributed to M L Blue.

At least 19 recordsLinked to original sources

PubMed2001

Cloning, expression, purification, and characterization of rat MMP-12.

Macrophage metalloelastase (MMP-12) is implicated in the pathology of many diseases such as emphysema, aortic lesions and cancer. Recently, MMP-12 was cloned and purified from mouse and human macrophages. We report here the expression of the full-length and catalytic domain of rat MMP-12 in Escherichia coli and characterization of the purified enzyme. Inclusion bodies of expressed rat MMP-12 catalytic domain were denatured and refolded using a new method, and then affinity purified to near homogeneity with zinc-chelating Sepharose. The purified rat MMP-12 catalytic domain was highly active in digesting substrates, having a K(m) of 12 microM and optimal pH of 7.5--8.5. During investigation of natural substrate specificity, we found that rat MMP-12 catalytic domain was able to completely degrade collagen-V, partially degrade collagen-I, but it was unable to digest collagen-IV. The enzyme could also degrade osteonectin, vitronectin, and fibronectin, but not laminin and albumin. The catalytic properties and natural substrate specificity of rat MMP-12 catalytic domain differed from those of human MMP-12 catalytic domain.

Animals↗

PubMed1998

T-cell alterations in cardiac allograft recipients after B7 (CD80 and CD86) blockade.

BACKGROUND: T-cell activation requires engagement of the T cell receptor with the antigen-MHC and simultaneous ligation of the coreceptor CD28. CD28 binds both the CD80 (B7-1) and CD86 (B7-2) ligands on antigen-presenting cells. The functional role of these costimulatory pathways in transplantation is not completely understood. We tested the hypothesis that in vivo blockade of the CD28 pathway via the anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) would prolong allograft survival. METHODS: Neonatal C57BL/6J (H2b) hearts were transplanted to CBA/J (H2k) recipients in a heterotopic nonvascularized model, with anti-CD80 and/or anti-CD86 mAbs being administered intravenously at the time of allografting (day 0) and on the following day (day 1). RESULTS: Anti-CD80 mAb (29.8+/-1.5 days) and anti-CD86 mAb (30.8+/-0.5 days) alone significantly prolonged allograft survival compared with the isotype control (10.7+/-0.4 days, P < 0.01, Wilcoxon rank sum). The concurrent (days 0 and 1) and sequential administration of anti-CD86 mAb on days 0 and 1 plus anti-CD80 mAb on days 2 and 3 prolonged allograft survival to >80 days. Simultaneous administration of anti-CD80 and anti-CD86 mAbs significantly suppressed donor-specific cytotoxic T lymphocyte responses to alloantigen. Anti-CD86 mAb suppressed intragraft interleukin (IL)-4, IL-10, IL-12 p40, and IL-15 mRNA expression. CONCLUSIONS: Anti-CD80 and/or anti-CD86 mAbs are potent immunosuppressants in prolonging allograft survival. Combined blockade of the B7 (CD80 and CD86) ligands seems to be the most effective in prolonging allograft survival and suppressing donor-specific allogeneic cytotoxic T lymphocyte responses. In vivo blockade of CD86, in comparison to CD80, had the greatest immunosuppressive effect on day 7 intragraft cytokines, suggesting its role on early allogeneic immune responses.

Animals↗

PubMed1998

Divalent cations and ligands induce conformational changes that are highly divergent among beta1 integrins.

Here we show striking differences in conformational regulation among beta1 integrins. Upon manganese stimulation, a beta1 epitope defined by monoclonal antibody (mAb) 9EG7 was induced strongly (on alpha4beta1), moderately (on alpha5beta1), weakly (on alpha2beta1), or was scarcely detectable (on alpha6beta1 and alpha3beta1). Comparable results were seen for the beta1 epitope defined by mAb 15/7. Likewise, soluble ligands caused strong (alpha4beta1), moderate (alpha5beta1), weak (alpha2beta1, alpha6beta1), or minimal (alpha3beta1) induction of the 9EG7 epitope. Exchange or deletion of alpha chain cytoplasmic tails did not alter Mn2+-induced 9EG7 epitope levels. Upon removal of calcium by EGTA or EDTA, the hierarchy of 9EG7 epitope induction was similar (alpha5beta1 > alpha2beta1 > alpha6beta1 > alpha3beta1), except that EGTA reduced rather than induced 9EG7 expression on alpha4beta1. Thus in contrast to other beta1 integrins, calcium uniquely supports constitutive expression of the 9EG7 epitope on alpha4beta1. Likewise, calcium supported vascular cell adhesion molecule-stimulated 9EG7 appearance on alpha4beta1, whereas calcium inhibited ligand-induced 9EG7 epitope on other integrins. Constitutive expression of 9EG7 on alpha4beta1 was eliminated by a D698E mutation in alpha4, suggesting that Asp-698 may play a key role in maintaining atypical alpha4beta1 response to calcium. In conclusion, our results (i) demonstrate that mAb such as 9EG7 and 15/7 have limited diagnostic utility as reporters of ligand or Mn2+ occupancy for beta1 integrins, (ii) indicate pronounced differences in conformational flexibilities (alpha4beta1 > alpha5beta1 > alpha2beta1 > alpha6beta1 > alpha3beta1), (iii) allow us to hypothesize that beta1 integrins may differ markedly in conformation-dependent inside-out signaling, and (iv) have uncovered an atypical alpha4beta1 response to calcium that requires alpha4 Asp-698.

Amino Acid Sequence↗

PubMed1997

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.

Amino Acid Sequence↗

PubMed1996

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.

Animals↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1993

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.

Baculoviridae↗

PubMed1993

Cell contact between T cells and synovial fibroblasts causes induction of adhesion molecules and cytokines.

Human activated T cells adhere to synovial fibroblast-like cells in vitro. The present study was conducted to investigate the consequences of T cell-synovial fibroblast interactions with regard to induction of adhesion molecules and proinflammatory cytokines. A sensitive Western blot technique, polymerase chain reaction (PCR) amplification, and fluorescence-activated cell sorter (FACS) analysis were used to analyze the induction of VCAM-1 and ICAM-1 expression in T cell-synovial fibroblast cocultures. VCAM-1 and ICAM-1 expression could be induced in synovial fibroblast-like cells by 2 h. PCR amplification showed that both forms of VCAM-1 mRNA are found after the interaction of synovial fibroblasts with T cells. Up-regulation of VCAM-1 and ICAM-1 was confined to synovial fibroblasts; T cells did not express VCAM-1 or increased ICAM-1. In contrast to the T cell-synoviocyte interaction, the interaction between T cells and dermal fibroblasts resulted in the up-regulation of ICAM-1 but not VCAM-1, suggesting tissue-specific regulation of VCAM-1. The T cell-synovial fibroblast interaction also resulted in increased levels of tumor necrosis factor (TNF), interferon-gamma, and interleukin-6 in coculture supernatant. Of the neutralizing antibodies used against these cytokines, only anti-TNF could significantly inhibit VCAM-1 and ICAM-1 expression. When T cells were separated from synoviocytes by a chamber that allowed medium exchange but no cell contact, VCAM-1 and ICAM-1 failed to be up-regulated and cytokine accumulation in cocultures was drastically reduced. Our results demonstrate mutual cell activation of T cells and synoviocytes upon cell contact as shown by the release of T cell- and synoviocyte-specific cytokines and suggest a cell contact-mediated and T cell-initiated mechanism for the chronic accumulation and retention of mononuclear cells via VCAM-1/ICAM-1 by synovial fibroblasts in the rheumatoid synovium.

Arthritis, Rheumatoid↗

PubMed1993

Interacting monocytes and synoviocytes induce adhesion molecules by a cytokine-regulated process.

Monocytes/macrophages and synovial fibroblast-like cells are in intimate contact in the synovium and are believed to play a critical role in the development of rheumatoid arthritis. We investigated the effects of monocyte-synoviocyte interactions in vitro on cytokine release and the expression of adhesion molecules. Using a sensitive Western blot assay, we found that VCAM-1 and ICAM-1 expression were up-regulated in synoviocytes following coculture. The interaction also resulted in the accumulation of TNF and IL-6, but not IFN-gamma in the culture medium. Culture supernatant from monocyte-synoviocyte samples effectively induced adhesion molecules in synoviocytes. Anti-TNF partially inhibited the increase in VCAM-1 and ICAM-1 expression, indicating that TNF in part mediates VCAM-1 and ICAM-1 expression. Interestingly, the induction of cytokines and adhesion molecules did not require cell contact between monocytes and synoviocytes, suggesting cell communication via soluble factors. T cell cytokines enhanced the induction of adhesion molecules induced by the monocyte-synoviocyte interaction. IFN-gamma and IL-4, which are produced by distinct T helper subsets, had differential effects on monocyte-synoviocyte interactions. IFN-gamma had a minimal effect on VCAM-1 expression by synovial fibroblasts, but synergized with monocytes to dramatically up-regulate ICAM-1 expression. IL-4 had no effect on ICAM-1 expression but enhanced monocyte-induced expression of VCAM-1. Our results demonstrate that the up-regulation of adhesion molecules following monocyte-synoviocyte interactions is mediated by soluble factors and can be regulated by specific T cell cytokines.

Antigens, CD↗

PubMed1993

Specific cleavage of the alpha 4 integrin associated with activation of peripheral T lymphocytes.

The heterodimeric VLA-4 integrin has been implicated in lymphocyte migration to inflamed peripheral tissues, lympho-haemopoiesis and autoimmune disease. To determine the structure and function of VLA-4 in physiological processes, molecular forms of the VLA-4 alpha-chain were analysed during T-cell activation. The results showed that prolonged activation of human peripheral T cells was associated with increased cleavage of the 150,000 MW alpha 4 chain into 80,000 and 66,000 MW fragments. Similar-sized alpha 4 fragments could also be generated from 150,000 MW alpha 4 on intact resting T cells by brief trypsinization, whereas trypsin treatment of isolated 150,000 MW alpha 4 resulted in nearly complete protein degradation. Native 80,000 and 66,000 MW alpha 4 chains on activated T cells could not be digested further by trypsin. These results indicated that specific cleavage of 150,000 MW alpha 4 was largely dependent on the tertiary structure of native alpha 4 chains. To determine the specific cleavage site in alpha 4 on peripheral T cells, VLA-4 was isolated and purified from in vitro-activated T cells and the N-terminus of the 66,000 MW fragment was partially sequenced. The sequence SKR/STE was identified as the specific alpha 4 cleavage site on T cells. These results indicate that T cells, upon activation, acquire an enhanced dipeptidase processing activity, which cleaves alpha 4 at a specific site.

Amino Acid Sequence↗

PubMed1992

Mechanism of peripheral T cell activation by coengagement of CD44 and CD2.

A number of CD44 antibodies are known to augment peripheral T cell proliferation stimulated with suboptimal concentrations of activating pairs of CD2 mAb. These findings have implicated the CD44 adhesion receptor in the activation of peripheral T cells via CD2. We have investigated early events after CD44 and CD2 coengagement on peripheral T cells. CD44 and CD2 coengagement resulted in enhanced [Ca2+]i mobilization. However, the increase in [Ca2+]i mobilization did not occur until at least 3 min after CD2 and CD44 coengagement, suggesting that other events precede the elevation in [Ca2+]i. Using a T cell/fibroblast adhesion assay, we could demonstrate a dramatic increase in T cell adhesiveness after about 1 min after CD44 and CD2 coengagement. The increase in T cell adhesiveness was comparable to that induced by PMA. In the absence of antibodies or treatment with mAb directed to other T cell surface Ag, there was little if any adhesion between unstimulated peripheral T cells and fibroblasts. Enhancement of T cell adhesiveness through CD44 engagement was not mediated by a direct effect on lymphocyte-function associated Ag-3, the known ligand of CD2. However, cross-linking of CD44 resulted in epitopic modulation of CD2 as demonstrated by the increased expression of the T11(3) activation epitope. Furthermore, anti-CD44 could substitute for anti-T11(2) in the activation of peripheral T cells via CD2. These results suggest that CD44 ligation has profound effects on CD2-mediated events by inducing epitopic modulation of CD2.

Antigens, CD↗

PubMed1992

Expression of vascular cell adhesion molecule-1 in fibroblastlike synoviocytes after stimulation with tumor necrosis factor.

Rapid expression of mRNA encoding vascular cell adhesion molecule-1 (VCAM-1) was induced by tumor necrosis factor (TNF) in fibroblast-like cells obtained from synovial tissue. Both alternatively spliced forms of VCAM-1 mRNA were detected by polymerase chain reaction in TNF-stimulated fibroblast-like synoviocytes. Western blotting analysis showed that two distinct proteins, reactive with an anti-VCAM-1 anti-sera, were expressed by 2 hours of TNF stimulation in both synoviocytes and human umbilical cord vein endothelial cells (HUVEC). The majority of HUVEC and synoviocytes displayed VCAM-1 surface expression after several hours of TNF stimulation. In contrast, dermal fibroblasts upregulated intercellular adhesion molecule-1 (ICAM-1) but not VCAM-1 expression in response to TNF. These results indicate that VCAM-1 and ICAM-1 expression can be differentially regulated and suggest tissue specific regulation of VCAM-1 expression. Furthermore, these findings may provide an explanation for the chronic retention and activation of long-lived lymphocytes and monocytes, which express VLA-4 (the receptor for VCAM-1), in the synovium in rheumatoid arthritis.

Cell Adhesion Molecules↗

PubMed1991

Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.

Animals↗

PubMed1991

Expression and tyrosine phosphorylation of the T cell receptor zeta-subunit in human thymocytes.

Recent evidence suggests that the zeta-subunit of the TCR complex plays a critical role in transducing signals initiated by the Ag receptor heterodimer. Because thymic maturation involves specific interactions between the TCR complex and thymic stromal cells, the zeta-subunit has been postulated to also play a role in this process. To assess the potential for zeta to contribute to thymocyte maturation, we have used an anti-zeta mAb (TIA-2) to quantitate its expression in mature (CD3bright) and immature (CD3dim and CD3-) populations of human thymocytes. Using both flow cytometric and immunoblotting analysis, we found that the relative expression of TCR-zeta varied directly with the surface expression of CD3. Importantly, TCR-zeta was detected in the majority of CD3- thymocytes, indicating that its expression precedes the surface appearance of CD3:TCR. In thymocytes, TCR-zeta was found to be constitutively phosphorylated on tyrosine residues. The relative expression of phospho-zeta varied directly with the maturational stage of the thymocyte, with the mature (CD3bright), single positive cells accounting for most of the phospho-zeta found in the human thymus. The expression of phospho-zeta could be significantly increased by activating thymocytes with mAb reactive with either CD3 or CD2. These results suggest that TCR-zeta is functionally linked to the major thymocyte activation receptors.

Cell Differentiation↗

PubMed1991

Enhancement of CD2-mediated T cell activation by the interaction of VLA-4 with fibronectin.

Human fibroblasts were shown to enhance the proliferation of peripheral T cells in the presence of suboptimal concentrations of anti-CD2 antibodies (anti-T112 and anti-T113) and agonistic anti-VLA-4 antibody. Evidence is provided that the interaction of VLA-4 with immobilized fibronectin can enhance the proliferation of T cells subjected to suboptimal stimulation via CD2. Our results suggest that the fibroblast-stimulated T cell proliferative response to low doses of anti-T112 and T113 antibodies is due to the interaction of VLA-4 with fibroblast fibronectin. These findings also suggest a role for the fibronectin/VLA-4 interaction in the inflammatory process.

Antibodies, Monoclonal↗

PubMed1990

Differential regulation of Ca2+ mobilization in human thymocytes by coaggregation of surface molecules.

Variations in intracellular Ca2+ levels in developing thymocytes are likely to play a major role in both the activation-associated differentiation of thymocytes and in the selection or clonal deletion of cells. Here we examine the role of CD4, CD8, CD2, and CD45 in the regulation of intracellular Ca2+ levels in mature and immature thymocytes. Mature and immature thymocytes, distinguished on the basis of their CD5 expression, were analyzed simultaneously for their ability to mobilize Ca2+ after coaggregation of their CD3/TCR with other thymic surface Ag. Flow cytometric analysis by using Indo-1 showed that coaggregation of CD4, CD8, and CD2 with CD3/TCR clearly enhances a minimal signal delivered via CD3/TCR on immature thymocytes. Coaggregation with class I MHC had no discernible effect. The responsiveness of immature thymocytes correlated strictly with CD3 surface expression, such that loss of responsiveness occurred with reduced CD3 cell-surface density. However, even thymocytes with very low CD3 expression were able to respond to triggering via CD3 under optimal conditions, indicating that the CD3 signal-transducing mechanism is functional on early thymic cells. Intracellular increases in Ca2+ concentrations induced via CD3, could effectively be inhibited by cross-linking of CD45 and CD3 on immature thymocytes. Although triggering via CD2 alone induced a strong Ca2+ flux, prolonged incubation with activating anti-CD2 antibodies made thymocytes refractory to subsequent triggering. Refractoriness was associated with partial loss of surface CD3 and CD3 zeta. Our results indicate that thymic surface Ag are differentially involved in the regulation of intracellular Ca2+ levels in immature as well as mature thymocytes.

Antigens, CD↗

PubMed1990

A monoclonal antibody reactive with a 15-kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes.

We have recently described a novel method for the production and characterization of mAb reactive with T cell-restricted intracellular antigens. From a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells, we have selected one, designated TIA-1, that reacts with 20 to 36% of digitonin permeabilized peripheral blood T lymphocytes. Flow cytometric analysis of purified CD4+ and CD8+ subsets showed TIA-1 to recognize a subpopulation of 49 to 64% of CD8+ lymphocytes. Little or no reactivity with CD4+ resting T lymphocytes was observed. TIA-1 did not react with any of a panel of T cell lines, B cell lines, or monocytoid cell lines. TIA-1 reacted strongly with NK cell clones and CD8+ cytolytic T cell clones, and less strongly with CD4+-activated T cell clones, suggesting a preferential expression in cells possessing cytolytic potential. Cell fractionation experiments showed TIA-1 to be membrane associated. Furthermore, Percoll gradient fractionation of a cytolytic T cell clone (T4T8C1) showed the majority of TIA-1 to be contained in a low density membrane fraction that also contained serine protease activity. Immunoelectron microscopy showed TIA-1 to decorate the membranes of electron lucent and electron dense cytoplasmic granules in this same cytolytic T cell clone. Biochemical analysis showed TIA-1 to be a 15-kDa protein in unstimulated T cells. Upon activation with Con A or anti-CD3 antibodies. TIA-1 was induced to form disulfide linked dimers, trimers, and tetramers of the basic 15-kDa unit. Taken together, our data suggest that TIA-1 is a cytolytic granule associated protein that may define a subpopulation of resting CD8+ T lymphocytes possessing cytolytic potential.

Antibodies, Monoclonal↗

PubMed1989

Monoclonal antibodies reactive with the T cell receptor zeta chain: production and characterization using a new method.

We have developed a novel method for the production and characterization of monoclonal antibodies reactive with lineage-restricted intracellular Ag. Using this technique, we have produced a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells. One of these antibodies, designated TIA-2, was found to react with greater than 98% of peripheral blood T lymphocytes. Immunoblotting experiments showed TIA-2 to recognize a 32 kd protein that was reduced to 16 kDa in the presence of 2-mercaptoethanol. Immunoprecipitates analyzed on non-reducing/reducing diagonal polyacrylamide gels showed the homodimeric structure recognized by TIA-2 to be associated with additional structures whose pattern closely resembled that of the T cell receptor complex. When immunoprecipitates formed using antibodies reactive with CD3 epsilon were immunoblotted with TIA-2, the homodimeric TCR zeta chain was specifically recognized. Using TIA-2 as a TCR zeta specific reagent, we show that whole cell expression of this TCR subunit is dramatically reduced following exposure to mAb reactive with CD3. mAb reactive with activating epitopes of CD2 were also capable of down-modulating the expression of TCR zeta, but to a lesser degree. Exposure to Con A or IL-2, on the other hand, did not reduce the whole cell expression of TCR zeta. Given the central importance of TCR zeta in the expression of a functionally competent Ag receptor, its reduced expression in response to certain activating stimuli is likely to play an important role in regulating T cell responsiveness.

Animals↗